Virtual Library

Background:
This report is on behalf of the Mesothelioma Domain (MD) of the IASLC International Staging and Prognostic Factors Committee (ISC). The ISC MD previously developed the largest international staging database in MPM and analyzed outcomes and prognostic factors. (JTO 2012:1631-1639 and 2014:856-864).These results indicated the need for more granular TNM data to inform revisions of the staging system for the upcoming 8th edition of the AJCC/UICC staging manuals. We report analyses of this new MPM database.

Methods:
The MD established a new data dictionary with more detailed information about TNM descriptors and permitting electronic data capture. Minimum case submission requirements: complete clinical and/or post-surgical TNM stage with anatomical descriptors to support stage designation, accurate survival information, no conflict between descriptors and reported stage, and node positivity recorded by individual station. Overall survival analyzed by Kaplan-Meier and significance of individual T,N, and M descriptors evaluated by logrank and Cox regression.

Results:
3,519 cases treated 1995-2014 were submitted from 31 centers or consortia. 1,069 cases were excluded due to timing of presentation (244), missing dates (196), conflicting or incomplete stage information (615) or incorrect cell type (14). Geographic source for remaining 2,450 cases was: Europe 33%, North America 36%, Turkey 12%, Asia 10%, Australia 9%. Stage available: clinical (cTNM) only 34%; post-surgical (pTNM) only 33%; both 34%. A total of 1,982 cases (81%) underwent surgery (43% EPP, 23% PD, 8% partial pleurectomy, 26% exploration without resection). 5 year overall survival (OS) for any N, M0 showed no difference for T1a versus T1b or for post-surgical T2 versus T3. 5 year OS for any T, M0 showed no difference for N1 versus N2 (Table 1). Median and 5 year OS by stages I-IV were similar to those reported from original database. Table 1. Median overall survival times (MST), 2-year, and 5-year overall survival rates for pre-treatment and post-surgical stage categories. Figure 1



Conclusion:
While additional analyses are ongoing, these initial results suggest some changes in the current MPM staging system are warranted, especially regarding T categories.

Background:
Uncertainty surrounds the long term benefits of extended pleurectomy decortication(EPD). In the absence of randomized controlled evidence enabling informed consent for such a major procedure with little prospect of cure is challenging. We have reviewed the largest series of EPD procedures in the UK to provide existing selected evidence for decision making and future research surrounding radical surgery for mesothelioma.

Methods:
We retrospectively analysed the case notes and pathological reports of 266 patients who underwent EPD over the last 15 years to determine length of hospital stay, complication rates and survival.

Results:
Overall survival was: 48.0% at 1 year, 10.3% at 3 years and 2.7% at 5 years. In the most favourable subgroup, those with epithelioid pN0 pathology, the 1, 3 and 5 year survivals were 64.9%, 17.5%, and 5.2% respectively. Overall median survival was 12.2 months, ranging from 23.1 months in those with epithelioid pN0 disease to 6.2 months in those with non-epithelioid, node positive tumours. Post-operative mortality was 3.8% at 30-days and 9% at 90 days. Median length of hospital length of stay was 13 (5-70) days. Re-operation was required in 20 patients (11.9%). A significant increase in postoperative hospital stay was associated with: postoperative atrial fibrillation(14 vs. 20 days p=0.037); persistent air leak(19 vs. 13 days p<0.001); postoperative empyema(40 vs.14 days p<0.001) and subsequent removal of the prosthetic neodiaphragm(21 vs. 14 days p=0.013). Postoperative 30-day mortality was significantly higher in those patients who developed pneumonia(15.8% vs. 3.2% p=0.048). Postoperative 90-day mortality was significantly increased in those who developed a pleural empyema(71.4 v. 8.6% p=0.001), similarly overall survival was reduced in this group(3.1 vs. 12.7 months p=0.072). Duration of intercostal drainage was significantly associated with the development of an empyema(p<0.001) and with the incidence of prosthetic dehiscence of the neodiaphragm(p=0.042). Revisional surgery to remove an infected prosthesis had no detrimental effect on 30 or 90-day mortality, or on overall survival Adjuvant chemotherapy significantly increased overall survival (18.1 vs. 8.2 months p<0.001), but 22.7% patients with neodiaphragm dehiscence, and 28% of those with empyema, did not receive this due to these complications.

Complication	Rate (%)
Persistent air leak	31.0
Atrial Fibrillation	16.7
Pneumonia	8.7
Diaphragmatic patch dehiscence	8.7	Mechanical	22.9 %
		Infection	77.1 %
Empyema	4.8
Wound infection	4.4
Thromboembolic	6.3
Chylothorax	3.6

Conclusion:
Extended pleurectomy decortication(EPD) can be performed in high volume centres with acceptable risk. In all but a selected subgroup it remains a palliative procedure. Thus, reducing postoperative air leak, which increases pleural sepsis and perioperative risk and decreases adjuvant chemotherapy, is paramount. The true role of EPD can only be answered by a prospective randomized comparison with non-surgical treatment.

Background:
Clinical staging of malignant pleural mesothelioma (MPM) is challenging due to the unique morphology of the tumor, macroscopic resolution and lack of radiographic contrast between tumor and adjacent structures and the number and complexity of anatomic features comprised by the descriptors. Recent analysis of a large IASLC MPM database revealed discrepancy between clinical (cTNM) and pathological (pTNM) staging (J Thorac Oncol 2012;7: 1631–1639). The current study examined in a retrospective cohort the concordance between cTNM and pTNM stage, the accuracy of individual clinical T and N features in predicting corresponding pathological features, and the prognostic significance of each feature.

Methods:
An IRB approved MPM registry was queried to identify patients who had undergone extrapleural pneumonectomy with complete pathological evaluation and who had preoperative CT or PET-CT scans available for review. All scans were assigned binary scores at the level of individual features by a single chest radiologist (R.G.) with significant experience with MPM. Corresponding scores for pathological features were obtained from the registry database along with histological subtype and overall survival (OS). cTNM and pTNM stage were assigned according to AJCC/UICC 7[th] edition criteria. Taking pTNM as gold standard, each case was scored as concordant, understaged or overstaged by cTNM. Sensitivity, specificity and univariate hazard ratio (HR) for death were determined for individual cT and cN features.

Results:
Inclusion requirements were met for 390 patients. Available preoperative imaging comprised CT scan for 240 (62%) and integrated PET-CT for 150 (38%) patients. MPM was left-sided in 196 (50%) cases. Histology was epithelioid in 234 (60%), biphasic in 141 (36%), sarcomatoid in 13 (3%) and desmoplastic in 2 (<1%) cases. Staging by pTNM was: I, 7 (2%); II, 33 (8%); III, 225 (58%); IV, 125 (32%). Staging by cTNM was: I, 30 (8%); II, 39 (10%); III, 250 (64%); IV, 71 (18%). cTNM was concordant with pTNM staging in 188 (48%), overstaged in 139 (36%), and understaged in 63 (16%) cases. Concordance rate was not substantially modulated by type of scan, use of contrast, prior sclerosis or presence of pleural effusion. The most predictive and prognostic features included (N, sensitivity, specificity, HR, p-value): T2: Interlobar fissures (297, 85%, 71%, 1.4, 0.02); T3: Endothoracic fascia (158, 48%, 64%, 1.4, 0.004), Mediastinal fat (105, 28%, 73%, 1.8, <0.0001); T4: Diffuse/multifocal chest wall (21, 12%, 96%, 1.8, 0.01).

Conclusion:
Data-driven modification of cTNM criteria may improve concordance between cTNM and pTNM staging. Despite inherent sensitivity limitations of cTNM, improved prognostic performance may be achievable by 1) incorporating a size criterion (e.g. radiographic tumor volume), and 2) emphasizing features with high specificity and significant prognostic value when defining T descriptors.

Abstract not provided

Background:
There is little evidence to support the use of radiotherapy in treating pain in malignant pleural mesothelioma (MPM), however it is widely used. The aim of the present study was to assess the role of radiotherapy in palliating pain in MPM.

Methods:
A multi-centre, single arm, phase II study was conducted in the UK. Eligible patients met the following criteria: a diagnosis of MPM; worst pain score of > 4/10; performance status 0-2; CT scan within eight weeks of radiotherapy; due to receive radiotherapy for pain. Patients who had received anti-cancer therapy in the previous 6 weeks were ineligible. The following key assessments were performed at study baseline: pain (Brief Pain Inventory), Quality of Life (EORTC QLQ-C30) and inflammation (CRP). Following this, all patients were treated with 20 Gray in five fractions to the area of tumour felt to be responsible for the pain. The primary endpoint was a 30% drop in the BPI score five weeks after radiotherapy. Patients were followed up for 12 weeks after radiotherapy.

Results:
Forty patients were recruited between June 2012 and December 2013. Mean age was 71 with a male to female ratio of 7 : 1. Histological diagnosis was present in 85% of patients; 52.5% epithelioid, 25% sarcomatoid, 7.5% biphasic and 15% unspecified. The mean response to radiotherapy at five weeks was 35% (95% CI 20.6-51.7%). 37 patients started radiotherapy and 35 patients completed the full course. Fourteen patients had received prior chemotherapy. No association between baseline CRP and response was observed (p=0.958). Only one patient had a radiological response on CT with stable disease seen in a further 13 patients. There was no significant change in quality of life (QoL) score at any timepoint (p=0.680 week 1, p=0.765 week 5, p=0.384 week 12).

Conclusion:
Radiotherapy provides effective pain relief in a proportion of patients with MPM and should be considered for all patients with MPM related pain. Randomised dose escalation studies are now warranted and funding has been secured for such a study, SYSTEMS 2.

Background:
Use of radiotherapy (RT) to treat malignant pleural mesothelioma (MPM) has been limited due to reported significant morbidity and risk of fatal pneumonitis when treating large pleural volumes. To date, RT for MPM has generally been limited to palliation, prophylaxis of surgical tract sites, and adjuvant therapy generally after extrapleural pnuemonectomy. Reports of RT for MPM have employed photons and electrons nearly exclusively. Proton therapy (PT) can significantly reduce irradiation to lung and other critical organs, possibly reducing treatment toxicities and enabling novel RT indications. To date, only a single case series of 4 patients has reported on PT for MPM. We report our prospective experience using PT as adjuvant or definitive therapy for MPM and hypothesized that PT will have low rates of esophagitis and pneumonitis, while providing excellent local control.

Methods:
All consecutive patients diagnosed with MPM from 2011-2015 and treated at the Penn Mesothelioma and Pleural Program with PT on a prospective registry study were included for this Institutional Review Board-approved analysis. Local control, defined as lack of tumor progression in the RT portal, and overall survival were measured from PT completion to last follow-up or death. Toxicities were scored using CTCAEv4.

Results:
Sixteen patients treated to 17 PT courses were included. Patients were predominantly male (81%) and Caucasian (100%) with epithelial histological subtype (82%) and stage III-IV disease (94%). Patients were a median of 69.8 years old at PT start, which was delivered at a median of 11.1 months (range 3.5-69.3 months) after diagnosis. All patients received pemetrexed plus cisplatin or carboplatin prior to (n=15) or concurrent with (n=1) PT. PT was administered as adjuvant therapy following lung-sparing radical pleurectomy (n=8), to sites of gross disease following progression on systemic therapy (n=8), or as initial definitive therapy with concurrent chemotherapy (n=1). Patients were treated to a median dose of 51.75Gy (CGE) in 2.0Gy daily fractions (range 50.0-75.0Gy/1.8-2.5Gy). At a median follow-up of 5 months from PT completion, all patients had durable local control throughout the study period. Five patients died at a median of 5.4 months following PT. Median overall survival for the cohort has not yet been reached, and 6- and 12-month survival rates were 35% and 24%, respectively. No patients experienced grade ≥3 acute or late toxicity. Across the 17 PT courses, acute grade 2 toxicities included radiation dermatitis (n=8), dysphagia/esophagitis (n=4), anorexia (n=3), fatigue (n=2), and cough (n=1). Late grade 2 toxicity included a single patient with radiation pneumonitis (6%). Overall, patients experienced no significant change in ECOG performance score from PT beginning to end (mean 0.82 to 0.88).

Conclusion:
This is the largest report of PT for MPM and demonstrated PT is well tolerated with a favorable toxicity profile compared with photon reports. As such, PT may better allow for integration of RT in multimodality therapy for MPM. This study also demonstrated the efficacy of PT, with local control achieved following all 17 treatment courses. Longer follow-up and additional patients are needed to assess late toxicities and overall survival after PT.

Background:
Recently we demonstrated that members of the miR-15/16 family of microRNAs are implicated as tumor suppressors in malignant pleural mesothelioma (MPM) (Reid et al, Ann Oncol, 2013). MesomiR 1 is a first-in-man study testing TargomiRs (miR-15/16-derived mimics packaged in EDV[TM]nanocells [EDVs] targeted with EGFR antibodies) in MPM patients.

Methods:
In this phase I study (ClinicalTrials.gov: NCT02369198) a standard 3-6 patient dose escalation cohort design examining weekly/twice weekly administration of TargomiRs is followed. Patients tolerating weekly/twice weekly TargomiR infusions well are allowed to continue experimental therapy for at least 8 weeks. Fifty percent of the MTD previously established for EDVs was chosen as the first dose level to be studied and corresponded to 5 billion EDVs containing 1.5 μg miR-15/16 mimics. Based on prior experience with EDVs, patients who presented with elevated IL-6 levels were given a dose adaptation period of two weeks before receiving phase I doses. Premedication consisted of dexamethasone, promethazine and paracetamol and patients were monitored for a minimum period of 3 hours after TargomiR infusion. Response assessment (CT, FDG-PET, pulmonary function) was scheduled for patients completing 8 weeks of treatment. Quality-of-Life (QoL) questionnaires (EORTC) were requested on a weekly basis.

Results:
Ten MPM patients have enrolled to date. The majority of patients receiving 5 billion TargomiRs experienced a period of shivering/rigor 80-90 minutes after the start of the infusion, sometimes associated with burning/painful sensations in the area of disease. Overall TargomiR treatment was well tolerated and no patient failed to complete the first (8 weeks) treatment period. Laboratory examination revealed a steep but transitory rise in inflammatory cytokines, neutrophilia and lymphopenia shortly after TargomiR infusion, sometimes accompanied by mild elevation of liver enzymes. QoL assessment (9 patients) showed improving scores in 3 patients, stabilization in 4 and slightly lower scores in 2 patients. Response assessment (modified RECIST) in the 6 patients completing 8 weeks of treatment to date: 1 PR (see Figure 1, reconfirmed after 12 and 16 weeks), 4 SD and 1 PD. Figure 1. FDG-PET scintigraphy before (left) and after (right) 8 weeksFigure 1 of TargomiR treatment (patient 5)



Conclusion:
Early MesomiR 1 data revealed that infusions with 5 billion TargomiRs were well tolerated. Transient inflammatory (cytokine-mediated) reactions were noted shortly after TargomiR administration. One objective response was recorded while stable disease and stable QoL scores were noted in the majority of patients completing 8 weeks of experimental treatment.

Abstract not provided

Background:
The PD-1 and PD-L1 pathway is an immune checkpoint, which protects normal tissues from immune attack by curbing the effector T-cell response but can also prevent anti-tumor immune response. However their role in mesothelioma is not well understood. The present study aimed to understand the PD-1 and PD-L1 expression levels and their interactions in mesothelioma patients.

Methods:
Sections of formalin-fixed, paraffin-embedded pleural and peritoneal mesothelioma tumor samples from patients who were evaluated for various clinical trials at the NCI Center for Cancer Research were tested for PD-L1 expression (Anti-PD-L1 rabbit monoclonal recombinant primary antibody MKP-1B-196-10; Merck-Serono). PD-L1 expression on primary and established mesothelioma cell lines, malignant pleural effusions and ascites of mesothelioma patients were assessed using a commercial anti-PD-L1 antibody and analyzed by flow cytometry. Paired malignant effusion and peripheral blood samples were tested for PD-1 and PD-L1 expression on immune cells. Co-cultures of autologous tumor cells and T cells grown from malignant effusions of a mesothelioma patient were evaluated to understand the PD-1 and PD-L1 interaction.

Results:
Tumor samples from 65 patients included 44 peritoneal and 21 pleural mesotheliomas; 55 with epithelioid histology and 10 of other subtypes (sarcomatoid 2, biphasic 3, uncategorized 5). 41 of 65 (63%) tumors were positive for PD-L1 expression (defined as >5% PD-L1 expression on tumor cells, of any intensity) with levels of expression ranging from 5% to 80% of tumor cells, and intensities from 1+ to 3+. 24 (37%) were negative including 10 with focal staining. A higher proportion of males had tumor PD-L1 expression than females (73% vs. 46%; p=0.04). There was no association between PD-L1 expression and primary site of disease, age, race, histology and distant metastasis. Patients with PD-L1 positive tumors had a numerically inferior overall survival than patients with PD-L1 negative tumors (23.0 months vs.33.3 months; p=0.35). All 6 primary and 4 established mesothelioma cell lines tested showed basal expression of PD-L1. This was enhanced on treatment with IFN-γ. The fraction of cells expressing PD-L1 in malignant effusions ranged from 17 to 43%. Malignant effusions from 2 of 3 patients had high PD-1 expression on both CD4[+] and CD8[+] T cells. In addition, CD8[+] T cells in malignant effusions had significantly higher levels of PD-L1 expression compared to CD8[+] T cells in peripheral blood (7.47±2.75 % T cells versus 1.97±1.22% T cells; p=0.03). Autologous lymphocytes when co-cultured with tumor cells from malignant effusion, recognized tumor cells and induced IFN-γ mediated PD-L1 expression on their surface.

Conclusion:
High PD-L1 expression in mesothelioma patient tumor samples and tumor cells derived from malignant effusions, as well as presence of PD-1[+] T cells in these effusions indicates the prominent role of PD-1/PD-L1 pathway in maintaining an immunosuppressive milieu in mesothelioma. Thus, inhibiting this pathway could be useful therapeutically.

Background:
Whole exome sequencing has revealed key genetic events in several cancer types that have been successfully translated into clinical benefits. These advances are still lacking in malignant mesothelioma (MM), a highly aggressive malignancy with limited effective therapy. Frequent BAP1 mutations occur in a subset of this disease but the full molecular landscape of MM is still poorly characterized.

Methods:
We have therefore conducted whole exome sequencing of tumours from the pleura for 36 cases of MM. DNA from matched blood was available for 7 of the cases and was also sequenced. The variants were identified with GATK tools and annotated with ANNOVAR. Variants were filtered with the following criteria: quality score ≤ 50, present in dbSNP138, 1000 genomes variants and NHLBI ESP 6500 variants. Mutations with deleterious functional consequences predicted by Polyphen-2, SIFT and Mutation Taster tools were confirmed by Sanger sequencing.

Results:
A total of 9,064 variants (3,256 somatic) were identified. We confirmed mutations in genes previously described to be mutated in MM in 5 cases: BAP1 (R227C, Q684X, H141P), NF2 (76_76del, R221X) and TP53 (I195N). In BAP1 wt tumours (6 of the 7 cases with matched blood), we confirmed somatic mutations in 5 genes encoding components of either MAPK or WNT signaling pathways. In addition, we validated somatic mutations in 12 genes across 4 of the 6 cases, many of which are novel in MM and are involved in chromatin modification. We also observed these genes to be mutated in BAP1 wt tumours in the 29 additional unmatched MM cases.

Conclusion:
Thus our data suggests that in addition to BAP1, mutations in genes associated with MAPK, WNT signaling and the chromatin remodeling complex may represent a consistent pattern of molecular alterations in MM.

Background:
The potential of immunotherapy in mesothelioma has recently been demonstrated in multiple (pre)clinical studies. The success of immunotherapy relies on the induction of an anti-tumor immune response which has to overcome the local immunosuppressive environment in established tumors. Tumor-associated macrophages (TAMs) are an important part of the suppressive environment in mesothelioma and reprogramming these TAMs towards a more pro-inflammatory phenotype using a CD40-agonist has shown promising results in multiple solid tumors. Dendritic cell (DC) immunotherapy has been shown to elicit anti-tumor T-cell responses and is currently being studied in mesothelioma patients at our institution. We hypothesize that the combination treatment with a CD40-agonist and DC therapy has synergistic effects and the aim of the current study is to investigate the efficacy of this combinatorial approach.

Methods:
Wildtype Balb/c mice were injected intraperitoneally (i.p.) with the AB1 murine mesothelioma cell line. Different treatment regimens were compared as follows: untreated control group (n=6), monotherapy with CD40-agonist (FGK4.5 monoclonal antibody, n=5), monotherapy with DC immunotherapy (n=5) and combination therapy of DC immunotherapy followed by treatment with the CD40-agonist (n=5). Three days after completion of the treatment regimens, blood was drawn and analyzed using flow cytometry to investigate peripheral immune activation. All mice were monitored and sacrificed when showing signs of severe illness. After sacrifice, tumors are investigated using flow cytometry to determine the local immunological composition.

Results:
Blood analysis revealed that peripheral monocytes of the CD40-agonist group and the combination therapy group showed an increase in expression of MHC-II and PD-L1 compared to the mice in the control group and the DC immunotherapy group. In addition, the combination therapy induced a profound increase in effector CD8 T-cells and proliferating CD8 T-cells compared to the monotherapies. The interim survival analysis at day 40 post tumor cell injection demonstrates a 17% survival of the control group, 80% survival of the monotherapies and 100% survival of the combination therapy. The final survival analysis will be presented at the conference.

Conclusion:
Combination therapy of DC immunotherapy and a CD40-agonistic antibody induces synergistic immune activation in the peripheral blood of mesothelioma-bearing mice compared to the monotherapies. Although the final survival data are awaited, the presented data demonstrate the potential of the combination of cellular immunotherapy and targeting of the local tumor microenvironment in mesothelioma.

Abstract not provided

Background:
Malignant pleural mesothelioma (MPM) is an aggressive tumor in the pleural lining of the lung and peritoneum usually resulting from prior exposure to asbestos. Median overall survival with standard of care (SOC) chemotherapy is only 12 months from diagnosis. This poor prognosis may be attributable at least in part to cancer stem cells (CSCs) that are resistant to chemotherapy and can mediate cancer recurrence and progression. VS-6063 (defactinib) is an oral small molecule that targets cancer stem cells through the inhibition of focal adhesion kinase (FAK). VS-6063 has demonstrated tolerability, target inhibition, and preliminary signs of clinical activity as a single agent and in combination with paclitaxel in Phase 1 clinical trials (Jones et al., J Clin Oncol 29: 2011 (suppl; abstr 3002); Patel et al., J Clin Oncol 32:5s, 2014 (suppl; abstr 5521)). Currently, VS-6063 is being tested in a randomized, double-blind, placebo-controlled trial in malignant pleural mesothelioma immediately following front-line therapy (COMMAND Trial, NCT01870609). In an effort to identify additional mesothelioma patients who may benefit from a CSC targeting agent, we sought to identify compounds that show synergistic anticancer activity with VS-6063. PI3K/mTOR inhibitors were previously demonstrated to show activity in mesothelioma. VS-5584 is a potent oral small molecule that selectively kills CSCs by targeting multiple PI3K isoforms and mTORC1/2 (Kolev et al, Cancer Res; 75:1, 2014). VS-5584 is currently being investigated as a single agent in a Phase 1 clinical trial, (NCT01991938).

Methods:
The synergy between VS-6063 and VS-5584 was demonstrated in vitro using cell viability assays analyzed by CalcuSyn, HSA and Loewe models. CSCs from mesothelioma cell lines were assessed by the Aldefluor+ flow cytometric assays. The anti-tumor activity of the VS-6063 and VS-5584 combination treatment was tested with in vivo mouse mesothelioma xenograft models.

Results:
A dual PI3K/mTOR inhibitor VS-5584 showed synergistic activity with a FAK inhibitor, VS-6063. VS-5584 further enhanced reduction of mesothelioma CSCs by VS-6063 measured by the Aldefluor+ assay in Mero-14 mesothelioma cells. In a 3D matrigel cell viability assay, the combination of VS-6063 and VS-5584 displayed synergistic reduction in cell viability based on multiple combination analysis models. In a MM87 mesothelioma xenograft model in vivo, the single agent treatment with either VS-6063 or VS-5584 was active in inhibiting mesothelioma tumor growth. Combination treatment further enhanced the antitumor efficacy of either agent alone (p <0.0001).

Conclusion:
VS-6063 (defactinib) and VS-5584 exhibit synergistic anticancer activity in preclinical models of mesothelioma. These data provide a strong preclinical rationale for the open dose-escalation Phase I clinical trial of VS-6063 and VS-5584 in patients with relapsed mesothelioma (NCT02372227).

Background:
Malignant pleural mesothelioma (MPM) is a highly aggressive tumour with a high local recurrence rate and often a poor prognosis despite multimodal treatment. We evaluated the prognostic impact of morphological and immunohistochemical changes in sequential biopsies obtained from patients with MPM during disease progression.

Methods:
Tissue microarrays were constructed from paraffin-embedded tissue samples of 36 MPM patients (26 epithelioid, 6 biphasic and 4 sarcomatoid) taken before induction-chemotherapy, during surgery and at the time point of tumour recurrence. Immunohistochemical staining for calretinin, cytokeratin 5/6 (CK5/6) and Wilm’s tumor-1 (WT-1) as well as the biomarkers mesothelin, osteopontin, and fibulin-3 was performed, and staining intensity and percentage of positively stained tumour cells scored semi-quantitatively. The results were correlated with clinico-pathological characteristics of the patients including overall survival (OS). To determine the prognostic value of the markers at the different time points, a multivariate analysis including all factors that were significant in univariate analysis was performed.

Results:
In 28% of patients with epithelioid or biphasic MPM, a transition towards biphasic or sarcomatoid growth pattern during disease progression was observed (Figure 1). This dedifferentiation was associated with significantly decreased immunoreactivity for WT-1 (p=0.03), calretinin (p=0.005), mesothelin (p=0.01) as well as a shorter OS (p=0.04). Figure 1 Overall, patients with epithelioid or biphasic MPM in the diagnostic biopsy had a significantly better OS (29 months; 95% confidence interval (CI): range 27-32 months) in comparison to patients with sarcomatoid MPM (5 months; 95% CI: 3-7 months) (p<0.0005). On multivariate analysis, male gender (p=0.04) and high fibulin-3 (p=0.02) in the pre-chemotherapy samples were found to be associated independently with shorter OS.



Conclusion:
In patients with epithelioid or biphasic MPM, high fibulin-3 expression in pretreatment samples and gender are independent predictors of shorter OS. In up to one third of patients disease progression is accompanied by sarcomatoid differentiation, suggesting that factors such as molecular alterations involved in epithelial-to-mesenchymal transition (EMT) are contributing to disease course and clinical outcome. Alternatively, induction chemotherapy might contribute to this transition by promoting selection and outgrowth of therapy resistant tumor cells. Eventually, the different tumor biology of this subgroup of patients may be taken into account for the consideration of alternative patient handling.

Background:
Growing evidence suggests that nuclear–cytoplasmic transport is frequently dysregulated in cancer cells, and is involved in promoting carcinogenesis, cell survival, drug resistance and tumor progression. In particular, enhanced nuclear export is one mechanism by which malignant cells inactivate tumor suppressor proteins (TSPs). Inhibition of XPO1 (CRM1), the main karyopherin involved in the nuclear export of TSPs, restores nuclear localization and function of TSPs in several preclinical models. Selinexor(KPT-330) is an XPO1 inhibitor being tested clinically in solid tumors and hematological malignancies that showed some activity in patients with thymic epithelial tumors (TETs). Here, we describe the activity of selinexor in preclinical models of TETs.

Methods:
Thymoma (IU-Tab1, T1682), thymic carcinoma (Ty82, T1889, MP57) and immortalized normal thymic epithelial cells (TEC84) treated with selinexor or vehicle were assayed by CellTiter-Glo and flow cytometry. Western blot analysis of nuclear and cytoplasmic protein fractions and immunofluorescence assays were used to study the cellular sublocalization of XPO1 cargoes before and after treatment. The effect of selinexor on cell migration was determined using a wound-healing assay. A selixinor-resistant cell line was generated by growing selinexor-sensitive IU-Tab1 cells at increasing concentrations of the drug. Mutational status and copy number of the XPO1 gene was assessed by Q- PCR and Sanger sequencing.

Results:
All TET cell lines were sensitive to selinexor (IC~50~ 90-250 nM) with the exception of T1682 (thymoma type B), which showed intrinsic drug resistance (IC~50~ > 1000 nM). In the sensitive cell lines, selinexor treatment induced G1 (MP57) or G2 (IU-Tab1, Ty82) cell-cycle arrest at 24 hours, and induced apoptosis 2-5 fold over untreated cells by 72 hours. The cytotoxic effects of selinexor were not observed in immortalized normal TEC84 cells at nanomolar concentrations, and required higher concentrations (IC~50 ~800nM) to induce a cytostatic effect. Drug treatment led to increased nuclear concentrations of several TSPs involved in cell cycle regulation (e.g. p21, p27), genomic stability (p53) and induction of apoptosis (FOXO3a) and also reduced the total cellular expression of the oncogenic protein NF-kB. These results were confirmed with siRNA knockdown of XPO1. In addition,selinexor treatment impaired tumor cell migration and had cytotoxic synergistic effect in combination with doxorubicin or etoposide in T1889 and IU-Tab1 cell lines, increasing nuclear accumulation of the XPO1 cargo protein, Topoisomerase IIα. Furthermore, we demonstrated that selinexor-resistant cell line has similar growth rates to their parental cells, however overexpress XPO1 due to gene amplification, confirming the importance of aberrant XPO1 activity in TET survival.

Conclusion:
Our data show the importance of XPO1 in TETs biology and demonstrate activity of selinexor in preclinical models, further supporting the planned Phase II trial in patients with TETs.

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