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J. Minna
Moderator of
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MO19 - Lung Cancer Immunobiology (ID 91)
- Event: WCLC 2013
- Type: Mini Oral Abstract Session
- Track: Biology
- Presentations: 13
- Moderators:Y. Sekido, J. Minna
- Coordinates: 10/30/2013, 10:30 - 12:00, Bayside 201 - 203, Level 2
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MO19.01 - Epigenetic Targeting of CD1d increases anti-tumour iNKT activity in Non-Small Cell Lung Cancer (ID 3373)
10:30 - 12:00 | Author(s): É.F. Dockry
- Abstract
- Presentation
Background
Immunotherapy is now accepted as the fourth most important modality for malignant tumours, and is currently being applied in the treatment of non-small cell lung cancer (NSCLC). CD1d, a MHC class-like molecule, presents glycolipids to natural killer T (NKT) cells. In doing so, CD1D contributes to their anti-tumour activity. CD1d is expressed in certain tumour types, and there is increasing evidence that CD1d acts as a target for NKT-mediated cell killing, however most human and mouse solid tumours are CD1d-negative. Recently evidence has indicated that CD1d expression is epigenetically regulated through HDAC1/2 and Spl.Methods
CD1d levels were examined at the mRNA level by RT-PCR. To determine whether CD1d expression is subject to epigenetic regulation, non-small cell lung cancer (NSCLC) cell lines were treated with various epigenetic modifying agents. The A549 (adenocarcinoma) and SK-MES-1 (Squamous cell carcinoma) cell lines were treated with HDACi, (a) SAHA at a dose of 5 µM for 24 h. Treatments with other epigenetic modifyng agents such as trichostatin A (TSA) and DNA methyltransferase inhibitors are currenly ongoing. iNKT cells were co-cultured wiht NSCLC cells to examine their antitumourigenic activity using a CD107a externalised assay.Results
Using RT-PCR it was possible to confirm that SAHA significantly induced CD1d expression in both A549 and SKMES cell lines (p ≤ <0.005). RT-PCR was also performed on A549 and SKMES cell lines that had been allowed to recover, following treatment with SAHA. A549 cells showed a statistically significant induction in recovered SAHA-treated cells (p < 0.05). Using flow cytometry the cytotoxic T-lymphocytes (CTL) activity of iNKTs was measured. While there was a slight induction of CD107a-bearing iNKT cells, it is believed that treatment with HDACi will increase the anti-tumour activity of these cells. This work is ongoing at present.Conclusion
CD1d expression is significantly induced using the HDACi, SAHA. This induction is present in A549 cells treated with SAHA and allowed to recover; indicating that SAHA may possibly be used to increase the anti-tumourigenic activity of iNKT cells. These results may have important consequences for treating patients with combined epigenetic targeting agents and immunotherapy.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO19.02 - The Tumor Immune Microenvironment in Octogenarians with Stage I Non-Small Cell Lung Cancer: Implications for Immunotherapy (ID 3155)
10:30 - 12:00 | Author(s): M. Lee, K. Kadota, H. Ujiie, N.P. Rizk, W.D. Travis, V.W. Rusch, M. Sadelain, P.S. Adusumilli
- Abstract
- Presentation
Background
The elderly have less-robust immune responses to infections, immunizations, and tumors, compared with younger people. Furthermore, preclinical studies have indicated that immunotherapeutic interventions are less effective in older animals. Considering the effects of age-associated changes in immune function, most clinical trials of cancer-related immunotherapy have been conducted in relatively young patients. With the increasing focus on immunotherapy for non-small cell lung cancer (NSCLC), we investigated the relationship between patient age and tumor immune parameters in stage I NSCLC.Methods
Tissue microarrays from patients with stage I NSCLC (n=1371; 1995-2009; median follow-up, 3.5 years) were constructed, and immunohistochemical analyses for immune cell infiltration (CD3, CD4, CD8, CD20, FoxP3) were performed. Patients were categorized into 3 groups: (1) ≤65 years old, (2) 66-79 years old, and (3) ≥80 years old. Stains were analyzed for immune cell infiltration (low vs high) in the tumor nest. The Chi-Square test was used to analyze the association between immune parameters and age group. The Kaplan-Meier method was used to estimate recurrence-free survival (RFS).Results
In total, 1116 patients with stage I lung adenocarcinoma and 255 patients with stage I squamous cell carcinoma were enrolled. Patients aged ≥80 years did not have a significantly poorer prognosis (n=155; 5-year RFS, 76.0%) than the patients in the two younger groups (p=0.65; Figure 1A). There were no statistically significant differences in numbers of tumor-infiltrating lymphocytes in the tumor nest between the three groups (Figure 1B), nor was there a statistically significant difference between the elderly group and the younger patients when effector regulatory immune response ratios were compared (FoxP3/CD3 ratio; high vs low, p=0.85). Figure 1Conclusion
In this large cohort of stage I NSCLC patients selected for surgical resection, the tumor microenvironment among elderly patients resembles other age groups. Our study provides important information while considering immunotherapy in elderly patients with lung cancer.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO19.03 - The effects of epidermal growth factor (EGFR) receptor inhibitors on the immune system in patients with advanced non-small cell lung cancer (NSCLC) (ID 3152)
10:30 - 12:00 | Author(s): T. Meniawy, R. Lake, A.K. Nowak
- Abstract
- Presentation
Background
EGFR tyrosine kinase inhibitors (TKIs) have an important role in the treatment of NSCLC, particularly in the context of activating mutations, but resistance invariably develops. Recently, the immune checkpoint blockers anti-PD1 and anti-PDL1 were the first immunotherapies to demonstrate activity in advanced lung cancer. As immunotherapies such as anti-CTLA4, anti-PD1 and anti-PD-L1 antibodies enter clinical practice, there is potential to combine immunotherapies with TKIs to improve patient outcomes. The immune effects of EGFR-TKIs have not been elucidated, and the aim of this study is to examine the effect of TKIs on the immune response in patients with NSCLC, to provide a rationale and pilot data to underpin future clinical development of combinations of TKIs and immunotherapy.Methods
Patients with advanced NSCLC who were commencing an EGFR-TKI were included in this prospective study. Eligible patients had a confirmed diagnosis of NSCLC, were treated with a single-agent EGFR-TKI , had no concurrent autoimmune disease and received no chemotherapy within 21 days, or corticosteroid therapy within 3 days of study entry. Peripheral blood samples were collected before commencing a TKI and 7 days, 21 days and 8 weeks after start of treatment. Peripheral blood mononuclear cells (PBMCs) were isolated and immediately frozen for subsequent analysis by 8-colour flow cytometry for relevant surface and intracellular marker expression. 4 panels were developed to examine the activation and proliferation status of effector CD8[+] T and CD4[+] T-regulatory cells (Tregs), enumeration of dendritic cells and B-cells, as well as the inhibitory pathway programmed death-1 (PD-1) receptor and its ligands PD-L1 and PD-L2 on T cells and antigen-presenting cells. Changes in immune parameters will be correlated with overall survival (OS) and radiological response (RECIST 1.1 criteria) at 8 weeks post-treatment.Results
33 eligible patients were prospectively enrolled. Histopathology was adenocarcinoma (n=23), squamous cell carcinoma (n=7) and NSCLC not otherwise specified (NOS, n=3)). 12 patients had an activating EGFR mutation, 11 were EGFR wild type, and mutation-status was unknown in 10. 6 patients received the TKI gefitinib and 4 received erlotinib as first-line treatment for EGFR-mutation positive disease. 22 patients received erlotinib and 1 patient received afatinib as second or subsequent line therapy. At time of this report, 22/33 patients were deceased. Median OS from study entry was: all patients (7.7 months); mutation-positive (11.1 months); and mutation negative (4.4 months). Samples for 13 patients (2 were mutation-positive) have been analysed for effector T cell and Treg panels and no significant changes were seen between baseline and subsequent time points. Data will be presented for all samples.Conclusion
This is the first study to explore to the immune effects of EGFR-TKIs. Initial results have not revealed a significant effect on peripheral T-cells, and analysis of remaining patient samples and other panels is in progress. An understanding of the immune effects of targeted therapies will be crucial in the rational development of strategies for incorporating immunotherapy into the anti-EGFR treatment paradigm, in an era of promising immunotherapy and checkpoint blockade approaches.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO19.04 - DISCUSSANT (ID 3902)
10:30 - 12:00 | Author(s): D.P. Carbone
- Abstract
- Presentation
Abstract not provided
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- Abstract
- Presentation
Background
EGFR-tyrosine kinase inhibitor Gefitinib interrupts signaling through the EGFR in non-small-cell lung cancer (NSCLC) cells with EGFR driver mutation and has shown impressive activity in terms of clinical benefit for patients with NSCLC, however, almost all patients develop resistance to this drug. Although much research is focusing on the mechanisms of drug resistance in tumor cell, the role of EGFR signaling in tumor escape from the host immune system is poorly understood. NK cell activity is promoted via NK group 2, member D (NKG2D) on NK cells. The engagement between NKG2D and its ligands enhances cell-mediated cytotoxicity and cytokine production against transformed cells. Both MHC class I-related chain A and B (MICA/B) and UL16 binding protein (ULBP)s are expressed by intestinal epithelial and at low levels also by other non-malignant cell types. Primary tumor cells and tumor cell lines frequently express NKG2D ligands, but the mechanisms responsible for the induction of NKG2D ligands during oncogenesis are also poorly understood. Here we demonstrate Gefitinib downregulates NK group 2 member D (NKG2D) ligand MICA/B and ULBPs, resulting in attenuation of NK cell-mediated cytotoxicity in NSCLC cells.Methods
Possible influences of Gefitinib on expressions of MHC class I, MICA/B and ULBP1-3 in 5 NSCLC cell lines (A549, PC-9, RERF-LC-KJ, RERF-LC-AI, and LC2/ad) were investigated by flow cytometry. We also assessed whether genetic silencing of EGFR using siRNA of EGFR affected on the expression of NKG2D ligands. To ask the main downstream pathway of EGFR regulating the expression of NKG2D ligands, the cells were treated with PI3K-AKT inhibitor LY294002 or MEK inhibitor PD98059 then the expression of NKG2D ligands was analyzed. NK cell-mediated cytotoxicity against cancer cells was assessed by [51]Cr release assay or flow cytometry based CD107a degranulation assay.Results
Gefitinib downregulated NKG2D ligands in NSCLC cell lines. In line with these results, siRNA-mediated silencing of EGFR downregulated the expression of NKG2D ligand. Among the major downstream pathways activated by EGFR signaling, the expression of NKG2D ligands was mainly regulated by the PI3K-AKT pathway. Treatment with EGF promoted MICA/B expression in 2 of 5 cell lines, while EGF decreased MICA/B in 1 of 5 cell lines. These observations further emphasize that EGFR signaling is one of the major pathways regulating the expression of NKG2D ligands in NSCLC cells. As expected, inhibition of EGFR signaling-downregulated NKG2D ligands attenuated NK cell-mediated cytotoxicity.Conclusion
We conclude that EGFR signaling directly regulates the expression of NKG2D ligands and that this may influence the recognition of tumor cells by the innate immune system.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO19.06 - The role of fibroblast growth factor-9 in the regulation of the tumour-specific immune response in malignant mesothelioma (ID 3237)
10:30 - 12:00 | Author(s): S. Lansley, A.L. Tan, J. Varano, S. Karabela, G. Stathopoulos, J. Creaney, Y.G. Lee
- Abstract
- Presentation
Background
Identifying key molecules in the pathobiology of malignant mesothelioma is needed to develop new therapies and biomarkers. Fibroblast growth factor-9 (FGF-9) is an exciting and novel target uncovered from our global gene profiling of human MPM samples. Recently FGF-9 has been implicated in cancer development and neoplastic transformation of embryonic fibroblasts. We have verified over-expression of FGF-9 in MPM over other cancers and benign pleuritis in five separate cohorts of human pleural tissues and effusions. Our preliminary in vitro work demonstrated that FGF-9 induces mesothelioma cell proliferation and matrix invasion. We therefore hypothesised that antagonising FGF-9 may reduce tumour aggressiveness, growth and induce tumour regression in vivo.Methods
To study the ‘necessity’ of FGF-9 in MPM development in vivo we transfected the mouse MM cell line, AB1, with shRNA directed against murine FGF-9 (or control vector expressing a scrambled sequence). For the heterotopic model murine AB1-FGF-9 knock-down cells (or controls) were injected (5x10[5 ]cells) subcutaneously into the flank of Balb/c mice. Tumour dimensions were measured thrice weekly and animals sacrificed when tumours reached 100mm[2] and tumour tissues harvested. FGF-9 expression in tumour tissue was determined by immunohistochemistry. For orthotopic experiments, Balb/c mice received a single intraperitoneal injection of 5x10[5 ]AB1-FGF-9 knock-down cells (or controls). At day 13, animals were sacrificed and the number of peritoneal tumour nodules enumerated by blinded investigators. To determine whether the immune system plays a role in the regulation of AB1 MM tumour growth, 5x10[5 ]AB1-FGF-9 knock-down cells (or controls) were injected subcutaneously into nude mice. To elucidate the immune cells involved in AB1 MM tumour growth regulation, T cells were depleted in Balb/c tumour bearing mice using specific antibodies to CD4 and CD8 and tumour growth monitored. T cell depletion was confirmed using flow cytometry.Results
Heterotopic tumour growth was significantly retarded in mice inoculated with AB1-FGF-9 knockdown cells compared to the scrambled vector and parent MM cells (p<0.001). A significant reduction in the number, and hence tumour burden, of tumour nodules was also observed for AB1-FGF-9 knockdown tumours in the orthotopic peritoneal model compared to controls (p<0.001). When grown in nude mice, which lack a functional T cell repertoire, AB1-FGF-9 knockdown tumours grew at a similar rate to that of the parent and vector controls which was suggestive of a role of the immune response in the regulation of MM tumours lacking FGF-9. AB1-FGF-9 knockdown tumours demonstrated significantly greater tumour burden in mice depleted of CD4+ and CD8+ T cells, either alone or in combination, when compared to saline controls which is highly suggestive of a T cell-mediated immune response to these tumours. These results also suggest that FGF-9 inhibits the tumour-specific immune response in MM.Conclusion
In combination with our previous in vitro data which clearly demonstrated the proliferative and invasive properties of FGF-9, we suggest that FGF-9 has an important role in the pathobiological characteristics of MM in vivo and represents a novel therapeutic target.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO19.07 - Lung Cancer-Initiating Cells Avoid Immune Recognition (ID 3314)
10:30 - 12:00 | Author(s): J.C. Steel, B.J. Morrison, J.C. Morris
- Abstract
- Presentation
Background
Increasing evidence supports the concept of a unique population of cells within a tumor with stem cell-like characteristics. These cancer-initiating cells (CICs) are thought to be responsible for tumor organization, maintenance, progression and recurrence, as well as contributing to radiation and chemotherapy resistance. In this study we outline a new characteristic of CICs, one in which the CIC can subvert the host immune response. We pose that this characteristic is essential for the establishment of a tumor in an immunocompetent host and may represent a mechanism in which lung tumors may gain resistance to immune based therapies.Methods
We examined whether lung CICs could be enriched from murine and human cell lines through the use of tumorsphere culture assays. We vaccinated mice against the HPV E7 antigen and challenged mice with HPV E6/7 expressing murine lung cancer cells (TC-1) enriched for CICs. We also examined ex-vivo the capacity of CD8 and NK cells derived from vaccinated mice to lyse CICs. We examined by flow cytometry the expression of MHC-I and NK activating ligands. We examined the effect of interferon gamma (IFN-γ) on MHC-I expression by CICs and determined whether increased MHC-I by CICs would inhibit tumor formation in an immunocompetent animal.Results
We showed that both murine and human lung cancer cells can be grown and maintained in tumorsphere culture conditions. The resulting cells were enriched for CICs as evidenced by increased OCT4, NANOG, and/or SOX2 expression. Additionally, tumorsphere cultures of murine tumor lines had increased tumor take in immunocompetent animals, however this was significantly less than that seen in immunodeficient mice; indicating that true CICs must be able to thwart the immune response to establish tumors. In order to further examine this, we vaccinated mice with HPV E7 peptides and challenged with TC-1 non-CICs or CICs. We showed that mice challenged with CICs had no survival advantage compared to non-vaccinated animals; whereas those animals challenged with non-CICs exhibited a vaccine induced survival advantage. Further we showed ex-vivo that CICs were resistant to lysis from CD8+ T-cells compared to the non-CICs. Next, we showed that both murine and human lung CICs have down-regulated expression of MHC-I as well as a number of ligands for NK cell activating receptors, essentially making them “opaque” to the immune system and therefore less susceptible to CTL or NK cell lysis. Further, we demonstrate that IFN-γ increases MHC-I expression on CICs and restores sensitivity to CTL killing. Finally, we demonstrate that decreased MHC-I expression is a critical component of the ability of CICs to establish tumor in immunocompetent hosts.Conclusion
Increasing evidence indicates that CICs are critical players in the development and establishment of cancers. In this study we demonstrated that these cells also subvert tumor immune surveillance and play a key role in the resistance of tumors to cancer vaccines through their ability to escape host immune responses. Our results demonstrated that modulation of MHC-I on CICs can alter their susceptibility to T-cell mediated lysis thus opening a new target for cancer vaccine strategies.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO19.08 - DISCUSSANT (ID 3903)
10:30 - 12:00 | Author(s): E. Quoix
- Abstract
- Presentation
Abstract not provided
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MO19.09 - Molecular correlates of PD-L1 status and predictive biomarkers in patients with non-small cell lung cancer (NSCLC) treated with the anti-PDL1 antibody MPDL3280A (ID 1653)
10:30 - 12:00 | Author(s): S.N. Gettinger, M. Kowanetz, J. Soria, L. Gandhi, L. Horn, M.S. Gordon, D. Spigel, C. Cruz, P. Conkling, S. Antonia, H. Koeppen, Y. Xiao, A. Mokatrin, X. Shen, G. Fine, R.S. Herbst
- Abstract
- Presentation
Background
In NSCLC, antitumor immune response may be inhibited by PD-L1 expression. MPDL3280A, a human monoclonal antibody containing an engineered Fc-domain designed to optimize efficacy and safety, aims to restore tumor-specific T-cell immunity by blocking PD-L1 binding to its receptors, PD-1 and B7.1.Methods
Patients with squamous or nonsquamous NSCLC received MPDL3280A IV q3w up to 1 year as part of a phase I dose escalation/expansion study. Objective response rate (ORR) was assessed by RECIST v1.1 and included unconfirmed/confirmed responses. EGFR and KRAS status was initially assessed locally by investigators. Archival tumor tissues were evaluated centrally by IHC for PD-L1 and CD8. A qPCR-based gene expression panel measuring ≈90 immune-related genes was used to characterize the tumor immune microenvironment at baseline and during MPDL3280A treatment.Results
41 NSCLC patients first dosed at 1-20 mg/kg prior to Aug 1, 2012, were evaluable for efficacy with an ORR of 22%. Baseline tumor samples were available for IHC (n=33) and for gene expression analysis (n=29). Of patients with available tissue, 5 were PD-L1 tumor status positive and 28 were PD-L1 tumor status negative. Relationship between PD-L1 status and EGFR/KRAS status is described below (table). Elevated baseline PD-L1 expression was associated with response to MPDL3280A (80% ORR vs 14% ORR for PD-L1negative patients), and PD-L1 expression coordinated with CD8+ T cells. A Th1-type T-cell gene signature (including CD8, Granzyme-B and EOMES) was associated with treatment response. Non-responders exhibited at least a 2-fold higher ratio over CD8 of genes associated with immunosuppression, including RORC, FOXP3, TGFb1 and IL10 compared with responders. On treatment, responding tumors across indications showed increasing PD-L1 expression and a Th1-dominant immune infiltrate, providing evidence for adaptive PD-L1 up-regulation.Conclusion
PD-L1 expression and a Th1 driven T-cell gene signature correlated with response to MPDL3280A in NSCLC, and MPDL3280A therapy led to T-cell reactivation and restored antitumor immunity. Additionally, expression of immune suppressive factors in NSCLC tumors is associated with a lack of benefit from MPDL3280A. These data provide mechanistic insights into immunotherapy and patient selection for MPDL3280A monotherapy. Preliminary observations suggest clinical activity and molecular characteristics may be associated with PD-L1 tumor expression. Updated data will be presented. Table: Relationship between PD-L1 status and EGFR/KRAS mutational statusPD-L1-Positive (n = 5) PD-L1-Negative (n = 28) PD-L1 Unknown (n = 7) Overall (n = 40)* EGFRm, n 1 2 1 4 EGFR WT, n 2 20 4 26 EGFR Unknown, n 2 6 2 10 KRASm, n 1 4 1 6 KRAS WT, n 2 8 3 13 KRAS Unknown, n 2 16 3 21 * 1 patient had missing data. Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO19.10 - Prevalence and prognostic association of PD-L1 protein and immune gene expression in NSCLC (ID 2437)
10:30 - 12:00 | Author(s): M. Kowanetz, D.S. Shames, H. Koeppen, Y. Xiao, C. Behrens, R. Desai, L. Fu, C. Chappey, A. Mokatrin, E.E. Kadel, A. Do, O.T. Brustugun, M. D’arcangelo, T. Boyle, D.S. Chen, G. Hampton, L.C. Amler, F.R. Hirsch, P.S. Hegde, I.I. Wistuba
- Abstract
- Presentation
Background
Programmed Death Ligand 1 (PD-L1, CD274, B7-H1) is an immune checkpoint molecule that binds to the receptors PD-1 and B7.1 on activated T cells. Binding negatively regulates T-cell function in both physiological and pathological conditions. Recent clinical studies have suggested that numerous cancers, including NSCLC, may utilize PD-L1 expression to escape T-cell mediated cytotoxic activity. Inhibition of PD-L1 can restore anti-tumor immunity, leading to clinical responses. A better understanding of PD-L1 expression patterns, co-expression with other immune markers and actionable disease associated biomarkers may provide insight into the future design of cancer immunotherapy trials in NSCLC.Methods
Expression of PD-L1 was measured by immunohistochemistry (IHC) in archival tumors and, in some cases, in paired metastases in 2 FFPE NSCLC tumor tissue collections. Set 1 (N=561) was collected from patients who were eligible for surgery with curative intent from 2003 to 2005 at MD Anderson Cancer Center. The samples from Set 2 (N=300) contained surgically resected NSCLC tissue collected between 2006 and 2011 (UCCC and Norwegian Radium Hospital). PD-L1 expression was analyzed in both malignant and non-malignant cells (e.g., infiltrating immune cells). In addition, a multiplex qPCR assay that measures ≈90 immune-related genes was used to characterize the tumor immune microenvironment in the NSCLC tumor samples. Disease associated biomarkers, including the mutation status of EGFR and KRAS, as well as expression of MET (by IHC) were also evaluated.Results
Prevalence of PD-L1 was comparable between adenocarcinoma and squamous cell carcinoma (≈30% in tumor cells; ≈45% and ≈50%, respectively, in immune cells). PD-L1 prevalence varied depending on the pathological stage, and was higher in Stages I-IIIA than in Stages IIIB-IV. Similarly, the prognostic value of PD-L1 varied by both stage and histology. In adenocarcinoma, tumors with PD-L1–positive tumor cells had a higher frequency of KRAS mutation and high Met expression, and a lower frequency of EGFR mutation compared with PD-L1–negative tumors. In contrast, tumors with PD-L1–positive and PD-L1–negative immune cells had a comparable frequency of high Met expression. Expression of PD-L1 was frequently co-localized with CD8+ T-cell infiltrates. Gene expression profiling revealed differences in the tumor immune environment, including genes associated with cytotoxic T-cells, between adenocarcinomas and squamous cell carcinomas. PD-L1 protein and immune gene expression associations with patient characteristics will be described in further detail.Conclusion
These data provide a comprehensive description of PD-L1 expression in the context of disease biology utilizing large independent cohorts of well-characterized lung cancer tissues. The results highlight the complexity of the tumor immune environment in NSCLC with particular emphasis on the association with factors such as pathological stage, histology and oncogenic mutational status. These analyses may help guide future development of immunotherapy trials in NSCLC.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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- Abstract
- Presentation
Background
Programmed Death Ligand-1 is a ligand for Programmed cell death protein 1 which is a key receptor regarding one of the immune checkpoints. PD-L1 expression in tumor is known to correlate with post-operative survival in different set of cancer patients. In the present study, we investigated PD-L1 expression of tumor cells in specimens acquired from non-small cell lung cancer patients and analyzed the correlation between the PD-L1 expression and clinicopathological characteristics and postoperative prognosis of 208 Non-Small Cell Lung Cancer patients.Methods
PD-L1 expression in 208 specimens of NSCLC was assessed through an immunohistochemical process and inspected double-blinded.Results
PD-L1 expression is associated with clinicopathological features (histology, P=0.047 and differentiation, P=0.023). No significant association observed between PD-L1 expression and post-operative prognosis. However, subgroup analysis of squamous carcinoma patients with positive PD-L1 protein expression showed a tendency of poor overall survival and disease-free survival compared with those with negative PD-L1 protein expression.Conclusion
we have shown for the first time that PD-L1 expression is associated with clinicopathological features (histology and differentiation). In the subgroup analysis of squamous carcinoma patients, patients with positive PD-L1 expression showed a tendency to be associated with poorer survival compared with those with negative PD-L1 expression, which suggested that patients with squamous carcinoma might be the most benefit population in the anti-PD-1 immunotherapy.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO19.12 - Prognostic Impact of Tumor-Infiltrating Immune Cells in Lung Squamous Cell Carcinoma (ID 2896)
10:30 - 12:00 | Author(s): A.J. Bograd, K. Kadota, J. Nitadori, L. Cherkassky, V.W. Rusch, W.D. Travis, M. Sadelain, P.S. Adusumilli
- Abstract
- Presentation
Background
The prognostic significance of the tumor immune microenvironment in lung adenocarcinoma has been established by us (CCR 2011, JCO 2013, Oncoimmunology 2013) and others. Here, we investigate whether tumor-infiltrating immune cells correlate with prognosis, independent from TNM staging, in lung squamous cell carcinoma (SCC).Methods
All available tumor slides from therapy-naive, surgically resected solitary lung SCCs (n=485; 1999-2009) were reviewed. Tissue microarrays were constructed using 451 cases (stage I, 255; II, 131; III, 65) from 3 representative tumor areas. Immunostaining for CD3 (pan T cell marker), CD45RO (memory T cell), CD8 (cytotoxic T cell), CD4 (helper T cell), FoxP3 (regulatory T cell), CD20 (B cell), CD68 (macrophage), and CD10 (neutrophil) was performed. For each case, the average number of cells positive for T cell markers was recorded as the ratio to CD3+ lymphocytes, and classified as low or high by use of the median. CD20, CD68, and CD10 were classified as low or high by the number of positive cells (≥20, ≥50, and ≥10, respectively) as our recent publication (JCO 2013). Overall survival (OS) was estimated using the Kaplan-Meier method; multivariate analyses were performed using the Cox proportional hazards model.Results
Five-year OS was 59% for the entire cohort and 68% for stage I patients. Analysis of single immune cell infiltration revealed that high CD10+ neutrophil count was correlated with lower OS (5-year OS, 53%; n=160) than low CD10+ count (5-year OS, 61%; n=286; p=0.006). Analysis of biologically relevant immune cell combinations identified 2 significant factors of prognosis: (1) patients with high CD4+ and high FoxP3+ T cell ratios had worse prognosis (5-year OS, 52%; n=140) than the other groups (5-year OS, 62%; n=304; p=0.008), and (2) patients with high CD10+ neutrophil and low CD20+ B lymphocyte counts had worse prognosis (5-year OS, 43%; n=102) than the other groups (5-year OS, 63%; n=340; p<0.001). These results were confirmed in a subgroup analysis limited to stage I patients (p=0.020 for high CD4/high FoxP3+ ratios; p=0.007 for high CD10+/low CD20+ counts). In multivariate analysis, high CD4+/high FoxP3+ ratios (HR=1.58; p=0.001) and high CD10+/low CD20+ counts (HR=1.71; p<0.001) remained significantly associated with poorer survival (Table).Table. Multivariate analysis for overall survival
Variable HR 95% CI p High CD4+/high FoxP3+ ratios 1.58 1.21–2.06 0.001 High CD10+/low CD20+ counts 1.71 1.28–2.27 <0.001 Age (>65 years old) 1.51 1.09–2.09 0.014 Sex (male vs. female) 1.31 1.01–1.69 0.043 Smoking pack years (>90) 1.01 1.00–1.01 0.003 Stage (II and III vs. I) 1.53 1.16–2.02 0.002 Lymphovascular invasion 1.38 1.02–1.88 0.040 Conclusion
High CD4+/high FoxP3+ ratios and high CD10+/low CD20+ counts are significant factors of prognosis for lung SCC, independent of TNM staging. Targeting regulatory T cells or enhancing tumor-specific B-cell responses may thus have applicability for the treatment of lung SCC.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO19.13 - DISCUSSANT (ID 3904)
10:30 - 12:00 | Author(s): J. Lesterhuis
- Abstract
- Presentation
Abstract not provided
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MS02 - Stem Cells and Epigenetics in Lung Cancer (ID 19)
- Event: WCLC 2013
- Type: Mini Symposia
- Track: Biology
- Presentations: 4
- Moderators:G. Sozzi, J. Minna
- Coordinates: 10/28/2013, 14:00 - 15:30, Bayside 103, Level 1
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MS02.1 - Stem Cells & the Cell of Origin of Lung Cancer (ID 462)
14:00 - 15:30 | Author(s): K. Sutherland
- Abstract
- Presentation
Abstract
The cellular hierarchy of the lung is quite complex and it is believed that different progenitor cells and stem cell populations residing within distinct spatial regions of the lung are responsible for orchestrating lung development, regeneration and repair. These different stem cell populations are also likely to be instrumental in the development of the various cancers in lung as particular lung tumour subtypes are almost exclusively found in distinct compartments within the lung. Squamous cell carcinomas are thought to arise from the proximal airways, small cell lung cancer are predominantly located in the bronchioles while adenocarcinomas are more frequently detected the in the distal part of the lung. To investigate the cellular origin of lung cancer, we utilized Cre-loxP recombination technology, which is an effective method for expressing or deleting a target gene in Cre-expressing cells. We generated a series of recombinant adenoviruses expressing Cre recombinase from specific lung epithelial gene promoters. For these studies we chose to target Clara cells, alveolar type 2 (AT2) cells and neuroendocrine (NE) cells. Comprehensive experiments performed in Rosa26R-lacZ and mT/mG reporter animals showed that these viruses exhibit a high level of cell selectivity in the adult mouse lung. To address the cellular origins of small cell lung cancer (SCLC) we have utilised a sporadic mouse model of small cell lung cancer based on the conditional inactivation of the tumour suppressor genes Tp53 and Rb1. We infected Tp53F/F;Rb1F/F animals with our cell type-restricted Adeno-Cre viruses: Ad5-CC10-Cre, Ad5-SPC-Cre and Ad5-CGRP-Cre. Results from these studies show that inactivation of Trp53 and Rb1 can efficiently transform neuroendocrine (CGRP-positive) and to a lesser extent, alveolar type 2 (SPC-positive) cells leading to SCLC. In contrast, CC10-expressing cells were largely resistant to transformation. The results clearly indicate that neuroendocrine cells serve as the predominant cell-of-origin of SCLC in this model. Interestingly a different, cell type specificity was observed when a K-rasG12D oncogene-driven non-small cell lung cancer (NSCLC) model was used to reveal the cell of origin of NSCLC (mostly of adenomas and adenocarcinomas). In this case we noted a difference between K-rasLSL-G12D/+ and K-rasLSL-G12D/+;Trp53F/F animals following infection with our cell type-restricted adenoviruses. In K-rasLSL-G12D/+ mice alveolar type 2 cells appeared to be the most effective target cell for inducing adenomas, whereas in K-rasLSL-G12D/+;Trp53F/F mice multiple cell types had the capacity to give rise to adenomas and adenocarcinomas. Moreover, preliminary data from these experiments indicates that the cell-of-origin may also influence the characteristics and behaviour of the resulting tumours. Taken together, our data show that both cell specific features and the nature of the genetic lesion(s) are critical factors in determining the tumour initiating capacity of lung (progenitor) cells to give rise to various lung cancer subtypes.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MS02.2 - Targeting Hedgehog Signaling in Small Cell Lung Cancer (ID 463)
14:00 - 15:30 | Author(s): N. Watkins
- Abstract
- Presentation
Abstract
Small cell lung cancer (SCLC) is a malignant neuroendocrine tumour responsible for 20% of all lung cancer deaths. Despite the effectiveness of platinum-based chemotherapy, the overwhelming majority of patients succumb to a chemoresistant recurrence within 2 years of diagnosis. Therefore, the discovery of novel strategies to prevent disease recurrence may have a significant impact on outcome. Many groups, including our own, have identified the importance of embryonic signaling pathways in promoting tumor-regeneration through the regulation of self-renewal. Activation of self-renewal pathways such as Notch, Hedgehog (Hh) and WNT is also thought to contribute to the survival of cancer cells with innate resistance to chemotherapeutic agents. Since the naturally occurring Hh inhibitor cyclopamine can block self-renewal in SCLC cells in vitro, we postulated that this pathway might be targetable following platinum-based chemotherapy. The Hh pathway is a highly conserved signaling system that specifies cell fate and self-renewal in development and homeostasis. The Hh ligand Sonic Hh (Shh) binds to, and inactivates, the receptor Patched (Ptch). This prevents Ptch from inhibiting Smoothened (Smo), the molecular target of the small molecule Hh inhibitors. Smo activation requires translocation to the tip of the primary cilium, a single, immotile, tubulin-based organelle present on most vertebrate cells. The ciliary motor protein Kif3a is essential for Smo translocation to the ciliary tip, and is required for Smo signaling in development. The importance of primary cilia in cancer is poorly understood. In addition, the formation of cilia is normally restricted to cells in the G0 of G1 phases of the cell cycle. In order to better understand how Hh signaling is regulated in SCLC, we investigated pathway activation in the context of cilia formation, and the expression of Smo protein in these cilia. Using a genetic mouse model of SCLC, we observed that approximately 25% of tumour cells express primary cilia, with a variable number expressing Smo at the cilia tip. Clonal growth of these tumour cells could be inhibited by blocking the Shh ligand with a monoclonal antibody, or by inactivating Smo with the small molecule LDE225 (Novartis). These data suggest that activation of Smo by Hh ligand at the level of the primary cilium is a crucial step in the initiation and self-renewal of SCLC. By contrast, cilia were rarely observed in human SCLC cells, both in vitro and in vivo. Based on our hypothesis that Hh signaling may be important in the regeneration of SCLC stem-like cells following chemotherapy, we employed a primary xenograft model in which we could identify minimal residual disease following treatment with single-agent carboplatin. Following chemotherapy, more than 50% of the residual tumour cells expressed primary cilia, and in the majority of these, Smo could be detected in the tip. In addition, marked upregulation of Shh ligand expression was observed. However, when these tumours were allowed to regrow to their original size, expression of Shh and cilia returned to the same pattern as seen in the treatment naive tumour, once again supporting a role for cilia-dependent activation of Smo by Hh ligand in SCLC stem-like cells. Furthermore, treatment of mice following chemotherapy with several small molecule inhibitors of Smo, including LDE225, delayed the regeneration of these tumours in vivo. One major controversy surrounding the role Hh signaling in cancer relates to the role of tumour stroma as a target of Shh signaling. To exclude this as a potential mechanism, we used two approaches. First, we crossed the mouse genetic model of SCLC referred to above with a reporter mouse in which LacZ activity can be used to measure Hh pathway activation. In this model, heterogeneous LacZ expression was clearly seen in tumour cells, but not stromal cells. Second, we recreated the carboplatin regeneration model of human SCLC in vitro using a three dimensional, stroma-free clonogenic assay. Transient manipulation of Hh signaling at all levels of the pathway by antibody blockade, siRNA, transfection or small molecule treatment dramatically affected long term cloning capacity in SCLC cells. Moreover, SCLC cells that survived an LD~95~ treatment with carboplatin in vitro demonstrated a marked increase in clonal capacity that was even more sensitive to Hh pathway blockade. Confocal immunofluoresence imaging of these cells revealed expression of Smo in the tips of numerous primary cilia, demonstrating that cell autonomous Hh pathway activation could be observed in a subset of innately chemoresistant, stem-like cells. In the last 5 years, targeted deletion of different components of the primary cilium in mice has dramatically expanded our understanding of the role of these structures in cell signaling. Once considered vestigial, cilia are now recognised as key signaling nodes for Hh, WNT, Notch, PDGF and mTOR signaling. In addition, we have identified heterogeneous expression of primary cilia in several other tumour models, most strikingly in osteosarcoma. More recently, we have shown that knockdown of KIF3a, an essential component of cilia assembly, causes a more dramatic loss of cloning capacity than inhibition of Hh signaling alone. These data suggest that in a subset of self-renewing tumour cells, cilia-dependent signaling pathways in addition to Hh are of importance, and may represent novel therapeutic targets. We are currently using genetically modified mouse models of cancer in which we can conditionally knockout Kif3a to address this question.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MS02.3 - Micro-RNA in Lung Cancer (ID 464)
14:00 - 15:30 | Author(s): G. Sozzi, M. Boeri, U. Pastorino
- Abstract
- Presentation
Abstract
Lung cancer, for its high incidence and mortality, is the most common cause of death from cancer in many developed countries. In contrast to other cancers, there has been almost no improvement in the 5-year survival rates of lung cancer in the past 30 years, rate just above 10% in Europe, primarily because lung cancer is detected in most cases in an advanced stage. Detecting lung cancer at an earlier stage and, ideally, predicting who will develop the disease and particularly the most aggressive forms of cancer are the biggest challenge. Imaging via low-dose computed tomography (LDCT) scanning is being actively evaluated as a screening tool for early detection of lung cancer in high risk patients but, although the positive results in mortality reduction reported in the large NLST trial (1) were very promising, at present, the real efficacy of LDCT lung cancer screening in heavy smokers remains a controversial issue (2). Nonetheless, the high false positive rates of LDCT, leading to multiple screening rounds, the issue of over-diagnosis, the unnecessary and sometimes harmful diagnostic follow-up and the costs underscore the need for non-invasive complementary biomarkers for standardized use. MicroRNAs are small, non coding, endogenous single–stranded ribonucleic acids with regulatory functions that are involved in tuning of many important pathways, including developmental and oncogenic pathways. Because of their fundamental role in development and differentiation, their involvement in the biological mechanisms underlying tumorigenesis, as well as their low complexity, stability and easily detection, they represent a promising class of tissue and blood-based biomarkers of cancer (3). We explored miRNA expression profiles of lung tumors and normal lung tissues from cases with variable prognosis identified in a completed spiral-CT screening trial with extensive follow-up (4). We found a panel of deregulated miRNAs discriminating normal lung tissue versus lung cancer and significant association of miRNA expression profiles in both tumor and non-involved lung tissue with clinical-pathological characteristics of the patients such as tumor histotype, tumor growth rate, disease free survival. miRNA expression profile in tumor and normal lung tissues from patients identifed in the first two years of the screening, including mainly Stage Ia ADC with excellent survival, was found to be significantly different from the profile of subjects with more aggressive tumors appearing in later years of screening, independently from tumor Stage. Overall these results indicate that, both in tumors and in non involved lung tissues, miRNA signatures are able to discriminate patients according to tumor aggressiveness, independently from Stage and type. We have then investigated mirRNA profiles in plasma samples from cases and controls belonging to two independent LDCT screening trials with extensive follow-up where multiple plasma samples, collected before and at time of disease detection were available. We reported that miRNA profiling in plasma samples collected 1–2 yrs before the onset of disease, at the time of lung cancer detection by LDCT and in disease-free smokers, resulted in the generation of four miRNA signatures with strong predictive, diagnostic, and prognostic potential (4). Overall, these results suggest that plasma miRNA profiles might be helpful in pinpointing those early stage tumors at high risk of aggressive evolution that would need additional treatments. We recently completed a large validation study where the diagnostic performance of the plasma-based miRNA test was retrospectively evaluated in samples prospectively collected from smoker subjects within the MILD trial. In this study, 1,000 consecutive MILD plasma samples collected from June 2009 to July 2010 among lung cancer-free individuals enrolled in the trial and all patients with lung cancer diagnosed by September 2012 (n=85) were obtained. In patients we analyzed plasma samples collected both pre-disease (four to 35 months before lung cancer detection, median lag time of 15 months) and at the time of diagnosis. Custom-made microfluidic cards containing the 24 microRNAs composing the signatures identified in the exploratory study were created, and on each card eight plasma samples were analyzed per time. Since the goal of this study was to combine the plasma miRNA assay with LDCT results, in order to have a clinical useful tool to classify plasma samples, we developed a three-level miRNA signature classifier (MSC) of Low, Intermediate, or High risk of disease with subject categorization to one of these three risk groups based on pre-defined cut-points of positivity for the four different expression signatures of the 24 miRNAs previously identified. The results of this large validation study indicates that MSC is a significant diagnostic instrument for lung cancer detection with prognostic performance and support the combined use of MSC and LDCT to improve the efficacy of lung cancer screening (5). References 1. Kramer BS, Berg CD, Aberle DR et al. Lung cancer screening with low-dose helical CT: results from the National Lung Screening Trial (NLST). J Med Screen. 2011;18:109-111. 2. Pastorino U, Rossi M, Rosato V, Marchianò A, Sverzellati N, Morosi C, Fabbri A, Galeone C, Negri E, Sozzi G, Pelosi G, La Vecchia C. Annual or biennial CT screening versus observation in heavy smokers: 5-year results of the MILD trial. Eur J Cancer Prev. 2012 May;21(3):308-15 3. Boeri M., Pastorino U. and Sozzi G. Role of MicroRNAs in Lung Cancer: MicroRNA Signatures in Cancer Prognosis. Cancer J. 2012 May;18(3):268-74 4. Boeri M, Verri C, Conte D, Roz L, Modena P, Facchinetti F, Calabrò E, Croce CM, Pastorino U, Sozzi G. MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3713-8. 5. Sozzi G, Boeri M, Rossi M, Verri C, Suatoni P, Bravi F, Roz L, Conte D, Grassi M, Sverzellati N, Marchiano’ A, Negri, La Vecchia C, Pastorino U. Clinical Utility of a Plasma-based microRNA Signature Classifier within Computed Tomography Lung Cancer Screening: A Correlative MILD Trial Study. SubmittedOnly Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MS02.4 - Targeting Epigenetic Changes in Lung Cancer (ID 465)
14:00 - 15:30 | Author(s): C.M. Rudin
- Abstract
- Presentation
Abstract
The process of carcinogenesis is driven by clonally maintained genetic and epigenetic events that lead to aberrant cell proliferation, inhibit cell death, promote cell dissemination, and affect other key pathways. Research in the past decade has led to new insights into the epigenetic mechanisms controlling gene expression, and into the multiple ways in which these mechanisms are specifically disrupted in cancer. Epigenetic control of gene expression is dependent on modifications of the DNA itself, primarily methylation at CpG dinucleotides, and also by a host of site-specific protein modifications of histones, histone modifiers, and transcriptional machinery. Progress in understanding the multiple layers of epigenetic control is leading to the development and clinical testing of anti-cancer agents specifically targeting these aberrant pathways. DNA methylation and histone acetylation are two well established epigenetic control mechanisms that are known to be aberrantly regulated in essentially all cancers, including lung cancer. We conducted an exploratory phase I/II trial combining an inhibitor of DNA methyltransferase, azacitidine, and an inhibitor of histone deacetylase, entinostat, in patients with recurrent metastatic non-small cell lung cancer. DNA methylation of gene promoters, and loss of histone acetylation, are coordinately regulated processes that can lead to selective silencing of gene expression: this mechanism has been implicated in silencing key tumor suppressor genes in cancer. Treatment with the combination of azacitidine and entinostat led to rare but impressive objective responses, including a complete response in a patient with extensively pretreated disease. In addition, a surprising fraction of patients experienced objective responses to the immediate subsequent therapy, including standard cytotoxic agents and investigational agents targeting the PD-1/PD-L1 immune checkpoint pathway. Preclinical data offer some potential explanations for this observation: many relevant immunoregulatory pathways in both tumor cells and immune effectors are markedly affected by azacitidine. We are now following up on the priming hypotheses suggested by these data, in randomized phase II studies assessing whether limited duration epigenetic therapy can enhance subsequent chemotherapy or immunotherapy efficacy in patients with advanced non-small cell lung cancer. This study represents an initial foray into combinatorial epigenetic strategy in lung cancer: many other strategies are now possible and are being pursued. “Second generation” agents targeting DNA methyltransferase, including an oral formulation of azacitidine and a prodrug, SG-110, are in early phase clinical development. So too are newer histone deacetylase inhibitors differing in specificity, selectivity, route of administration, and pharmacokinetics. Among the exciting new horizons in epigenetic therapy are new agents targeting more recently defined modifiers of epigenetic control, including many of the readers, writers, and erasers of histone modification. The recent remarkable expansion in knowledge about epigenetic regulatory pathways, and how they become dysregulated in cancer, is opening new therapeutic opportunities in lung cancer and other diseases.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO16 - Prognostic and Predictive Biomarkers IV (ID 97)
- Event: WCLC 2013
- Type: Mini Oral Abstract Session
- Track: Medical Oncology
- Presentations: 1
- Moderators:S. Toyooka, J.C. Yang
- Coordinates: 10/29/2013, 16:15 - 17:45, Parkside Auditorium, Level 1
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MO16.10 - DISCUSSANT (ID 3916)
16:15 - 17:45 | Author(s): J. Minna
- Abstract
- Presentation
Abstract not provided
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O12 - Lung Cancer Biology II (ID 87)
- Event: WCLC 2013
- Type: Oral Abstract Session
- Track: Biology
- Presentations: 1
- Moderators:Y. Nakanishi, B. Solomon
- Coordinates: 10/29/2013, 10:30 - 12:00, Parkside 110 A+B, Level 1
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O12.08 - DISCUSSANT (ID 3897)
10:30 - 12:00 | Author(s): J. Minna
- Abstract
- Presentation
Abstract not provided
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P1.01 - Poster Session 1 - Cancer Biology (ID 143)
- Event: WCLC 2013
- Type: Poster Session
- Track: Biology
- Presentations: 1
- Moderators:
- Coordinates: 10/28/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
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P1.01-002 - Clinicopathological and biological significance of epiregulin expression in non-small cell lung cancer (ID 755)
09:30 - 16:30 | Author(s): J. Minna
- Abstract
Background
KRAS mutations are one of the most common driver mutations in non-small cell lung cancer (NSCLC) and efficient therapeutic stratergies for oncogenic KRAS-driven NSCLC are urgently needed. We recently identified epiregulin (EREG) as one of several putative transcriptional targets of oncogenic KRAS signaling in KRAS-mutant NSCLC cells and immortalized bronchial epithelial cells expressing ectopic mutant KRAS. In the present study, we assessed clinicopathological and biological significance of EREG expression in NSCLC.Methods
Seventy-eight lung cancer cell lines (23 small cell lung cancers [SCLCs] and 35 NSCLCs), five noncancerous bronchial epithelial cell lines and 174 surgical specimens from NSCLC patients (136 adenocarcinomas and 38 squamous cell carcinomas) were used for EREG expression analysis by real-time RT-PCR methods. In vitro cell growth was evaluated by MTT assay, colony-formation assay in liquid culture and soft agar assay. Apoptosis was evaluated by the DNA fragment detection method and the annexin-V-fluorescein staining method. The Kaplan-Meier method was used for analysis of disease-free survival (DFS) and overall survival (OS) and log-rank test was used for survival differences. Cox proportional hazards model was used to identify independent prognostic factors for PFS and OS.Results
EREG is predominantly expressed in NSCLC lines harboring KRAS, BRAF or EGFR mutations whereas most SCLC lines lack EREG expression. Small interfering RNAs (siRNAs) targeting against these mutations resulted in down-regulation of EREG expression in NSCLC cells. EREG expression was significantly reduced by treatments with the inhibitors of MEK or ERK in EREG-overexpressing NSCLC cell lines, irrespective of mutation status of KRAS, BRAF and EGFR. EREG expression significantly correlated with KRAS copy number in KRAS-mutant NSCLC cell lines whereas EREG expression significantly correlated with EGFR copy number in NSCLC cell lines with wild-type KRAS/BRAF/EGFR. In the analysis of surgical specimens from NSCLC patients, EREG was predominantly expressed in lung adenocarcinomas. In a subgroup of adenocarcinomas, EREG expression was significantly higher in the tumors from elderly patients (≥70-year-old), males and smokers and was higher in the tumors with pleural involvement-, lymphatic permeation- or vascular invasion-positive. EREG was highly expressed in lung adenocarcinomas with KRAS mutation compared to those with EGFR mutation or wild-type EGFR/KRAS. Lung adenocarcinoma patients with high EREG expression had significantly shorter DFS and OS compared to those with low EREG expression. When the patients were divided into four groups according to EREG expression levels and KRAS mutation status, DFS and OS were significantly shorter in the patients with KRAS-mutant/EREG-high than those with wild-type KRAS/EREG-low. Cox regression analysis demonstrated that elevated EREG expression was an unfavorable prognostic factor. siRNA-mediated EREG silencing inhibited anchorage-dependent and -independent growth and induced apoptosis in KRAS-mutant and EREG-overexpressed lung adenocarcinoma cells.Conclusion
Our findings suggest that oncogenic KRAS-induced EREG overexpression contributes to an aggressive phenotype and unfavorable prognosis in lung adenocarcinoma patients, and EREG could be a promising therapeutic target in oncogenic KRAS-driven NSCLC.
