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C. Brambilla
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ORAL 40 - Biology 1 (ID 154)
- Event: WCLC 2015
- Type: Oral Session
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 8
- Moderators:C. Brambilla, R. Bueno
- Coordinates: 9/09/2015, 16:45 - 18:15, 702+704+706
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ORAL40.01 - PD-L1 Is Highly Expressed in Malignant Mesothelioma and PD-1<sup>+ </sup>Lymphocytes within Malignant Effusions Induce PD-L1 Expression (ID 553)
16:45 - 18:15 | Author(s): S. Khanna, A. Thomas, D. Abate Daga, B. Morrow, J. Zhang, S.M. Steinberg, A. Orlandi, P. Ferroni, J. Schlom, F. Guadagni, R. Hassan
- Abstract
- Presentation
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.
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ORAL40.02 - Molecular Landscape of Malignant Mesothelioma from Whole Exome Sequencing (ID 2439)
16:45 - 18:15 | Author(s): S.K. Lu, H. Anbunathan, S. Popat, M.E.R. O'Brien, E. Lim, A. Montero Fernandez, A.G. Nicholson, M. Lathrop, A.M. Bowcock, M.F. Moffatt, W.O.C. Cookson
- Abstract
- Presentation
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.
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ORAL40.03 - Combination Therapy with a CD40-Agonist and Dendritic Cell Immunotherapy Has Synergistic Effects in a Murine Mesothelioma Model (ID 2643)
16:45 - 18:15 | Author(s): L. Lievense, F. Dammeijer, M. Lambers-Kaijen, R. Hendriks, M. Van Nimwegen, J. Hegmans, J.G. Aerts
- Abstract
- Presentation
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.
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ORAL40.04 - Discussant for ORAL40.01, ORAL40.02, ORAL40.03 (ID 3466)
16:45 - 18:15 | Author(s): Y. Sekido
- Abstract
- Presentation
Abstract not provided
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ORAL40.05 - The Cancer Stem Cell Inhibitors VS-6063 and VS-5584 Exhibit Synergistic Anticancer Activity in Pre-Clinical Models of Mesothelioma (ID 2753)
16:45 - 18:15 | Author(s): D. Weaver, V. Kolev, Y. Wang, J. Testa, J. Pachter
- Abstract
- Presentation
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).
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ORAL40.06 - Sarcomatoid Differentiation During Progression of Malignant Pleural Mesothelioma (ID 1161)
16:45 - 18:15 | Author(s): B. Vrugt, E. Felley-Bosco, S. Simmler, M. Storz, M. Friess, M. Meerang, A. Soltermann, H. Moch, R. Stahel, W. Weder, I. Opitz
- Abstract
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.
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ORAL40.07 - Xpo1 Inhibition: A Promising Therapeutic Strategy in Thymic Epithelial Tumors (ID 1230)
16:45 - 18:15 | Author(s): F. Conforti, T. De Pas, A.T. Alberobello, G. Rao, Y. Wang, G. Giaccone
- Abstract
- Presentation
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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ORAL40.08 - Discussant for ORAL40.05, ORAL40.06, ORAL40.07 (ID 3467)
16:45 - 18:15 | Author(s): T.M. Jahan
- Abstract
- Presentation
Abstract not provided
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ORAL 06 - Next Generation Sequencing and Testing Implications (ID 90)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:G. De Lima Lopes, V. Miller
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 1a-1f
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ORAL06.01 - Genomic Characterization of Large-Cell Neuroendocrine Lung Tumors (ID 1667)
10:45 - 12:15 | Author(s): C. Brambilla
- Abstract
Background:
Neuroendocrine lung tumours account for 25% of all lung cancer cases, and they range from low-aggressive pulmonary carcinoids (PCA) to highly malignant small-cell lung cancer (SCLC) and large-cell neuroendocrine lung carcinoma (LCNEC). The last two are strongly associated with heavy smoking and are typically detected at a clinically advanced stage, having a poor survival. Comprehensive genomic analyses in lung neuroendocrine tumours are difficult because of limited availability of tissue. While more effort has been done in the context of SCLC, the detailed molecular features of LCNEC remain largely unknown.
Methods:
We conducted 6.0 SNP array analyses of 60 LCNEC tumours, exome sequencing of 55 tumor-normal pairs, genome sequencing of 11 tumour-normal pairs, transcriptome sequencing of 69 tumours, and expression arrays on 60 tumors. Data analyses were performed using in house developed and published pipelines.
Results:
Analyses of chromosomal gene copy number revealed amplifications of MYCL1, FGFR1, MYC, IRS2 and TTF1. We also observed deletions of CDKN2A and PTPRD. TTF1 amplifications are characteristic of lung adenocarcinoma (AD); CDKN2A deletions are frequent alterations in both AD and squamous-cell lung carcinoma (SQ); FGFR1 amplifications are found in SQ and, less frequently, in SCLC; and MYCL1 and IRS2 amplifications are frequent events in SCLC. Similar to the copy number data, we found patterns of mutations characteristic of other lung cancer subtypes: TP53 was the most frequently mutated gene (75%) followed by RB1 (27%), and inactivation of both TP53 and RB1, which is the hallmark of SCLC, occurred in 20% of the cases. Mutations in STK11 and KEAP1-NFE2L2 (frequently seen in AD and SQ) were found in 23% and 22% of the specimens, respectively. Interestingly, mutations in RB1 and STK11/KEAP1 occurred in a mutually exclusive fashion (p-value=0.016). Despite the heterogeneity observed at the mutation level, analysis of the pattern of expression of LCNEC in comparison with the other lung cancer subtypes (AD, SQ, SCLC, and PCA) points to LCNEC as being an independent entity. An average mutation rate of 10.7 mutations per megabase was detected in LCNEC, which is in line with the rate observed in other lung tumours associated with smoking. We found that, similar to SCLC, the mutation signatures associated with APOBEC family of cytidine deaminases, smoking, and age (based on Alexandrov et al 2013) were the predominant ones in LCNEC. However, the contribution of the individual SCLC and LCNEC samples to these three signatures was quite different, and we are currently exploring it.
Conclusion:
Taking into account somatic copy number and mutation data, we distinguished two well-defined groups of LCNEC: an SCLC-like group, carrying alterations in MYCL1, ISR2, and in both RB1 and TP53; and a group resembling AD and SQ, with alterations in CDKN2A, TTF1, KEAP1-NFE2L2, and STK11. Although these results suggest that LCNEC might be a mix of different lung cancer subtypes, mutation clonality and expression analyses show that they are likely to be a separate entity, sharing molecular characteristics with the other lung cancer subtypes. Their heterogeneity suggests that LCNEC might represent an evolutionary trunk that can branch to SCLC or AD/SQ.
