MPM Patients Research Articles

Clinical evaluation of circulating miR-548a-3p and -20a expression in malignant pleural mesothelioma patients.

miRNAs may act as promising diagnostic and prognostic biomarkers of mesothelioma. This study integrates serum miR-548a-3p and miR-20a expression based on in silico data analysis followed by clinical validation in malignant mesothelioma patients (malignant pleural mesothelioma [MPM]). Serum miR-548a-3p andmiR-20a level was assessed in the serum of patients with MPM, chronic asbestos exposure and healthy volunteers by quantitative real-time PCR. The expression of serum miR-548a-3p andmiR-20a was positive in 91.6 and 96.7% MPM patients, respectively. Both miRNAs were able to segregate between cases and controls. The sensitivity of the combined chosen serum miRNAs reached 100% in the diagnosis of MPM. The current work revealed that sera miR-548a-3p and miR-20a may serve as promising novel diagnostic tools for MPM.

Breath analysis by gas chromatography-mass spectrometry and electronic nose to screen for pleural mesothelioma: a cross-sectional case-control study.

RationaleMalignant pleural mesothelioma (MPM) is mainly caused by previous exposure to asbestos fibers and has a poor prognosis. Due to a long latency period between exposure and diagnosis, MPM incidence is expected to peak between 2020-2025. Screening of asbestos-exposed individuals is believed to improve early detection and hence, MPM management. Recent developments focus on breath analysis for screening since breath contains volatile organic compounds (VOCs) which reflect the cell’s metabolism.ObjectivesThe goal of this cross-sectional, case-control study is to identify VOCs in exhaled breath of MPM patients with gas chromatography-mass spectrometry (GC-MS) and to assess breath analysis to screen for MPM using an electronic nose (eNose).MethodsBreath and background samples were taken from 64 subjects: 16 healthy controls (HC), 19 asymptomatic former asbestos-exposed (AEx) individuals, 15 patients with benign asbestos-related diseases (ARD) and 14 MPM patients. Samples were analyzed with both GC-MS and eNose.ResultsUsing GC-MS, AEx individuals were discriminated from MPM patients with 97% accuracy, with diethyl ether, limonene, nonanal, methylcyclopentane and cyclohexane as important VOCs. This was validated by eNose analysis. MPM patients were discriminated from AEx+ARD participants by GC-MS and eNose with 94% and 74% accuracy, respectively. The sensitivity, specificity, positive and negative predictive values were 100%, 91%, 82%, 100% for GC-MS and 82%, 55%, 82%, 55% for eNose, respectively.ConclusionThis study shows accurate discrimination of patients with MPM from asymptomatic asbestos-exposed persons at risk by GC-MS and eNose analysis of exhaled VOCs and provides proof-of-principle of breath analysis for MPM screening.

Abstract 1721: Detection of CTCs in thoracic malignant tumors with "universal" CTC-chip

Abstract Background: Circulating tumor cells (CTCs) are tumor cells shed from primary tumor and circulate in the peripheral blood. CTCs, as a surrogate of distant metastasis, can be potentially useful in diagnosis and monitoring therapeutic effects in malignant tumors. Among a variety of systems for detection of CTCs, the “Cellsearch” is the only approved system for clinical use. However, EpCAM-negative tumor cells, such as those originating from non-epithelial cells and those undergoing epithelial-mesenchymal transition (EMT) cannot be captured with the “CellSearch” that is an EpCAM-based isolation system. Therefore, we have developed a novel polymeric microfluidic device (“Universal” CTC-chip) that can capture CTCs with or without EpCAM expression (AACR 2015). In the present study, we examined CTCs-detection performance of the CTC-chip in patients with thoracic malignant tumors (lung cancer [LC] as an “EpCAM-positive” tumor and malignant pleural mesothelioma [MPM] as an “EpCAM-negative” tumor) in comparison with that of the CellSearch. Methods: Peripheral blood sampled from each patient was divided and subjected to quantitative evaluation of CTCs with the CTC-chip as well as with the “CellSearch”. The CTC-chip, coated with an anti-EpCAM antibody, was used to capture CTCs in the blood samples (n=19) from lung cancer patients. To capture CTCs in the samples (n=11) from MPM patients, the CTC-chip was coated with an antibody against podoplanin that is expressed on the mesothelioma. After immuno-staining for cytokeratin and CD45 on the chip, a captured cell containing Hoechst-positive nucleus and cytokeratin-positive/ CD45-negative cytoplasm was judged as a CTC. The CTC-count for each sample was represented as the number per 7.5mL of the blood. Results: The median CTC-count detected with the CTC-chip in LC was 50 (range, 0-270), which was significantly higher than that (the median CTC-count, 0; range, 0-47) with the CellSearch (p<0.01). In the peripheral blood sampled from MPM patients, CTC was detected in only one patient using the CellSearch, but was detected in all 11 patients with the median CTC-count of 144 (range 0-470). Conclusion: The “universal” CTC-chip achieved higher performance in detection of CTCs of thoracic malignant tumors as compared with the CellSearch. The updated data will be presented at the AACR annual meeting 2017. Citation Format: Kazue Yoneda, Taiji Kuwata, Yasuhiro Chikaishi, Kenichi Kobayashi, Sakiko Yura, Hiroki Matsumiya, Masatoshi Kanayama, Akihiro Taira, Yusuke Nabe, Shinji Shinohara, Masaru Takenaka, Soichi Oka, Ayako Hirai, Yuko Tashima, Naoko Imanishi, Koji Kuroda, Fumihiro Tanaka. Detection of CTCs in thoracic malignant tumors with "universal" CTC-chip [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1721. doi:10.1158/1538-7445.AM2017-1721

Abstract 25: Efect of nimotuzumab on malignant pleural mesothelioma cell lines

Abstract Malignant Pleural Mesothelioma (MPM) is one of the most clinically aggressive malignancies, with a median survival time of less than 12 months. The monotherapy or combination of therapy with platinum-antifolate chemotherapy agents, may provide survival and life quality improvement in MPM patients. EGFR has been a target for new development drugs that could be used as oncologyc treatment. Nimotuzumab is a humanized antibody, design by Centro de Inmunología Molecular (Habana, Cuba), for treatment EGFR overexpress neoplasms. This antibody was approved for treatment of head and neck cancer and metastasic colorectal cancer. MPM exhibit an important expression of EGFR. In this study, we analyzed the efect of nimotuzumab on two cell lines derived of MPM and a xenograph model of one of them, in atimic nude mouse model. MSTO-211 and H2452 mesothelioma cell lines; MRC-5 normal lung cell line and A431 as EGFR positive cell line were used for all assays. We analyzed the viability of cell lines using violet cristal assays. Apoptosis were analyzed with Anexin-V-Fluos Staining kit accordin to manufacturer instructions. We analyzed the EGFR expression by WB assays. Finally, we generated a xenograft model with MSTO cells in nu/nu mice, and we treated a group with nimotuzumab or cisplatin-gemcitabine one doses/week. Nimotuzumab at any concentration (50, 100, 400 μg/mL) no modified the cellular viability, in both tumor and normal cells, the assays was followed from 24 h to 96 h after exposition. With respect at apoptosis, we no found significant diferences between control and exposured samples at 48 and 72 h after nimotuzumab treatment. In order to analyzed the EGFR expression in mesothelioma cell lines, we obtained protein total extract and then a WB was realized. The total protein from MSTO-211, H2452, A431 and MRC-5 cultures in normal conditions and exposed at nimotuzumab 100 μg/mL by 24h, were obtained; then a PAGE-SDS were realized. We observed that both mesotelioma derived cell lines showed similar EGFR expresion when it compared with A431 in normal culture conditions; while MRC-5 no showed EGFR expresion. When cellular cultures were exposed to nimotuzumab 100 μg/mL by 24h, H2452 and A431 cells showed similar EGFR expression while MSTO-211 showed high expression. Mice nu/nu (n=3) were subcutaneously inoculated in the dorsal right flank with MSTO-211 cells. Treatment was applied to animals when tumors were 50 mm3. First mice group received nimotuzumab, second group received cisplatin-gemcitabine; and third group no received treatmed. Nimotuzumab induced a reduction in tumor volumen and mice survival by 4 weeks more that those in the other groups. In vitro exposition of nimotuzumab in mesotelioma cell lines no affect their viability or induce apoptosis. According with the treatment scheme used in this work, nimotuzumab treatment induce a major survival and low toxicity in mice with mesothelioma pleural malignant xenograph. Citation Format: Saé Muñiz-Hernández, Vanessa Izquierdo-Sánchez, Jorge A. Mendoza-Desión, Carolina González-Torres, Oscar Arrieta. Efect of nimotuzumab on malignant pleural mesothelioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 25. doi:10.1158/1538-7445.AM2017-25

Abstract 2460: Copy number alterations in NOTCH2 and PTP4A3 are associated with prognosis and support novel therapeutic strategies for malignant pleural mesothelioma

Abstract Malignant pleural mesothelioma(MPM) is an aggressive cancer, which incidence has constantly increased over the past two decades and is expected to peak in 2020. The overall prognosis is poor, and predictive biomarkers of drug activity are missing. Given the heterogeneous and complex nature of MPM, it is likely that genomic aberrations changing the expression of several genes, might affect therapeutic response. Therefore, the aim of the present study was to identify genes whose copy number alterations might predict the MPM prognosis. Recurrent copy number alterations of genes were analyzed by high-resolution whole-genome sequencing in DNA obtained from a “discovery cohort” of 26 resected MPM patients treated with pemetrexed-based chemotherapy (8 with progressive disease, vs. 10 with stable disease and 8 with partial response). Prognostic markers identified by Copy Number Variation analysis with Nexus, Control-FREEC and ReadDepth software were validated by PCR gene copy number and gene expression analyses both in the “discovery” and in two “validation” cohorts of pemetrexed-treated and untreated patients (N=45 and 40). The role of emerging genes was evaluated through siRNA and pharmacological studies using proliferation, migration and apoptosis assays in MPM cells. As reported previously we observed copy number loss of CDKN2A (15.4%) and BAP1 (7.7%). Interestingly, copy number gain of NOTCH2 was observed in 50% of samples of the patients who underwent progression, whereas losses of PTP4A3 were associated with clinical benefit (SD+PR). The prognostic relevance of NOTCH2 was confirmed by PCR analysis. NOTCH-2 silencing reduced MPM cell migration and enhanced apoptosis induction by pemetrexed, while a PTP4A3 inhibitor overcame pemetrexed resistance in MPM cells characterized by high NOTCH2 and PTP4A3 expression. These results support the role of NOTCH2 as a novel prognostic/predictive biomarker for MPM, prompting prospective randomized trials for its validation. Moreover, preclinical data suggest that NOTCH2 and PTP4A3 are oncogenes suitable for effective therapeutic targeting in pemetrexed-resistant MPM cells. Citation Format: Leticia G. Leon, Maria Gemelli, Paolo A. Zucali, Christos Zoumadakis, Elisa Giovannetti. Copy number alterations in NOTCH2 and PTP4A3 are associated with prognosis and support novel therapeutic strategies for malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2460. doi:10.1158/1538-7445.AM2017-2460

Cetuximab plus platinum-based chemotherapy in patients with malignant pleural mesothelioma: A single arm phase II trial.

e20030 Background: Cetuximab is a chimeric monoclonal antibody targeting the epidermal growth factor receptor (EGFR). Previous studies suggested that cetuximab enhances chemotherapy activity in the presence of EGFR overexpression, which is present in a majority of MPM specimens. We investigated the activity and safety of the combination of platinum-based chemotherapy and cetuximab in EGFR immunohistochemical positive (IHC+) MPM patients (pts). Methods: This is an open, single arm multicenter phase II study according to a Simon 2-step design with progression-free survival (PFS) rate at 18 weeks as primary endpoint. PFS rate at 18 weeks in MPM pts with cisplatin-pemetrexed is 0.67. We hypothesized that the combination of cetuximab and chemotherapy would increase the PFS rate with 30% to 0.87 (alpha = 0.05, power 90%). 18 chemotherapy-naïve pts with epithelioid MPM and PS 0-1, EGFR IHC+ (assessed by DAKO kit with at least 1% of cells showing staining), were included in step 1 of in the study. If ≥14 were alive and free of progression at 18 weeks, sample size was to be increased to 43 pts. Treatment consisted of cisplatin (75 mg/m², day 1 of each 3-week cycle) or carboplatin AUC 5 (day 1 of each 3-week cycle) and pemetrexed (500mg/m2, day 1 of each 3-week cycle) and cetuximab (loading dose of 400 mg/m² followed by 250 mg/m² weekly) during chemotherapy until disease progression. Chemotherapy was given up to 6 cycles. Secondary end points were overall survival (OS), toxicity and response rate. Results: We enrolled 18 eligible pts between June 2010 and June 2013; 89% males, median age 68 years (range 57-80), 83% PS 1. Median number of cycles was 4 (range 2-6). Median number of weekly administrations of cetuximab was 25 (range 8-51). 13 pts were alive and without progression at 18 weeks, median PFS was 24 weeks (95% CI 18.5-29.5), median OS 49 weeks (95% CI 38.6-59.4). 44% (8/18) of patients obtained a partial response. The treatment was well tolerated; the most common adverse event was skin rash (89%), mainly grade 1-2. Conclusions: As platinum-based chemotherapy in combination with cetuximab in pts with epithelioid MPM did not reach the predefined threshold, the study was not continued. No new safety signal was observed. Clinical trial information: NCT00996567.

MTE29.02 Advances in Malignant Pleural Mesothelioma

MTE29.02 Advances in Malignant Pleural Mesothelioma

OA22.06 Refinement of the Prognostic miR-Score for Use in Diagnostic Specimens from Chemo-Naïve Malignant Pleural Mesothelioma Patients

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. 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. 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). 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.

SC06.03 Intraoperative Therapies in Malignant Pleural Mesothelioma

SC06.03 Intraoperative Therapies in Malignant Pleural Mesothelioma

P1.01-049 Predictors of High Grade Toxicity of Chemotherapy among Malignant Pleural Mesothelioma Patients

Malignant pleural mesothelioma is an aggressive thoracic malignancy associated with exposure to asbestos, and its incidence is anticipated to increase during the first half of this century. Chemotherapy is the mainstay of treatment, yet sufficiently robust evidence to substantiate the current standard of care has emerged only in the past 5 years. A retrospective cohort study of 100 MPM patients referred to NCI, Cairo University in 3 years. Detailed data, Pearson's Chi (×2) square and Logistic regression model were used for statistical analysis. We found a statistical significant relation between age (0.005), male gender (0.002), endemic area residence (0.001), industrial workers (0.018), duration of exposure (0.04), smokers (0.009), Simian virus (0.019), P53 (0.001), RbP (0.001), PS (0.0.16) and development of high grade toxicity of platinum based chemotherapy. Median age = 46 years, only 17% of cases developed high grade toxicity complications of platinum based chemotherapy. Males were 59% of cases. PS, residence, smoking, occupation, history of asbestos exposure, family history, simian virus, P53, Rbp, dyspnea, chest pain, cough, expectoration, hemoptysis, weight loss, fatigue, metastatic symptoms, chronic lung infection, Tuberculous pleuritic, effusion, pleural thickening, Tracheal shift, TNM staging, surgical operations, pathological staging, radiotherapy, cause of death and chemotherapy toxicity are assessed in our patients. Many factors predict high grade chemotherapy toxicity. So, search for target therapy and immunotherapy instead of chemotherapy in this selected group can improve both quality of life and response rate.

P3.03-034 Comprehensive Immunophenotyping of the Blood and Pleural Fluid from Patients with Malignant Pleural Mesothelioma by Flow Cytometry: Topic: Mesothelioma Clinical

Malignant pleural mesothelioma (MM) remains an almost universally fatal disease with limited treatment options. Immunotherapy represents a rapidly emerging therapeutic strategy for multiple malignancies including MM. However durable therapeutic responses occur in a minority (∼30%) of patients. Better understanding of the quantities and immunophenotype of circulating and intrapleural immune cells is essential to design and apply personalized immunotherapeutic strategies. We used a comprehensive flow cytometry panel (PLoS One. 2015 Mar 23;10 (3):e0121546) to prospectively characterize circulating and pleural fluid immune cells in patients with MPM (n=12) and normal volunteers (circulating cells only, n=50). Matched blood and pleural samples were available from 11 patients, including samples from 9 patients enrolled into a Phase I study investigating the intrapleural administration of the modified vaccine strain measles virus (MV-NIS), MC1023. Pre- and post-treatments samples were available from 7 patients. The immune cell counts and cell fractions were compared using a false discovery rate (FDR) of 10% and the non-parametric Mann-Whitney test and the Wilcoxon matched-pairs signed rank test (blood versus pleural fluid and different time points) (p ≤ 0.05). Cell counts and immune phenotype of circulating immune cells differ between of patients with MPM differ from normal volunteers (31 of 86 cell types). Patients with MPM had fewer B-lymphocytes, more pro-inflammatory monocytes (CD14+CD16+) and exhausted CD4 and CD8 T-lymphocytes (CD4 and CD8 PD1+TIM3+ double positive cells). As expected we observed characteristic differences between blood and pleural fluid in MPM patients. For example there are a higher numbers of antigen experienced, PD-1 expressing CD4 and CD8-memory cells within the pleural fluid compared to the peripheral blood. The intrapleural administration of a modified vaccine strain measles (MV-NIS) triggered changes in pleural and circulating immune cells. While these changes varied among patients, we observed increased CD80 and CD86 expression on CD14+ monocytes in the pleural fluid down. Comprehensive flow-cytometric immunophenotying of blood and pleural fluid is feasible in patients with MPM. This approach may help to identify patients who may respond favorably to specific immunotherapeutic interventions such as immune checkpoint inhibitors or facilitate longitudinal immune monitoring during clinical trials. Additional data is needed and we are continuing to prospectively analyze samples from patients with MPM.

P3.03-029 EPP Followed by Hemithoracic IMRT - A Multicenter Study on Behalf of the Italian Association for Radiation Oncology Lung Cancer Study Group: Topic: Mesothelioma Clinical

The treatment of MPM is not well defined. Although it has been shown that extrapleural pneumonectomy (EPP) followed by hemithoracic RT may improve survival in early-stage disease, this therapeutic approach is declining. This might be due to the recent publication of a randomized trial (SAKK 17/04) which assessed the effect of high-dose hemithoracic RT after EPP. The study reported poor outcomes and the results did not support the use of hemithoracic RT. The major weakness of the SAKK study is the heterogeneity of the radiation techniques and schedules, and that only 12 patients received IMRT. On this background we conducted the present study to assess the outcome of MPM patients treated with EPP and adjuvant IMRT. This is a retrospective multicenter study, including 5 academic centers in Italy. Seventy patients treated with EPP and adjuvant IMRT were enrolled. The majority (95%) of patients had an epithelioid histology. Sixty patients were affected by stage III-IVA disease. Fifty-four (77%) patients received neoadjuvant/adjuvant chemotherapy. The IMRT dose ranged between 50-60 Gy in 25-27 fractions. Radiation was interrupted in two patients due to systemic progression of disease. Median follow-up was 14 months (range, 0-83 months). Rates of local control, loco-regional control, distant-metastases free survival (DMFS) and overall survival (OS) at 2 years were 73%, 63%, 39%, and 63%, respectively. Patients with stage I-II had a better 2-year OS and DMFS than those with advanced disease: 60% vs. 40% (p=0.09), and 78% vs. 32% (p=0.03), respectively. Three fatal pneumonitis were reported. Other major toxicities were: Grade 2-3 pneumonitis in 2 cases, 1 bronchial fistula, and 1 Grade 3 esophagitis. This multicenter study showed that EPP followed by hemithoracic IMRT is associated with high rate of local and loco-regional control, and showed that long-term survival can be achieved, even if some patients may experience life-threatening lung toxicity. Distant metastasis represent the predominant pattern of failure.

Prognostic and predictive aspects of the tumor immune microenvironment and immune checkpoints in malignant pleural mesothelioma

ABSTRACTMalignant pleural mesothelioma (MPM) is an aggressive cancer with a poor prognosis and an increasing incidence, for which novel therapeutic strategies are urgently required. Since the immune system has been described to play a presumed role in the protection against MPM, characterization of its tumor immune microenvironment (TME) and immune checkpoints can identify new immunotherapeutic targets and their predictive and/or prognostic value.To characterize the TME and the immune checkpoint expression profile, we performed immunohistochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) tissue sections from 54 MPM patients (40 at time of diagnosis; 14 treated with chemotherapy). We stained for PD-1, PD-L1, TIM-3, LAG-3, CD4, CD8, CD45RO, granzyme B, FoxP3 and CD68. Furthermore, we analyzed the relationship between the immunological parameters and survival, as well as response to chemotherapy. We found that TIM-3, PD-1 and PD-L1 were expressed on both immune and tumor cells. Strikingly, PD-1 and PD-L1 expression on tumor cells was only seen in unpretreated samples. No LAG-3 expression was observed. CD45RO expression in the stroma was an independent negative predictive factor for response on chemotherapy, while CD4 and TIM-3 expression in lymphoid aggregates were independent prognostic factors for better outcome. Our data propose TIM-3 as a promising new target in mesothelioma. Chemotherapy influences the expression of immune checkpoints and therefore further research on the best combination treatment schedule is required.

Abstract 2936: Molecular profiling of primary malignant pleuralmesothelioma histotypes cell lines reveals relationship between GDF15overexpression and cisplatin resistance both in in vitro and in vivo models

Abstract Malignant pleural mesothelioma (MPM) development is mainly correlated with exposure to asbestos and appears to be a trend toward an increase in its incidence in the years to come. Although novel therapeutic strategies are under investigation, alone or in combination with conventional therapy, MPM remains a cancer with high medical need. With the aim to identify putative novel therapeutic targets/biomarkers expressed in specific MPM histotypes we investigated 9 different primary MPM cell lines at molecular, biochemical and pharmacological levels. A transcriptomic analysis has been conducted comparing the gene expression in epitheliod (E-MM473), sarcomatoid (S-MM432) and biphasic (B-MM487) primary cell lines. We found more than 1000 genes differentially regulated and statistically validated (p< = 0.05). The hierarchical clustering of the top 100 expressed genes revealed an histotype specific expression while few of them have a similar expression among the histotypes. 14 out of 100 genes were chosen based on their role in cell survival (hk1; gdf15; ifnar1/2; bmpr1/2; tgfrb; cdca8; birc5; plk1; srsf6; pin1; nox1; wt1). Their expression was confirmed via RT-PCR in all the 9 MPM cell lines (E-MM473, E-MM288, E-MM317, E-MM404, E-MM481; S-MM432, S-MM472; B-MM487, B-MM491) vs normal mesothelium cells (MET5A). Among the validated genes, gdf15 and hk1 expression strongly correlated with the epithelioid and bifasic histotype, respectively. The tumor relevance of these genes has been confirmed by siRNA interference in which hk1 and gdf15 transcript reduction significantly affected S-MM432 (>90%) and E-MM473/B-MM487 cell survival (>50%). GDF15 belongs to the TGF-β superfamily and its role in different pathologies including cancer is controversial (Breit et al., 2011). Its overexpression has been associated to tumor chemoresistance (Chung-Ying et al.,2007) including Cisplatin (Cis) (Li et al., 2015; Yang et al. 2014). To this end, we investigated if GDF15 protein levels in MPM cells exposed to Cis correlate with its citotoxicity. Interestingly GDF15 protein level, which is high in E-MM473, medium in B-M M432 and low in S-MM487, is inversely correlated with the cells sensitivity to the treatment (i.e. E-MM473 are 2-5 times more resistant) and GDF15 silencing increases Cis-induced cytotoxicity. To assess the relationship between GDF15 levels/ Cis resistance, efficacy of Cis (4mg/kg; q7dx3w;i.v.) on stably expressing luciferase E-MM473(highGDF15) and B-MM487 (lowGDF15), orthotopically xenografted in nude mice, were investigated. While, E-MM473 showed a poor response to the Cis (TGI 58%), an impressive tumor response (TGI 95%) was observed in treated B-MM487. Our results suggest and support the need of further clinical investigation to correlate GDF15 plasma level to the chemotherapy response in MPM patients. Citation Format: Claudio Pisano, Gianluca Carletta, Fulvio D’Angelo, Antonietta Esposito, Erminia Bianchino, Assunta Riccio, Michela Festa, Francesco Cardile, Emanuele Carchia, Walter D’Acunto, Giacomo Signorino, Mario B. Guglielmi, Pasquale De Luca. Molecular profiling of primary malignant pleuralmesothelioma histotypes cell lines reveals relationship between GDF15overexpression and cisplatin resistance both in in vitro and in vivo models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2936.

Abstract 108: Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas

Abstract Malignant pleural mesothelioma (MPM) is a rare malignancy with a highly unfavorable prognosis. A strong link has been established between increased risk for MPM and exposure to asbestos or erionite. As asbestos had widely been used in different industries, the incidence of MPM in the United States is expected to steadily rise and peak with about 70,000 new MPM cases over the next 20 years. Median survival has ranged from 10-17 months. But the underlying genetic mechanism is not fully understood. Moreover, genetic alterations and causes for multiple primary cancer development including MPM are unknown. We used whole exome sequencing to identify somatic mutations in a patient with MPM and two additional primary cancers who had no evidence of venous, arterial, lymphovascular, or perineural invasion indicating dissemination of a primary lung cancer to the pleura. The development of multiple primary malignancies including a rare MPM led us to search for underlying genetic alterations. Interestingly, most mutations identified in the MPM patient were highly enriched for the mutant allele, suggesting a homozygous alteration or deletion of wild-type allele when minimal contamination of normal pleura in the MPM is taken into consideration. We found that most variants identified showed a high frequency of mutant alleles, except variants on chromosomes 7, 16 and 20. Analysis of tumor allelic ratio to normal using all variants in exome sequencing revealed that this mesothelioma showed genome-wide allelic loss or loss of heterozygosity (LOH), which appears to be distinct from other cancers that have many genetic alterations with focal allelic loss. To the best of our knowledge, this type of extensive genome-wide allelic loss has not been described in MPM. Whole exome sequencing analysis revealed the MPM had R282W, a key TP53 mutation, and genome-wide allelic loss or loss of heterozygosity, a distinct genomic alteration not previously described in MPM. We identified frequent inactivating SETDB1 mutations in this patient and in 77 additional MPM patients (mutation frequency: 9%, 7/78) by targeted deep sequencing. In summary, we identified genome-wide allelic loss in a patient who had MPM and two additional primary cancers, results which suggest that careful analysis in exome sequencing is needed to detect genome-wide deletion in MPM samples with or without multiple primary cancers. The high frequency of mutations in SETDB1 that we found suggests that this and other histone-related genes are important in MPM. Citation Format: Hio Chung Kang, Hong Kwan Kim, Sharon Lee, Pedro Mendez, James Kim, Gavitt Woodard, Kuang-Yu Jen, Li Tai Fang, Kirk Jones, David Jablons, Il Jin Kim. Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 108.

Abstract 3764: Trabectedin activity in patient-derived mesothelioma xenografts

Abstract Malignant pleural mesothelioma (MPM) incidence is increased over the past two decades and it is predicted to increase further in the next 20 years especially in developing countries where asbestos has not yet been banned. Surgery is not an option for the majority of patients due to the diffuse growth of MPM, making chemotherapy the treatment of choice, but prognosis remains very poor with a median survival time of 10-17 months. Trabectedin (ET-743, Yondelis) is a marine alkaloid used for the 2nd line therapy of ovarian cancer (combined with pegylated doxorubicin) and soft tissue sarcomas. It binds the DNA minor groove modulating transcription of cancer cells and has an anti-inflammatory effect modifying tumor microenvironment and reducing tumor associated macrophages. The aim was to test antitumor activity of trabectedin in newly established patient derived xenografts of MPM. MPM473, MPM484 and MPM487 xenografts were obtained in athymic nude mice by s.c. injection of 107 cells isolated from the pleural effusion of MPM patients and maintained by serial passages in mice. H&E staining and immunohistochemistry were performed. Mice were randomized when tumor reached about 200 mg to receive trabectedin, cisplatin, pemetrexed, gemcitabine, and imatinib. Antitumor activity was expressed as T/C% were T and C are the mean tumor weight of treated and control mice, respectively. The three xenografts reproduced the morphology and the histochemical profile of the human tumors. As reported in the table trabectedin was able to induce a 50% reduction in tumor growth in MPM473 and MPM 487 xenografts and only a marginal effect in MPM484 xenograft. TreatmentDose (mg/kg)ScheduleBest T/C% (Day)MPM473MPM484MPM487Cisplatin5i.v. q7dx361.9 (69)66.1 (69)67.3 (39)Gemcitabine100i.p. q4dx583.2 (41)67.7 (48)49.7 (56)Imatinib200p.o. qdx1568.9 (76)89.4 (55)87.1 (39)Pemetrexed250i.p. q4dx477.4 (52)76.8 (49)84.3 (39)Trabectedin0.15i.v. q7dx351.0 (73)63.6 (69)51.3 (56) In conclusion we report three new MPM xenografts able to mimic the pathological features of human MPM. Although trabectedin showed only a moderate antitumor activity in our experimental models, its efficacy is comparable or better than that of the other drugs currently used for this disease. Studies are in progress in syngenic models that are more appropriate to test also the ability of trabectedin to modulate the immune system. Citation Format: Simonetta Andrea Licandro, Roberta Frapolli, Ezia Bello, Roberta Libener, Sara Orecchia, Federica Grosso, Maurizio D’Incalci. Trabectedin activity in patient-derived mesothelioma xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3764.

WE-FG-206-06: Dual-Input Tracer Kinetic Modeling and Its Analog Implementation for Dynamic Contrast-Enhanced (DCE-) MRI of Malignant Mesothelioma (MPM)

Purpose: To use dual-input tracer kinetic modeling of the lung for mapping spatial heterogeneity of various kinetic parameters in malignant MPM Methods: Six MPM patients received DCE-MRI as part of their radiation therapy simulation scan. 5 patients had the epitheloid subtype of MPM, while one was biphasic. A 3D fast-field echo sequence with TR/TE/Flip angle of 3.62ms/1.69ms/15° was used for DCE-MRI acquisition. The scan was collected for 5 minutes with a temporal resolution of 5ߝ9 seconds depending on the spatial extent of the tumor. A principal component analysis-based groupwise deformable registration was used to co-register all the DCE-MRI series for motion compensation. All the images were analyzed using five different dual-input tracer kinetic models implemented in analog continuous-time formalism: the Tofts-Kety (TK), extended TK (ETK), two compartment exchange (2CX), adiabatic approximation to the tissue homogeneity (AATH), and distributed parameter (DP) models. The following parameters were computed for each model: total blood flow (BF), pulmonary flow fraction (γ), pulmonary blood flow (BF_pa), systemic blood flow (BF_a), blood volume (BV), mean transit time (MTT), permeability-surface area product (PS), fractional interstitial volume (vi), extraction fraction (E), volume transfer constant (Ktrans) and efflux rate constant (kep). Results: Although the majority of patients had epitheloid histologies, kinetic parameter values varied across different models. One patient showed a higher total BF value in all models among the epitheloid histologies, although the γ value was varying among these different models. In one tumor with a large area of necrosis, the TK and ETK models showed higher E, Ktrans, and kep values and lower interstitial volume as compared to AATH and DP and 2CX models. Kinetic parameters such as BF_pa, BF_a, PS, Ktrans values were higher in surviving group compared to non-surviving group across most models. Conclusion: Dual-input tracer kinetic modeling is feasible in determining micro-vascular characteristics of MPM. This project was supported from Cycle for Survival and MSK Imaging and radiation science (IMRAS) grants

Gender-Specific Molecular and Clinical Features Underlie Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive cancer that occurs more frequently in men, but is associated with longer survival in women. Insight into the survival advantage of female patients may advance the molecular understanding of MPM and identify therapeutic interventions that will improve the prognosis for all MPM patients. In this study, we performed whole-genome sequencing of tumor specimens from 10 MPM patients and matched control samples to identify potential driver mutations underlying MPM. We identified molecular differences associated with gender and histology. Specifically, single-nucleotide variants of BAP1 were observed in 21% of cases, with lower mutation rates observed in sarcomatoid MPM (P < 0.001). Chromosome 22q loss was more frequently associated with the epithelioid than that nonepitheliod histology (P = 0.037), whereas CDKN2A deletions occurred more frequently in nonepithelioid subtypes among men (P = 0.021) and were correlated with shorter overall survival for the entire cohort (P = 0.002) and for men (P = 0.012). Furthermore, women were more likely to harbor TP53 mutations (P = 0.004). Novel mutations were found in genes associated with the integrin-linked kinase pathway, including MYH9 and RHOA. Moreover, expression levels of BAP1, MYH9, and RHOA were significantly higher in nonepithelioid tumors, and were associated with significant reduction in survival of the entire cohort and across gender subgroups. Collectively, our findings indicate that diverse mechanisms highly related to gender and histology appear to drive MPM.

Abstract 4236: FAK inhibitor VS-6063 (defactinib) targets mesothelioma cancer stem cells, which are enriched by standard of care chemotherapy

Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor in the lining of the lung often resulting from prior exposure to asbestos. Median overall survival with standard of care chemotherapy is only 12 months from diagnosis. This poor prognosis may be attributable at least in part to cancer stem cells (CSCs) which are resistant to chemotherapy and can mediate cancer recurrence and progression. Focal adhesion kinase (FAK) has been shown to play an essential role in the survival, self-renewal and tumor-initiating capability of CSCs. Accordingly, the FAK inhibitor VS-6063 is currently being tested in patients with MPM following disease control on standard pemetrexed/platinum chemotherapy (COMMAND, ClinicalTrials.gov NCT01870609). Aldehyde dehydrogenase (ALDH) activity was validated as a CSC marker in MPM by assessment of tumor-initiating capability in mice. As compared to ALDH-negative cells, sorted ALDH-positive MM87 MPM cells showed 35-fold greater tumor initiating capacity when implanted in limiting dilutions into immunodeficient mice. Indeed, 50 ALDH-positive cells were sufficient to generate sizeable tumors in 3 weeks illustrating the aggressive nature of MPM CSCs. Treatment of a human MPM cell line with pemetrexed enriched ALDH-positive CSCs 6-fold, with similar CSC enrichment by cisplatin. In direct contrast, the FAK inhibitor VS-6063 markedly reduced the proportion of CSCs. The enrichment of MPM CSCs was similarly observed in samples from 11 tested patients following first line chemotherapy. Patient specimens post treatment with pemetrexed and cisplatin showed an elevated ALDH immunohistochemistry H-score and an increase in expression of CSC genes such as CD133 as compared to matched MPM biopsies taken from the same patients prior to chemotherapy. To assess drug effects on tumor-initiating capacity, MM87 merlin-low MPM and H28 merlin-high MPM cell lines were treated in vitro with VS-6063, pemetrexed or the combination and subsequently implanted into mice. While control and pemetrexed-treated MPM cells showed robust tumor initiation, cells treated with VS-6063 alone or VS-6063 plus pemetrexed showed little or no tumor initiating capacity. Accordingly, in tumor biopsies from MPM patients treated for 12 days with VS-6063, tumor pFAK (Y397) and expression of CSC genes such as CD133 were reduced. MPM patient-derived xenograft (PDX) tumors were employed to model the clinical scenario. Compared to control, tumor growth was blocked by 2-week treatment with pemetrexed/cisplatin. Tumors then grew rapidly upon cessation of pemetrexed/platinum treatment, whereas tumor growth was substantially delayed by FAK inhibitor treatment after cessation of chemotherapy. These data provide strong rationale for the current clinical testing of VS-6063 following treatment with pemetrexed plus platinum to potentially prolong time to progression in patients with mesothelioma. Citation Format: Jonathan A. Pachter, Vihren N. Kolev, Laurel Schunselaar, Irina M. Shapiro, Raphael Bueno, Paul Baas, Qunli Xu, David T. Weaver. FAK inhibitor VS-6063 (defactinib) targets mesothelioma cancer stem cells, which are enriched by standard of care chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4236. doi:10.1158/1538-7445.AM2015-4236

Abstract 184: miR-379/411 cluster regulates IL-18 and contributes to drug resistance in malignant pleural mesothelioma

Abstract Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence in Japan. In this study, we performed gene and microRNA (miRNA) expres¬sion profiling to identify novel therapeutic targets in MPM cells. Based on relative sensitivities to pemetrexed (PEM) and the histone deacetylase (HDAC) inhibitor, vorinostat (SAHA), 211H cells were determined to be the only sensitive MPM cell line out of the 6 tested. On the same series of cell lines, we performed whole genome transcriptomic profiling via DNA microarrays and pathway analysis of the derived data. Of particular note, IL-18 gene expression levels were signifi¬cantly higher in the cell lines that were either drug resistant or displayed intermediate sensitivity, compared to the sensitive 211H cell line. Pathway analysis revealed IL-18 as an impor¬tant gene associated with drug sensitivity of MPM cells. A relationship between IL-18 overexpression and drug resistance was also observed following targeted assessment of 10 cyto¬kine genes using quantitative RT-PCR. miRNA expression profiles were evaluated in the MPM cell line panel in order to discern the mechanism of IL-18 induction in the drug-resistant lines. We found that miR-379 and miR-411 belonged to the same cluster of miRNAs located on chromosome 14q32 that commonly target the IL-18 gene. Luciferase reporter assays revealed that miR-379 and miR-411 directly target the IL-18 gene. Introduction of miR-379 plus miR-411, as well as IL-18 silencing, significantly suppressed the invasive capacity of MESO1 cells in vitro. Furthermore, the use of either PEM or SAHA together with miR-379 plus miR-411 mimics mediated increased sensitivity to these drugs in MESO1 cells. These results suggest that the miR-379/411 cluster may provide new therapeutic opportunities for advanced MPM patients, depending on the nature of IL-18 gene expression. Citation Format: Akihiko Miyanaga, Masahiro Seike, Kazuo Yamamoto, Susumu Takeuchi, Rintaro Noro, Yuji Minegishi, Kaoru Kubota, Akihiko Gemma. miR-379/411 cluster regulates IL-18 and contributes to drug resistance in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 184. doi:10.1158/1538-7445.AM2015-184