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P.A. Bunn, Jr
Moderator of
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MINI 27 - Biology and Other Issues in SCLC (ID 152)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Small Cell Lung Cancer
- Presentations: 15
- Moderators:P.A. Bunn, Jr, J. Sage
- Coordinates: 9/09/2015, 16:45 - 18:15, 605+607
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MINI27.01 - Investigation of Chimeric Antigen Receptor T Cells as a Novel Immunotherapy for SCLC (ID 2901)
16:45 - 16:50 | Author(s): W.L. Denning, D. Crossland, K.A. Gold, S. Ang, S. Olivares, N. Belousova, B. Glisson, L. Cooper, J.V. Heymach
- Abstract
- Presentation
Background:
Small cell lung cancer (SCLC) is an aggressive malignancy with an average of 20,000 new cases per year and 16,000 deaths per year. SCLC accounts for about 10-15% of newly diagnosed lung cancers. Even in the face of extensive research, the standard of care- platinum-based combination chemotherapy- has not changed in decades. Yet even with modern chemotherapy formulations, the two year survival rate for advanced disease stages is less than 5%. Complicating treatment is that often at the time of diagnosis, SCLC as already metastasized to the patient’s surrounding lymph nodes. Therefore, a novel therapeutic strategy will have address three disease aspects: (1) reduce primary tumor growth and eliminate metastatic spread; (2) avoid resistance mechanisms used by SCLC to escape radio- and chemotherapies; (3) synergize with or supersede current therapeutic strategies. Chimeric antigen receptor T cells, little explored in SCLC, is well suited to address these aspects.
Methods:
Human SCLC cell lines were analyzed using a 90 gene signature to establish immunological targets. Western blot analysis confirmed the expression of CD56 and other targets on SCLC cell lines. For CAR T cell generation, PBMC were electroporated with the Sleeping Beauty transposase and a transposon containing a CD56R chimeric antigen receptor. CD56R-CAR transduced T cells were cultured for 4 weeks in the presence of K562 cells expressing CD56 and the cytokines IL-2/IL-21 to expand CD56R-CAR T cells. CAR T cells were tested in vitro for killing ability in the presence of three SCLC cell lines using a chromium release assay. CAR T cells were also analysed via FACS to assess CAR expression, T cell phenotype, and memory status.
Results:
An analysis of immune markers in SCLC cell lines revealed that, compared to NSCLC lines, there is a reduction in the expression of suppressive ligands and co-stimulatory ligands, antigen presentation, and natural killer ligands. SCLC cell lines, however, express high levels of CD56. When two CD56-positive and one CD56-negative cell line was tested, CD56-CAR T cells could kill efficiency CD56 expressing cell lines, however there was little killing of the CD56-negative cell line. An analysis of PBMCs cultured after electroporation revealed that a large percentage of CD3+ T cells expressed the CD56 CAR and even after 4 weeks in culture, the CAR T cells displayed a memory phenotype.
Conclusion:
An interrogation of SCLC cell lines versus NSCLC cell lines revealed that SCLC cell lines had reduced expression of checkpoint ligands, NK cell killing ligands, antigen presentation, but consistent with their origin, high expression of CD56. Our conclusion from this analysis is that expansion of SCLC-specific immune responses in vivo or elicitation of de novo responses in vivo will be hindered. Therefore, immunotherapies centered around adoptive transfer of T cell that can kill in an HLA-independent manner maybe better suited for SCLC. In that vein, CD56R-CAR T cells effectively targeted CD56-positive SCLC in vitro, but was unable to kill CD56-negative cells- which indicates a possible escape variant. Our lab is now moving toward testing CD56R-CAR T cell in vivo in both xenograph models and spontaneous ones.
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MINI27.02 - RPS25 Is Essential for the Translation of the Seneca Valley Virus Genome and Proliferative Capacity of Small Cell Lung Cancer Cell Lines (ID 3278)
16:50 - 16:55 | Author(s): L.A. Miles, T. Hitchman, J. Poirier, C.M. Rudin
- Abstract
- Presentation
Background:
Small cell lung cancer (SCLC) is an extremely aggressive and lethal disease for which there is a desperate need for novel and more effective treatments. A recently discovered oncolytic picornavirus, Seneca Valley Virus (SVV), infects tumors with neuroendocrine features, including SCLC with high selectivity. SVV is highly effective in the eradication of solid tumors in multiple in vivo models; however the mechanism of selective tropism for SVV is unknown. Because of the strong selectivity of the virus for SCLC, we hypothesize the host determinants of SVV permissivity could constitute future druggable targets for the treatment of SCLC.
Methods:
A retroviral gene trap mutagenesis screen was utilized in HAP1, a haploid human cell line permissive to SVV, HAP1. Once mutagenized, resistant cells, or cells with retroviral insertion in a gene essential to the viral life cycle, were selected for by incubation of the pool with SVV at a high multiplicity of infection (MOI). Hits from this screen were deconvoluted using an insertion mapping approach. Illumina sequencing provided quantitative counts of each insertion site in each gene. Hits from the screen were validated using various mechanistic approaches.
Results:
Our screen identified multiple unique insertion sites in the gene RPS25 on Chromosome 11. The RPS25 protein is a ribosomal protein that is a component of the 40S subunit of the ribosome. RPS25 has been previously shown to be important for IRES-dependent translation of multiple viral genomes as well as cellular mRNAs containing IRES elements. Using the CRISPR-Cas9 approach, we knocked out the RPS25 gene in the SVV-permissive SCLC cell line, NCI-H446. Upon total knock-down of RPS25, H446 cells become completely resistant to cell killing by SVV at high MOI. Surprisingly, these cells also show a severely marked decrease in doubling time and robustness in culture. In contrast, RPS25 CRISPR knock-down in HEK293T cells has been previously shown to have no distinguishable phenotype other than defects in IRES-dependent translation. Further studies to fully characterize the interaction of RPS25 with the SVV genome as well as the importance of RPS25 in other SCLC cell lines are ongoing.
Conclusion:
We have identified a host protein that is essential for SVV replication and infection using a genome wide mutagenesis screen. SCLC cells completely defective in RPS25 are resistant to SVV-dependent cell killing. RPS25 appears to not only be important for the life cycle of SVV but may be important in proliferative capacity in SCLC. As SVV is highly selective for SCLC, we hypothesize that the host determinants of SVV tropism may be very specific to SCLC cells. Proteins important in the SVV life cycle may be novel “druggable” targets for the treatment of SCLC.
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MINI27.03 - PD-L1 Expression in Small Cell Lung Carcinoma: An Immunohistochemical Analysis of 26 Cases Using Two Anti-PD-L1 Antibodies (ID 2936)
16:55 - 17:00 | Author(s): P.B. Illei, P. Forde, C. Hann, S. Yang, R. Kelly
- Abstract
- Presentation
Background:
Small cell lung carcinoma (SCLC) represents 15% of lung cancers and is treated using chemotherapy +/- radiation but despite initial responses most recur within a few months and become resistant to therapy. Novel immune checkpoint inhibition of programmed death-1 (PD1) targeted therapy has shown promise in other solid tumors including non-small-cell lung cancer (NSCLC) and malignant melanoma. In some tumor types correlation with response and significant expression of programmed death- ligand 1 (PD-L1), the lead candidate biomarker of anti-PD-1 therapy, has been described but no data is available regarding expression levels in SCLC. Here we report the rate of PD-L1 expression in SCLC and in associated tumor infiltrating immune cells lymphocytes and macrophages.
Methods:
Immunohistochemistry (IHC) for PD-L1 using two monoclonal antibodies (clone 5H1 and clone SP142) and for CD3 (clone PS1) was performed on standard formalin fixed paraffin embedded tissue sections of 21 resected SCLC specimens (median age: 67) and three additional tumors with pre- and post-therapy biopsies. Since there is no generally accepted scoring system for PD-L1 expression we chose to evaluate staining in tumor cells and immune cells infiltrating the tumor nests and in adjacent stroma using a 4 tier semi quantitative scoring system (score 0 -no or <1%, 1+ 1-<5%, 2+ 5-25% and 3+ >25% of cells staining). Both cytoplasmic and membranous staining was accepted as positive. The number of tumor infiltrating lymphocytes (TIL) were estimated utilizing a CD3 stain while macrophages were identified on corresponding H&E stains.
Results:
PD-L1 staining of tumor cells and Immune Cells (TIL & Macrophage) are shown in the table below. Membranous PD-L1 staining was only seen in two tumors and in variable number of immune cells with 2+ or 3+ PD-L1 scores. The majority of positive staining was cytoplasmic with both antibodies. The staining intensity was stroger with the 5H1 antibody. The paired pre- and post-therapy samples were all negative for PD-L1.
* Tumors with membranous staining; IC: immune cellsClone/score PD-L1 staining in 5H1 Tumor IC in tumor IC in stroma 0 (<1%) 19/21 (90%) 7/21 (33%) 5/21 (24%) 1+ (1-<5%) 1/21 (5%)* 11/21 (53%) 6/21 (29%) 2+ (5-25%) 1/21 (5%)* 3/21 (14%) 7/21 (33%) 3+ (>25%) 3/21 (14%) SP142 Tumor IC in tumor IC in stroma 0 (<1%) 20/21 (95%) 8/21 (38%) 10/21 (48%) 1+ (1-<5%) 1/21 (5%)* 11/21 (52%) 7/21 (33%) 2+ (5-25%) 2/21 (10%) 4/21 (19%) 3+ (>25%)
Conclusion:
Most SCLC are tumor membrane PD-L1 negative by IHC. A subset of SCLC contain PD-L1 positive TILs and/or macrophages in the tumor and the stroma. No up regulation of PD-L1 expression was seen in a small pilot sample of matched pre- and post-therapy biopsies. It is unclear whether PD-L1 expression assessed by IHC will be a predictive marker for PD-1 targeted therapy in SCLC. Preliminary data indicates single agent and combined checkpoint inhibitors (PD1 plus CTLA-4 inhibitors) are active in previously treated SCLC indicating additional research is required to understand their mechanism of action in a tumor type that has seen no therapeutic advances in the last two decades.
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- Abstract
- Presentation
Background:
Blocking the interaction between the programmed cell death (PD)-1 protein and one of its ligands, PD-L1, has been reported to have impressive antitumor responses. PD-L1 interaction is a major pathway often hijacked by tumors to suppress immune control. Studies on the roles of PD-L1 in non-small cell lung cancer (NSCLC) are controversial, but its roles in small cell lung cancer (SCLC) are rare and unclear. Moreover, MET/HGF axis seems to be the other one of the most aberrant signaling pathways in SCLC. The aim of our study was to investigate the expression and prognostic roles of PD-L1 and cellular-mesenchymal to epithelial transition factor(c-MET) in SCLC.
Methods:
The expression of PD-L1 and c-MET were evaluated by immunohistochemical analysis in 83 specimens of SCLC, including 47 limited disease (LD) and 36 extensive disease(ED). Tumors with PD-L1 staining in over 5% of tumor cells were scored as positive for PD-L1 expression. Tumors with c-MET strong staining in at least 10% or weak to moderate staining in at least 40% of tumor cells were scored as positive for c-MET expression. Survival analysis was performed using the Kaplan-Meier method.
Results:
The positive rate of PD-L1 and c-MET in SCLC specimens were 51.8% and 25.3% respectively. The higher expression level of PD-L1 in tumor specimens was significantly correlated with a limited disease (LD) stage (p=0.004), a normal serum LDH level (p=0.031), and a normal NSE level (p=0.005). No association was found between the expression level of c-MET and PD-L1 , or c-MET expression with the other clinical characteristics of SCLC patients. SCLC patients with PD-L1-positive tumors showed significantly longer overall survival (OS) than those with PD-L1-negative (median OS, 17.0 vs 9.0, p=0.018). SCLC patients with positive c-MET expression showed a trend of shorter overall survival (12.0 vs 15.0, p=0.186). But sub-analysis of Limited disease (LD)-stage patients showed that the c-MET negative group had a longer OS (25.0 vs 14.0; p=0.011). Multivariate analyses revealed that LD stage, good performance status but except for PD-L1 or c-MET immunoreactivity were independently predictive of better OS.
Conclusion:
In patients with SCLC, expression of PD-L1 was positively correlated with a LD stage and better OS, but was not an independently predictive factor of outcome. High expression level of c-MET revealed a trend of worse outcome. It was associated with poor prognosis especially in LD-Stage patients.
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MINI27.05 - Discussant for MINI27.01, MINI27.02, MINI27.03, MINI27.04 (ID 3379)
17:05 - 17:15 | Author(s): J.W. Neal
- Abstract
- Presentation
Abstract not provided
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- Abstract
- Presentation
Background:
As a subtype of lung cancer, small cell lung cancer (SCLC) remains a severe threat to human health. Although it is initially a chemosensitive disease, development of acquired resistance is a major problem. Studies in recent years revealed that cancer stem cell (CSC) could play a role in this process. However, the CSC specific marker and the detailed signal pathway associated with acquired resistance in SCLC is unknown yet. It was recently reported that the voltage-dependent calcium channel α2δ1 subunit positive cells is a CSC marker in hepatic cell cancer and that 1B50-1 is the specific monoclonal antibody of the α2δ1 subunit. In present study, we attempted to disclose that α2δ1 could play a role in acquired resistance of SCLC. Also we investigated possible molecular mechanism of α2δ1 mediated resistance in SCLC and finally provided the potential strategies overcoming the resistance.
Methods:
We screened for positive expression of 1B50-1and CD133 in SCLC cell lines and in patient-derived xenograft (PDX) models, and we used flow cytometry to verify the properties of CSCs. We recorded the expression of 1B50-1 before and after chemotherapy in PDXs in chemosensitive and resistant models to determine if α2δ1 subunit-positive cells were related to acquired resistance. We used exome and transcriptome sequencing to explore the expression of genes related to stem cell properties and drug resistance. We used Western blotting to verify the key molecules and pathways in the process of drug resistance. On the basis of these results, we explored the mechanisms of acquired drug resistance that are mediated by the α2δ1 subunit.
Results:
We observed a difference in the positive expression levels of 1B50-1 and CD133 in SCLC cell lines (H1048, H69, and H209) and PDX models. Both 1B50-1-positive and CD133-positive cells exhibited stem cell-like properties such as the capacity to self-renew in vitro, tumorigenesis in vivo, the potential for differentiation, and high expression levels of genes related to CSCs and drug resistance. Chemotherapy could induce the enrichment of 1B50-1-positive cells but not CD133-positive cells in PDXs. Also, high rates of 1B50-1-positive cells corresponded to high levels of resistance. Together, these findings indicated that the expression of 1B50-1 is related to chemoresistance. Exome and transcriptome sequencing revealed that the expressions of multiple pathway related genes in pathways, including MAPK, CAMs, TGFβ, and Notch, were increased in 1B50-1-positive H1048 cells. Western blotting revealed the activation of the Erk protein in the MAPK pathway and the over-expression of the Erk protein in 1B50-1-positive H1048 cells. The specific α2δ1 antibody 1B50-1 improved response to chemotherapy and delayed relapse when combined with chemotherapy or used y as maintenance therapy.
Conclusion:
The α2δ1 subunit positive SCLC cells (1B50-1+) displayed CSC properties, and were associated with acquired resistance. The Erk protein in the MAPK pathway was highly expressed in the 1B50-1-positive H1048 cell line, and might be the key molecule involved in resistance mediated by the α2δ1 subunit. The α2δ1 subunit-specific antibody 1B50-1 could improve response to chemotherapy and delay relapse when combined with chemotherapy or when used as sequential therapy.
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MINI27.07 - Targeting Cancer Stem Cells in Small Cell Lung Cancer (ID 2727)
17:20 - 17:25 | Author(s): V. Kolev, Y. Wang, M. Padval, J. Pachter, D. Weaver
- Abstract
- Presentation
Background:
Small cell lung cancer (SCLC) is an extremely aggressive cancer with limited treatment options and poor outcome. The majority of SCLC patients respond to frontline chemotherapy, but experience rapid recurrence with metastasis, that may be attributed to the prevalence of cancer stem cells (CSCs). We previously demonstrated that PI3K/mTOR signaling is key for CSCs in cell culture and solid tumor models. As shown with a dual PI3K/mTOR inhibitor, VS-5584, inhibition of multiple PI3K isoforms and mTOR is necessary to achieve preferential targeting of CSCs.
Methods:
Antitumor activity of VS-5584 was assessed by in vitro proliferation assay as well as in multiple xenograft models in vivo, including patient-derived xenograft models. Anti-CSC activity was measured by the side-population CSC assay in vitro and in limiting dilution tumor initiation assay in vivo.
Results:
VS-5584 inhibited SCLC growth in vitro at sub-µM IC50, and was synergistic with cisplatin and etoposide in reducing the viability of SCLC cells. In vivo, single agent VS-5584 (20 mg/kg, 3 days per week dosing, MWF) demonstrated robust anti-CSC activity in the NCI-H841 SCLC model by reducing tumor initiating potential 70-fold (p=5x10[-6]). In tandem, VS-5584 partially reduced tumor growth of the NCI-H841 xenograft tumors. Furthermore, a VS-5584 dose dependency was evident, both for tumor initiating potential and tumor growth reduction. When VS-5584 was combined with cisplatin and etoposide, the standard of care agents for SCLC, an increased tumor growth inhibition was observed whether VS-5584 was concurrently administered or added sequentially following the dual chemotherapy. In the SCLC PDX model, combination treatment also suppressed the regrowth of the tumor following cessation of chemotherapy for extended duration. VS-5584 was found to preferentially induce apoptosis in CSCs in multiple cell lines, indicating that these cells are eliminated through cell death-related mechanism. Importantly, we demonstrated that for eradication of CSCs it is necessary to inhibit simultaneously multiple PI3K isoforms and mTOR pathways.
Conclusion:
The VS-5584 pre-clinical findings support the preferential targeting of CSCs in SCLC models and provide an important rationale for advancing clinical development of the compound. A phase 1 dose finding clinical trial is on-going to establish a Phase 2 dose of VS-5584 and explore target inhibition. VS-5584 alone or in combination with standard of care chemotherapy may lengthen the time to relapse and improve outcome for patients with small cell lung cancer.
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MINI27.08 - NOTCH3 Protein Expression and Outcome in Small Cell Lung Cancer (SCLC) and Therapeutic Targeting with Tarextumab (Anti-Notch 2/3) (ID 2999)
17:25 - 17:30 | Author(s): A. Chiang, J. McLaughlin, M.C. Pietanza, A. Spira, R. Jotte, S. Gadgeel, A. Mita, L. Gluck, S. Liu, A. Kapoun, D. Hill, R. Herbst, L. Zhou, J. Dupont, D.R. Spigel
- Abstract
- Presentation
Background:
NOTCH expression is associated with cancer cell survival via effects on cancer stem/progenitor cells. Targeting NOTCH2 and 3 decreases growth and survival of SCLC patient-derived human tumor xenografts (PDX). Phase1b/2 trials testing Tarextumab (TRXT) anti-NOTCH2/3 therapy are underway (NCT01647828 and NCT01859741) and show promising anti-tumor activity. Here, we studied NOTCH3 protein expression using immunohistochemistry (IHC) in SCLC human tissues and correlated with survival. Also, we studied NOTCH3 gene expression in phase 1b patients (pts) treated with TRXT.
Methods:
For NOTCH IHC staining, murine monoclonal antibodies were generated by immunizing mice with a NOTCH3 extracellular domain (ECD) protein, then creating hybridomas. Clones were screened by FACS and western blots for specificity to NOTCH3.ECD. A lead clone was selected for NOTCH3 protein measurement in 47 SCLC samples represented in a tissue microarray from Yale Pathology Tissue Services (YPTS). NOTCH3 signal was determined in tumors using H-scores generated by Leica Aperio Scanscope IHC membrane image analysis. For survival analysis, NOTCH3 signal was binarized with cutoffs defined by X-tile software. For the phase 1b clinical trial, a standard 3+3 dose escalation design was employed with cohorts of 3 to 6 pts treated at each dose level. TRXT was given IV on Day 1 of each 21 day cycle with etoposide 100 mg/m[2] (Days 1-3) and cisplatin 80 mg/m[2 ]or carboplatin at AUC 5 (Day 1) for 6 cycles, followed by TRXT alone every 21 days until progression of disease or unacceptable toxicities. Then, the MTD TRXT plus etoposide and carboplatin was confirmed in a cohort of 6 subjects. All pts are required to submit tissues for Notch 3 gene expression and IHC staining.
Results:
A single hybridoma clone demonstrating specific reproducible membranous staining with a dynamic range for NOTCH3.ECD in control and PDX tissues was chosen for IHC analysis in SCLC human FFPE tissues (n=47). Forty cases (85.1%) demonstrated NOTCH3 signal, with eighteen (38.3%) having none to very low signal. Of the 31 cases with adequate follow-up, there was a strong trend with worse outcome and high NOTCH3 expression in the extensive stage (p=0.063), but not in limited stage (p=0.857). The level of significance was a function of the experimental cut-point and can only be considered exploratory. Finally, 27 pts were treated with TRXT in the phase 1b trial, with an overall response rate of 84%. The median duration of treatment was 128 days (6 cycles) with mPFS and mOS of 124 and 228 days, respectively. The median follow-up for PFS and OS was 86 and 107 days, respectively. Twenty-five pts have tissues evaluable for NOTCH3 gene expression and the analysis is underway.
Conclusion:
NOTCH3 IHC staining showed expression in most SCLC cases, with high NOTCH3 trending towards worse survival in extensive stage. This supports the rationale of targeting NOTCH3 by TXRT in SCLC pts. Further evaluation of the prognostic and predictive value of TRXT for anti-Notch therapies in SCLC is underway in an ongoing Phase 2 clinical trial.
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MINI27.09 - A DLL3-Targeted ADC Effectively Targets Pulmonary Neuroendocrine Tumor-Initiating Cells to Result in Sustained Tumor Regressions (ID 2533)
17:30 - 17:35 | Author(s): L.R. Saunders, A.J. Bankovich, W.A. Anderson, M. Aujay, S. Bheddah, K.A. Black, R. Desai, P. Escarpe, J. Hampl, A. Park, A. Laysang, D. Liu, J. Lopez-Molina, M. Milton, M. Pysz, H. Shao, M. Torgov, S. Williams, O. Foord, P. Howard, J.T. Poirier, M.C. Pietanza, P.P. Massion, C.M. Rudin, R.A. Stull, B.S. Slingerland, S.J. Dylla
- Abstract
- Presentation
Background:
Pulmonary neuroendocrine tumors such as small cell lung cancer (SCLC) and large cell neuroendocrine cancer (LCNEC) remain among the most deadly malignancies and are increasing in incidence. Patient-derived xenograft (PDX) tumors provide excellent models to study tumor biology and discover tumor-initiating cell (TIC) populations. Novel therapies that target and eradicate TIC represent a promising strategy to improve survival. An effectively targeted antibody-drug conjugate (ADC) carrying a cell-cycle independent toxin should result in significant anti-tumor activity and eliminate TIC.
Methods:
Whole transcriptome sequencing was performed using TIC isolated by fluorescence-activated cell sorting from SCLC and LCNEC PDX tumors. Quantitative RT-PCR, microarray analysis of PDX tumors and cell lines, and mining of publically available transcriptome and proteome datasets were executed to validate that prospective targets, such as Delta-like protein 3 (DLL3), were highly expressed in neuroendocrine tumors, but limited in their expression in normal tissues. DLL3-specific monoclonal antibodies were generated and used to determine protein expression by immunohistochemistry, flow cytometry and ELISA. Select DLL3-specific antibodies were conjugated to a cell-cycle independent pyrrolobenzodiazepine (PBD) dimer toxin and evaluated for their ability to internalize and mediate cell killing. Finally, established SCLC and LCNEC PDX tumors were treated in vivo with a lead anti-DLL3 ADC (i.e. SC16LD6.5). Limiting dilution assay (LDA) serial transplantation experiments were executed to assess the impact of SC16LD6.5 on TIC.
Results:
Elevated expression of DLL3 mRNA was discovered in TIC of SCLC and LCNEC PDX tumors and confirmed in additional distinct primary SCLC and LCNEC tumor samples and PDX tumors. In contrast, little to no mRNA expression was detected in vital organs and other normal tissues outside of the brain. DLL3-specific antibodies confirmed protein expression on the cell surface in both primary SCLC and LCNEC tumors and in PDX tumors initiated from patients with these diseases, whereas protein was scarce in normal tissues. SC16LD6.5 rapidly internalizes and localizes to late endosomes, and treatment of 10 SCLC and 2 LCNEC PDX tumor models resulted in significant and durable tumor regression with a median time to progression benefit of 75 days versus 16 days with standard-of-care (SOC: SCLC, cisplatin/etoposide; LCNEC, cisplatin). During the course of these in vivo studies, many mice were cured as tumors often did not recur despite being followed for 120+ days post-randomization and treatment. LDA experiments executed using tumors actively responding to SC16LD6.5 provided further functional evidence that the common lack of tumor recurrence following treatment resulted from effective targeting of DLL3-expressing TIC. In vivo efficacy strongly correlated with DLL3 protein expression, and responses were observed in PDX tumor models initiated from patients with both limited and extensive stage disease and independent of their sensitivity to SOC.
Conclusion:
The DLL3-targeted ADC, SC16LD6.5, effectively targets and eradicates TIC in SCLC and LCNEC PDX tumors. SC16LD6.5 (i.e. rovalpituzumab teserine) is currently concluding Phase 1b trials and is a promising first-in-class therapeutic for the treatment of high grade pulmonary neuroendocrine tumors.
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MINI27.10 - Discussant for MINI27.06, MINI27.07, MINI27.08, MINI27.09 (ID 3380)
17:35 - 17:45 | Author(s): D.L. Gibbons
- Abstract
- Presentation
Abstract not provided
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MINI27.11 - Comprehensive Mutation Analysis of Never-Smokers with Small Cell Lung Cancer (SCLC) (ID 3135)
17:45 - 17:50 | Author(s): M.C. Pietanza, H. Won, L.M. Krug, A.M. Varghese, G.J. Riely, N. Rekhtman, L. Wang, W.D. Travis, M. Zakowski, M. Ladanyi, M. Berger, M.G. Kris, C.M. Rudin
- Abstract
- Presentation
Background:
Although most patients with SCLC are current or former smokers, this disease has been reported in never-smokers. In our prospective genomic profiling of SCLC patients, we have identified four never-smokers. Here, we report next generation sequencing (NGS) results for these four SCLC patients and describe how they differ from those of smokers.
Methods:
We are evaluating pathologically confirmed SCLC tumors in patients undergoing treatment. Formalin-fixed, paraffin-embedded surgical resections, core biopsies, and fine needle aspirates are being evaluated using a targeted, hybrid capture-based, NGS assay, MSK-IMPACT, which identifies single nucleotide variants, indels, and copy number alterations in 341 cancer-associated genes. We determined never-smoking status prospectively: all smoked <100 cigarettes in their lifetime. Clinical data on stage [extensive (ES), limited (LS)], treatment, and response were collected.
Results:
Four never-smokers have been identified within the 50 patient samples that have undergone NGS evaluation thus far. The median age at diagnosis of the four never-smokers is 58 (range, 47-75); 50% are male; and one presented with LS-SCLC. None of these four patients developed SCLC as acquired resistance to EGFR tyrosine kinase inhibitors after treatment for EGFR-mutant lung cancers. The tumors from the four never-smokers displayed a median of 3 non-synonymous somatic mutations, while those from moderate (<20 pack years) and heavy (20+ pack years) smokers contained 4.5 and 8 mutations, respectively (P<0.05). None of the four never-smoker samples contained smoking associated G-to-T transversions (see Table). Inactivation of RB1 and TP53 occurred in 75% and 50% of the samples, respectively. Only patient 4 had platinum-refractory disease. The median survival of these patients was 20.7 months (range, 17 to 25).Sample Gene altered Alteration Present Protein Alteration Base Pair Alteration Patient 1 PHOX2B Missense Mutation P82L G-to-A NOTCH1 Frame-Shift Insertion P2485fs RB1 Splice Site R500_splice G-to-A TP53 Frame-Shift Deletion V218fs TP53 Frame-Shift Deletion V73fs TERT Amplification Patient 2 CBL Missense Mutation C401S G-to-C GNAS Missense Mutation M102V A-to-G MYCL Amplification Patient 3 TP53 Nonsense Mutation R342 G-to-A RB1 Frame-Shift Insertion T197fs CDKN2C Amplification MYCL Amplification Patient 4 RB1 Nonsense Mutation C666 ETV1 Amplification
Conclusion:
Using a targeted NGS assay, we have shown that the molecular characteristics differ between never-smokers and smokers, while the majority of the tumors demonstrate RB loss. Whole exome sequencing of the tumors from these never-smokers is underway. Ongoing comprehensive, multiplexed genotyping is needed to fully characterize the molecular diversity of SCLC in this unique population.
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MINI27.12 - Using a Cell Surface Antibody Screen to Identify Biomarkers of Drug Resistance in Small Cell Lung Cancer (SCLC) (ID 3002)
17:50 - 17:55 | Author(s): D. Meyers, A. Poeppl, V. Bhat, C. Peltier, A. Raouf, S. Banerji
- Abstract
- Presentation
Background:
SCLC is an aggressive malignancy that shows dramatic clinical responses in 70% of cases to platinum-based chemotherapy in the first line setting, while the remainder have de novo treatment resistance. Of initial treatment responders, almost all will relapse with drug-resistant disease within months of initial therapy. From a clinical perspective, the emergence of drug resistance remains one of the most important barriers to improving SCLC patient outcomes. Better models of in vitro and in vivo drug resistance are therefore required. Cell surface markers are proteins expressed on the outer surface of cells that can identify specific cell types and biologic states. Surface markers represent an attractive class of biomarkers in SCLC: 1) they are independent of cellular transport mechanisms that are known to play a role in SCLC drug resistance; and 2) they can be readily accessed for diagnostic and therapeutic purposes using commercial antibodies.
Methods:
NCI-H69 is a classic SCLC cell line derived from a patient prior to systemic treatment, and is chemotherapy sensitive. H69AR is an anthracycline-resistant derivative cell line generated through serial culture of NCI-H69 in increasing concentrations of Adriamycin. H69AR is also cross-resistant to other cytotoxic drugs commonly used to treat SCLC. We performed a pilot screen in both cell lines in duplicate using a human cell surface marker panel containing Alexa Fluor 647-conjugated antibodies against 242 unique cell surface proteins. High-throughput multiplexed flow-cytometry was performed to generate cell surface expression profiles for each antibody in each cell line.
Results:
A total of 53 markers were expressed in at least 20% of cells in either cell line, with 22 positive markers shared by both cell lines including CD44. NCI-H69 was uniquely positive for 24 markers including CD56, a neural progenitor marker used commonly to diagnose SCLC. Seven markers were uniquely positive in H69AR including CD9 and some of its known interactors. The percentage H69AR cells positive for each of the 7 markers ranged from 25% to 88%. CD9 is a member of the transmembrane 4 superfamily involved in many cellular processes including differentiation, adhesion, and signal transduction. Eighty-eight percent of H69AR cells were positive for CD9 compared to only 5.4% of H69 cells. CD9 has previously been implicated in a cell adhesion-mediated drug resistance mechanism in unrelated SCLC chemotherapy-resistant cell lines (S. Kohmo et al. Cancer Research 2010).
Conclusion:
Our pilot data provides a proof-of-concept for our surface biomarker screen-based approach to further understand mechanisms of chemotherapy-resistance in SCLC. We have expanded this screen to additional drug-sensitive and drug-resistant SCLC cell line pairs. Results of the expanded screen will be presented at the meeting.
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MINI27.13 - Progastrin-Releasing Peptide (ProGRP) as a Biomarker for Clinical Response in Small-Cell Lung Carcinoma (SCLC) (ID 1390)
17:55 - 18:00 | Author(s): T. Muley, X. Zhang, S. Holdenrieder, C.M. Korse, X. Zhi, R. Molina, Z. Liu, G. Hartmann, M. Van Den Heuvel, K. Qian, R. Marrades, C. Engel, B. Wehnl, F. Dayyani, F. Herth
- Abstract
Background:
Most cases of small-cell lung cancer (SCLC) are detected at extensive stage, where current guidelines recommend 4-6 cycles of chemotherapy. Although upfront progression to doublet platinum based chemotherapy in SCLC is <20%, current clinical practice often includes treatment monitoring with imaging, mainly with computed tomography, every two cycles. Since the purpose of imaging during treatment is identification of progression (to avoid further exposure to an ineffective but still potentially toxic regimen), we sought to evaluate whether monitoring SCLC patients during treatment with a blood-based biomarker (ProGRP) would correlate with response.
Methods:
Patients with SCLC treated with mainly standard chemotherapy at six centers worldwide (Europe and China) were included. ProGRP levels from serum or plasma were measured with a fully automated ProGRP assay in a prospective fashion prior to treatment start and repeatedly during treatment. Imaging was done and interpreted according to local guidelines at each institution. Percent change of ProGRP from baseline to the time of maximum response (‘best response’) was correlated with imaging findings, and patients were divided into two groups: Responders (= stable disease [SD] or better) vs. Non-Responders (= progression on scan). The ability of ProGRP to discriminate between Responders and Non-Responders was assessed by sensitivity and specificity.
Results:
215 patients with available CT result were included, of whom 145 had received first-line treatment (131 received platinum+VP-16 doublet). Clinical characteristics were as follows: 60.5% male, median age was 62 years, 72.1% were (ex)smokers, 93.5% with Stage IIIB or IV SCLC, and the majority of patients were either Caucasian (59.6%) or Asian (32.6%). Across all lines of treatment, 186/215 (86.5%) had SD or better as best response to treatment. There was a positive trend for higher ProGRP levels with clinical stage at presentation, and in general higher pre-treatment levels in 1[st] line compared to later lines of treatment. A decline in ProGRP levels was strongly correlated with response, whereby higher baseline levels were associated with subsequent higher relative declines of ProGRP during treatment. Using different cut-off levels for ProGRP decline during treatment (-50%, -70%, or -90%), we detected patients with no response to treatment based on ProGRP levels alone, with a sensitivity of 82.8%, 89.7% and 96.6%, and a specificity of 65.6%, 55.4%, and 39.8%, respectively. Specificity was increased by approximately 10% when only patients with baseline ProGRP levels exceeding 100 pg/ml were included.
Conclusion:
To our knowledge, this is the largest SCLC cohort to date with available ProGRP data for therapy monitoring. The data showed that ProGRP levels at baseline were positively correlated with advanced disease stage, and decline in ProGRP levels during treatment was associated with tumor control in SCLC. The ProGRP assay used in this study is currently not cleared or approved for use in the USA.
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MINI27.14 - The Aurora Kinase B Inhibitor AZD1152-HQPA Inhibitor in Small Cell Lung Cancer (SCLC) (ID 2161)
18:00 - 18:05 | Author(s): B. Helfrich, J. Kim, D. Gao, D. Chan, A. Tan, P.A. Bunn, Jr
- Abstract
- Presentation
Background:
Aurora kinase expression has been associated with a poor prognosis in non-small cell lung cancer (NSCLC) and aurora kinase inhibitors have activity in preclinical lung models. Aurora kinases are required for mitosis and cell division. Small cell lung cancer cells have rapid proliferation and higher rates of MYC family amplification, which makes aurora kinase inhibition a natural target.
Methods:
23-SCLC lines with known MYC family amplification and MYC family gene expression were exposed to varying concentrations of the specific aurora kinase B inhibitor AZD1152-HQPA. The percentage growth inhibition compared to control was determined in MTS assays at 120 hours. Cell lines were classified as “sensitive” if the GI50 concentration was < 50 nM and with ≥ 80% growth inhibition at 100 nM. Fisher’s exact test was used to determine the correlation between amplification of MYC family members and sensitivity of the cell lines to growth inhibition by AZD1152-HQPA. A two-group t-test (gene expression as a continuous variable) and an odds ratio estimate (dichotomized gene expression level) were used to determine a correlation between MYC family gene expression and growth inhibition by AZD1152-HQPA. To determine whether growth inhibition correlated with the published MYC-signature gene expression, we used Fisher’s exact test. In vivo growth inhibition by AZD1152 (prodrug) was evaluated on SCLC xenografts in nude mice.
Results:
Nine (39%) of the 23 cell lines were sensitive to AZD1152-HQPA with IC50 values < 50 nM. There was a significant association between sensitivity to growth inhibition by AZD1152-HQPA and cMYC amplification (p = 0.018). The odds of being sensitive is 16 (95% CI, 1.4, 183) times higher for cMYC amplified compared to non-cMYC amplified cell lines. By a two-group t-test, the mean cMYC gene expression of 10.9 (std 4) in sensitive lines compared to 7.2 (std 3.3) in resistant lines was also significant (p = 0.026). Cell lines were separated into two groups based on cMYC gene expression > 12.9 vs < 12.9. The odds of being sensitive is 11 (95% CI, 1.2, 103) time higher for cell lines with cMYC gene expression > 12.9 compared to cell lines with cMYC gene expression < 12.9. Sensitive cell lines were enriched in a published MYC-signature of gene expression (p = 0.042). AZD1152 (prodrug) caused significant growth delay in vivo in two of these lines. The doses of AZD1152-HQPA used in this study are within the range reported to be clinically achievable.
Conclusion:
Aurora kinase inhibitors have promise in SCLC therapy. Questions that currently need answering in translating aurora kinase inhibitors in the clinical setting are: (1) the dosing schedule to avoid myelosupression, (2) should aurora kinase inhibitors be used in maintenance therapy and (3) should the aurora kinase inhibitors be evaluating in combination with chemotherapy.
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MINI27.15 - Discussant for MINI27.11, MINI27.12, MINI27.13, MINI27.14 (ID 3381)
18:05 - 18:15 | Author(s): M. Peifer
- Abstract
- Presentation
Abstract not provided
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PRC 04 - Press Conference 4 (ID 199)
- Event: WCLC 2015
- Type: Press Conference
- Track: Other
- Presentations: 6
- Moderators:P.A. Bunn, Jr
- Coordinates: 9/09/2015, 09:45 - 10:45, 108+110+112
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Abstract – A Randomized, Phase III Study Comparing Carboplatin/Paclitaxel or Carboplatin/Paclitaxel/Bevacizumab with or without Concurrent Cetuximab in Patients with Advanced Non-Small Cell Lung Cancer (NSCLC): SWOG S0819 - Dr. Roy Herbst, Ensign Professor of Medicine, Professor of Pharmacology, Chief of Medical Oncology, Director, Thoracic Oncology Research Program, Associate Director for Translational Research, Yale Comprehensive Cancer Center, Yale School of Medicine, New Haven, Connecticut (ID 3638)
10:17 - 10:25 | Author(s): R. Herbst
- Abstract
- Presentation
Abstract:
Background: Cetuximab, a chimeric monoclonal antibody targeting the epidermal growth factor receptor (EGFR), moderately increases survival in patients with advanced NSCLC. Our prior work suggested that EGFR gene copy number measured by fluorescent in-suit hybridization (FISH) could identify those patients most likely to benefit.Methods: Patients eligible for this NCTN multicenter open-label, phase III trial had histologically or cytological confirmed Stage IV NSCLC that was newly diagnosed or recurrent after previous surgery or radiation. Patients with controlled brain metastases were allowed entry. All patients were required to have tumor tissue available for EGFR FISH testing. Randomization was stratified by appropriateness for bevacizumab treatment, smoking status and M-substage. The co-primary objectives were progression-free survival (PFS) in EGFR-FISH positive (FISH+) patients and overall survival (OS) in the overall study population (OSP). Secondary objectives were OS in FISH-positive patients and clinical outcomes (PFS and OS) comparison among bevacizumab-appropriate (BA) and inappropriate (BI) patients.Results: The final accrual was 1,333 total and 1,313 eligible patients (control arm: 657 total, 275/382 BA/BI; cetuximab-containing arm: 656 total, 279/377 BA/BI). EGFR FISH status was determined on 976 patients, with 400 (41%) FISH+. Squamous carcinoma (SCCA) represented 24-25% of patients. PFS and OS were not significantly different in the OSP (HR (95%CI) =0.98 (0.87-1.09) and 0.94(0.84-1.06), respectively). There is some indication of benefit in PFS and OS (HR 0.91 (0.74-1.12) and 0.83 (0.67-1.04), respectively) in FISH+ patients, albeit not statistically significant. However, among FISH+ BI and SCCA patients, effect estimates for OS were 0.75 (0.57-1.00) and 0.56 (0.37-0.84), respectively. Cetuximab was generally well tolerated with a spectrum of adverse events consistent with prior reportsConclusion: The addition of cetuximab had minimal effect in unselected advanced NSCLC patients. In FISH+ patients there is a suggestion of benefit, predominantly in SCCA and BI. These data support using a new marker for determining who should receive an EGFR antibody inhibitor with chemotherapy.Support: NIH/NCI/NCTN grants SWOG: CA180888, CA180819; ECOG/ACRIN: CA180820; Alliance: CA180821; and in part by Bristol-Myers Squibb Co.N PFS OS Analysis Group OSP FISH+ OSP FISH+ OSP FISH+ All Patients Cetuximab-Containing Arm, Median, 95% CI 656 199 4.6(4.2-5.2) 5.4(4.5-5.7) 10.9(9.6-12.0) 13.4(11.7-14.8) Control Arm, Median, 95% CI 657 201 4.5(4.2-4.9) 4.8(3.9-5.5) 9.4(8.7-10.3) 9.8(8.7-12.1) Hazard Ratio, 95% CI 0.98(0.87-1.09) 0.91(0.74-1.12) 0.94(0.84-1.06) 0.83(0.67-1.04) 2-sided p-value 0.68 0.37 0.34 0.10 BA Cetuximab-Containing Arm, Median, 95% CI 279 86 5.6(5.3-6.1) 6.2(5.7-7.4) 12.7(10.9-13.4) 15.5(13.4-18.4) Control Arm, Median, 95% CI 275 80 5.9(5.5-6.6) 6.7(5.7-8.0) 11.6(10.5-13.8) 13.2(11.2-19.1) Hazard Ratio, 95% CI 1.05(0.88-1.25) 1.07(0.77-1.47) 1.04(0.86-1.25) 0.97(0.69-1.38) 2-sided p-value 0.57 0.70 0.70 0.88 BI Cetuximab-Containing Arm, Median, 95% CI 377 113 4.1(3.6-4.4) 4.5(3.8-5.2) 9.2(8.2-10.9) 11.2(8.6-12.9) Control Arm, Median, 95% CI 382 121 3.8(3.1-4.2) 3.7(2.8-4.6) 8.2(7.3-8.7) 8.7(5.9-9.7) Hazard Ratio, 95% CI 0.93(0.80-1.07) 0.82(0.63-1.07) 0.88(0.76-1.03) 0.75(0.57-1.00) 2-sided p-value 0.31 0.14 0.12 0.05 SCCA Cetuximab-Containing Arm, Median, 95% CI 160 55 4.2(3.7-4.6) 4.5(3.8-5.2) 9.6(8.2-11.5) 11.8(8.6-13.5) Control Arm, Median, 95% CI 161 56 3.7(2.8-4.3) 2.8(2.6-4.1) 8.0(7.1-8.9) 6.4(4.2-8.7) Hazard Ratio, 95% CI 0.88(0.70-1.11) 0.68(0.46-1.01) 0.85(0.67-1.08) 0.56(0.37-0.84) 2-sided p-value 0.29 0.06 0.18 0.006
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Abstract – EGFR IHC and FISH Correlative Analyses (SQUIRE Trial): Necitumumab + Gemcitabine-Cisplatin vs Gemcitabine-Cisplatin in 1st-Line Squamous NSCLC - Dr. Fred R. Hirsch, IASLC CEO, Congress President, Professor of Medicine and Pathology, University of Colorado (ID 3639)
10:25 - 10:33 | Author(s): F.R. Hirsch
- Abstract
- Presentation
Abstract not provided
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Abstract – Multiregion Whole Exome and Transcriptome Sequencing Defines the Genomic Spectrum of EGFR+ NSCLC and Reveals Novel Mechanisms of TKI Resistance - Dr. Daniel Shao Weng Tan, Consultant, Medical Oncology, National Cancer Centre, Singapore (ID 3637)
10:09 - 10:17 | Author(s): D.S. Tan
- Abstract
- Presentation
Abstract not provided
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Abstract – Randomized Phase III Trial of Adjuvant Chemotherapy with or without Bevacizumab in Resected Non-Small Cell Lung Cancer (NSCLC): Results of E1505 - Dr. Heather Wakelee, Associate Professor of Medicine (Oncology) at the Stanford University Medical Center, Stanford, California (ID 3635)
09:53 - 10:01 | Author(s): H.A. Wakelee
- Abstract
- Presentation
Abstract not provided
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Abstract – Stopping Smoking Reduces Mortality in Low-Dose Computed Tomography (LDCT) Screening Volunteers - Dr. Ugo Pastorino, Director Thoracic Surgery, IRCCS Istituto Nazionale dei Tumori Foundation, Milan, Italy (ID 3636)
10:01 - 10:09 | Author(s): U. Pastorino
- Abstract
- Presentation
Abstract not provided
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Daily Theme: Science Drives Lung Cancer Advances - Dr. David R. Gandara, Professor of Medicine, Division of Hematology/Oncology, Director, Thoracic Oncology Program, Senior Advisor to the Director, UC Davis Comprehensive Cancer Center, Sacramento, California (ID 3634)
09:45 - 09:53 | Author(s): D.R. Gandara
- Abstract
- Presentation
Abstract not provided
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ED 11 - Extending Personalized Treatment Beyond EGFR (ID 11)
- Event: WCLC 2015
- Type: Education Session
- Track: Community Practice
- Presentations: 1
- Moderators:R. Perez-Soler, R. Camidge
- Coordinates: 9/09/2015, 14:15 - 15:45, 401-404
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ED11.01 - What Testing Is Needed for Clinical Treatment? (ID 1814)
14:20 - 14:40 | Author(s): P.A. Bunn, Jr
- Abstract
- Presentation
Abstract:
The current IASLC/CAP/AMP guidelines indicate that testing for EGFR mutations and ALK fusions is recommended before instituting therapy in patients with advanced lung cancers with adenocarcinoma features. The guidelines indicate that the guidelines would be updated as other therapies become available for specific drivers. Since these guidelines were published, considerable information has become available on molecular changes that cause resistance to the first generation EGFR and ALK tyrosine kinase inhibitors (TKIs) and new 2nd and 3rd generation drugs to treat molecular resistance are in the clinic are likely to be approved in 2015. New drugs are also in development for other molecular drivers that have been reported in lung cancer including fusions that activate ROS1, RET, NTRK, and FGFR; mutations that activate KRAS, BRAF,HER2, and FGFR; and amplifications that activate MET, FGFR and HER2. It is also likely that some of these agents will approved in the near future. These developments bring into focus the most appropriate methods to test for molecular drivers in lung cancer. The techniques include direct sequencing, PCR testing for mutation hot spots, Next generation whole exome sequencing for mutations, translocations and amplifications, immunohistochemical testing for activated proteins or specific mutations, FISH testing for specific translocations, fusions, and amplifications. The sequential testing of one gene at a time is very inefficient especially with respect to the time it takes to complete testing, with the total cost and with respect to the amount of tissue necessary to complete the testing. Thus, multiplex testing is becoming far more common. However, in the US, the FDA’s companion diagnostic tests are one a time for specific tests for specific drugs. This one off policy is threatening the ability to perform one test for multiple analytes simultaneously. Hopefully, these issues can be resolved in the near future. What are the molecular changes that appear to be drivers for which there exist therapeutic TKIs? We can first discuss the development of resistance to 1st generation EGFR and ALK TKIs. About half of all patients who progress on a 1st generation EGFR TKI will have a secondary gatekeeper mutation, T790M that limits binding of 1st generation EGFR TKIs to the EGF receptor. 3rd generation EGFR TKIs such as AZD9291 and CO1686 (rocelitinib) have been shown to produce objective clinical responses in about 60% of patients with T790M who progress on a first generation EGFR TKI. These responses are associated with a median PFS of about 10 months. These drugs bind irreversibly to the T790M receptor and to activating mutations such as del19 and L858R but do not bind to the wildtype receptor. Therefore they have far less skin rash and diarrhea compared to the 1st generation inhibitors. Rocelitinb may cause hyperglycemia in up to a third of patients that is believed to be caused by a metabolite that inhibits IGFR. The use of oral anti-hyperglycemic agents such as metformin may be required for glucose control in these patients. Rocelitinib may also cause prolongation of the QTc on EKGs and thus monitoring is required. This side effect is rare but requires monitoring. AZD9291 may be associated with slightly more skin rash and diarrhea and uncommon reports of pneumonitis have been reported. The mechanism of resistance to these agents is under investigation but most often appears to be activation of alternative signaling pathways or phenotypic switching to a mesenchymal state. There are multiple tests available to detect the T790M mutation and many studies are evaluating its presence in circulating free DNA. Such analyses seem to be quite specific but less sensitive to analyses of tumor tissue. Testing for mutations in circulating free DNA is quite appealing because it does not require another tumor biopsy. Crizotinib which was the first agent approved for ALK positive cases, is also a potent ROS1 and MET inhibitor. ROS1 may be activated by fusion to other genes on the same chromosome and detected by FISH or Next generation sequencing. Crizotinib has been reported to produce objective response is about 60% of cases with ROS1 fusions with median progression free survival of about 16 months. Crizotinib has also been reported to produce objective responses in about 2/3 of patients with high MET amplification although the number of patients studied is very low. Additional studies are ongoing. Because MET amplification is frequent in patients who progress on 1st generation EGFR TKIs that do not have T790M, MET inhibitors are also being studied on this setting. Both ceritinb and alectinib have been approved for patients with ALK fusions who have progressed on crizotinib. It is clear that the two drugs have different activity among various resistance mutations for ALK. Thus, rebiopsy of tumor or testing of circulation free DNA may become standard in patients progressing on ALK TKIs as it is in patients progressing on EGFR TKIs. BRAF mutations occur in about 2% of advanced NSCLC patients and the V600E mutation is the most common. BRAF TKIs such as vemurafinib have been reported to produce frequent responses in NSCLC patients with V600E BRAF mutations. In melanoma the combination of BRAF inhibitors with MEK inhibitors has been more effective than BRAF inhibitors alone and this combination is being studied in NSCLC patient with BRAF mutations. HER 2 may be activated by either mutation or amplifications and the response to various HER inhibitors may vary by the method of activation. About 2% of patients have activation by amplification and about 2% by mutation (usually exon 20 insertions). The irreversible pan HER TKIs such as neratinib, afatinib and dacomitinib have not produced high response rates in these patients. However a phase 1 trial of the combination of niratinib with temsirolimus produced higher responses in both breast and lung cancers and a phase 2 study of the combination in lung cancers with HER2 mutations/amplifications is in progress. Other HER2 inhibitors have been reported to produce occasional responses but we await formal study of such agents. RET and NTRK fusions have been reported I about 1% of patients and there are sporadic reports of responses to specific TKIS. Formal studies are in progress but may not be reported for some time due to the rarity of the abnormalities and the fact that multiple TKIs are available. FGFR may be activated by amplification, fusion and mutations and there are both quite specific FGFR TKIs and multi-TKIs that have been studied in small numbers of patients. It is fair to say that response rates to specific FGFR TKIs are low in patients with squamous cell carcinoma patients with FGFR amplification. Additional studies with other biomarkers and other agents are ongoing. The most frequent oncogenic driver in lung cancer is KRAS. No specific TKI has been developed. Downstream inhibitors such as MEK inhibitors and FAK inhibitors have the most study to date. Response rates as single agents are relatively low and combination studies are in progress. In summary, the increased numbers of TKIs specific for various molecular drivers in lung cancer is becoming far more important not only a diagnosis but also at the time of progression. Future studies will focus on multiplex testing and testing of circulating free DNA.
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MINI 04 - Clinical Care of Lung Cancer (ID 102)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:L. Gaspar, V. Westeel
- Coordinates: 9/07/2015, 16:45 - 18:15, Mile High Ballroom 2c-3c
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MINI04.08 - Malignant Pleural Effusions Are Predictive of Peritoneal Carcinomatosis in Patients with Advanced EGFR Positive Non-Small Cell Lung Cancer (ID 3191)
17:25 - 17:30 | Author(s): P.A. Bunn, Jr
- Abstract
- Presentation
Background:
Lung cancer is the most frequent cause of cancer death and metastatic disease at the time of initial diagnosis is common. Peritoneal carcinomatosis (PC) from lung cancer is a rare clinical event with a reported incidence of 1.2% (Satoh et al. 2001). However, there are limited data on what factors predict peritoneal progression in lung cancer. Over the last decade, molecular analysis of NSCLC has provided more detailed classification of patterns of metastatic spread. It has also been shown that oncogene-addicted subsets of NSCLC have different patterns of metastatic spread (Doebele et al. 2012). We investigated whether certain baseline patterns of metastatic spread in patients with advanced EGFR mutation positive (EGFR+) NSCLC can predict subsequent PC.
Methods:
We identified 156 patients with EGFR+ (Exon 19 or L858R) mutations from 2009 - 2014, of which 139 had metastatic NSCLC. 11 patients developed PC. This was defined as the presence of biopsy-proven adenocarcinoma from peritoneal fluid or radiographic patterns consistent with omental metastases. We identified areas of metastatic disease in predefined sites (brain, liver, lung, adrenal, soft tissue and pleura) at the time of diagnosis or metastatic recurrence. We noted if patients developed T790M, a resistance mutation to targeted therapy, in EGFR+ patients. A Fisher-Exact test was used to determine statistical significance between metastatic site and subsequent PC.
Results:Table 1 - Sites of metastasis and presence of T790M mutation in patients with PC and without PC
The presence of a pleural effusion was universal in all 11 EGFR+ patients who subsequently developed PC and this finding was statistically significant (P = 0.0001). 9 out of 11 patients with PC were identified to have a T790M mutation, a finding that was statistically significant (P = 0.0001). Except one patient, all EGFR+ patients developed PC following targeted tyrosine kinase therapy.Metastatic site / mutation PC No PC P value Lung 9.1% 38.6% P = 0.06 Liver 18.2% 15.8% P = 0.689 Bone 36.4% 46.8% P = 0.549 Brain 0% 23.7% P = 0.3570 Adrenal 0% 6.4% P = 0.123 Soft tissue 9.1% 2.2% P = 0.265 Pleural effusion 100% 26.6% P = 0.0001 T790M mutation 81.1% 34.5% P = 0.0001
Conclusion:
The presence of a malignant effusion is highly predictive of developing PC in patients with EGFR+ NSCLC. Although the underlying mechanism of PC is not entirely clear, it may be related to serosal communication with subsequent micrometastatic seeding of the peritoneal cavity. The T790M mutation, the most common acquired resistance mechanism to EGFR kinase inhibitors, was significantly more prevalent in the group that developed PC, although it remains unclear whether this mutation has any causative effect on spread to the peritoneum.
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MINI 09 - Drug Resistance (ID 107)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 2
- Moderators:L. Villaruz, J. Minna
- Coordinates: 9/07/2015, 16:45 - 18:15, 205+207
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MINI09.01 - Inhibiting Tankyrase Prevents Epithelial-To-Mesenchymal Transition and Synergizes with EGFR-Inhibition in Wnt-Dependent NSCLC Lines (ID 2850)
16:45 - 16:50 | Author(s): P.A. Bunn, Jr
- Abstract
Background:
Despite their promise, therapies targeting driver receptor tyrosine kinases (RTKs) rarely produce complete responses and have shown modest clinical benefit in NSCLC. This suggests the presence of escape mechanisms that allow cells to survive and proliferate despite inhibition of an oncogenic driver.
Methods:
Using a genome-wide shRNA screen, we identified that the canonical Wnt/β-catenin pathway contributes to the survival of NSCLC cells during inhibition of the epidermal growth factor receptor (EGFR). In order to evaluate the effects of inhibiting the Wnt pathway on EGFR-inhibited cells, we categorized NSCLC cell lines as “Wnt-responsive” or “Wnt-non-responsive” based on their ability to upregulate β-catenin-dependent targets in response to treatment with exogenous Wnt3a. Using both shRNA knockdown and a novel tankyrase inhibitor, AZ1366, we evaluated the ability of tankyrase inhibition to synergize with EGFR-inhibition in multiple Wnt-responsive and Wnt-non-responsive cell lines. We then evaluated the effects of the combination of gefitinib and AZ1366 on the survival and tumor progression in an orthotopic mouse model. In order to comprehensively query transcriptional changes brought about by treatment, we performed RNA-seq on cells treated with gefitinib, AZ1366, or the combination of the two drugs.
Results:
We have demonstrated that inhibition of tankyrase, a key player in the canonical Wnt pathway, significantly increases the induction of senescense and/or apoptosis mediated by EGFR-inhibitors in cell lines with a Wnt-responsive phenotype, and that the ability of the tankyrase inhibitor to synergistically eliminate NSCLC cells is dependent on its actions within the canonical Wnt pathway. In Wnt-non-responsive cell lines, tankyrase inhibition did not synergize with inhibition of EGFR. We have further demonstrated that Wnt-responsive cell lines show evidence of EMT in response to Wnt ligand stimulation, and that this can be prevented with tankyrase-inhibitor treatment. Additionally, we have shown that mice orthotopically implanted with Wnt-responsive cell lines and treated with a combination of a tankyrase inhibitor and an EGFR inhibitor have a substantially reduced tumor burden and a significant improvement in survival when compared to treatment with an EGFR inhibitor alone. When Wnt-non-responsive cell lines were used, we noted no improvement in survival or reduction in tumor burden. RNA-seq analysis revealed that while most transcriptional changes present in the combination were driven by gefitinib, AZ1366 had the effect of significantly amplifying many of the changes thought to be instrumental in resistance to EGFR inhibition including increased expression of TP53 and apoptosis signaling machinery, increased expression of NF-kB signaling components, and a strong decrease in cell cycle drivers. Furthermore, treatment with AZ1366 alone resulted in decreased expression of Axl and its ligand, Gas6, a known mechanism of resistance to EGFR inhibition.
Conclusion:
Taken together, these results indicate that tankyrase inhibition impinges on multiple mechanisms of escape from EGFR-inhibition, and that its ability to synergize with EGFR-inhibition is dependent on its actions within the canonical Wnt pathway. As the goal of these studies is the development of combination therapies with EGFR inhibition, this suggest tankyrase as a promising target in the subset of NSCLC with known dependencies on signaling through the canonical Wnt pathway.
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MINI09.06 - Oncogenic Drivers including RET and ROS1 plus PTEN Loss and MET by IHC in Patients with Lung Adenocarcinomas: Lung Cancer Mutation Consortium 2.0 (ID 2114)
17:15 - 17:20 | Author(s): P.A. Bunn, Jr
- Abstract
- Presentation
Background:
The Lung Cancer Mutation Consortium (LCMC) 1.0 demonstrated multiplexed genomic platforms can assay 10 oncogenic drivers in tumor specimens from patients with lung adenocarcinomas. 28% of the patients with oncogenic drivers could be effectively targeted. The survival of these 275 patients treated with targeted agents was longer than the patients who were not treated with a targeted agent (Kris and Johnson JAMA 2014). The efficiency of Next-Generation Sequencing enables more comprehensive testing of additional aberrations with less tumor tissue. LCMC 2.0 was initiated to test tumor specimens for 12 oncogenic drivers and to provide the results to clinicians for treatment decisions and research purposes.
Methods:
The 16 site LCMC 2.0 is testing tumors from 1000 patients with lung adenocarcinomas in CLIA laboratories for mutations in KRAS, EGFR, HER2, BRAF, PIK3CA, AKT1, and NRAS, MET DNA amplification, and rearrangements in ALK as done in LCMC 1.0. The new genes that were added because of emerging information about potential therapeutic targets include MAP2K1 mutations, RET and ROS1 rearrangements, PTEN (MAb 138G4) loss and MET (MAb SP44) overexpression by immunohistochemistry (IHC). All patients were diagnosed with stage IIIB/IV lung adenocarcinoma after May 2012, had a performance status 0-2, and available tumor tissue.
Results:
Of 1073 patients registered, data is now reported for 759. The median age of the patients is 65 (23-90). The population includes 369 (55%) women; 164 (24%) never smokers, 399 (59%) former smokers, and 73 (11%) current smokers; 26 (4%) Asians, 58 (9%) African American, 548 (81%) Caucasian, and 43 (6%) of other races. As of April 2015 information on genomic and immunohistochemical changes for 675 eligible patients were recorded in our database. Alterations in oncogenic drivers were found in 45% of samples as follows: 159 KRAS (24%), 88 EGFR (13%), 25 ALK (4%), 19 BRAF (3%), 17 PIK3CA (3%), 9 HER2 (1%), 4 NRAS (1%) 0 AKT1, 28 had ≥ 2 findings (4%) and 25 MET DNA amplification (4%). The new genes studied in LCMC 2.0 revealed 1 MAP2K1 mutation (<1%), 19 RET (3%) and 9 ROS (1%) rearrangements, 94 had PTEN loss (14%), and 362 with MET overexpression (54%). As expected, PIK3CA mutations and PTEN loss by IHC were mutually exclusive in 109 of 111 (98%) patients’ tumors. Seventeen of the 23 (74%) with MET DNA amplification studied thus far with IHC had MET overexpression. Next-Generation platforms were used at 13 of 16 LCMC 2.0 sites.
Conclusion:
Next-Generation Sequencing is rapidly becoming routine practice at LCMC 2.0 centers with use going from 0 to 81% of sites since 2012. LCMC 2.0 identified additional targets (RET and ROS1 rearrangements and PTEN loss). PIK3CA and PTEN were largely mutually exclusive and an actionable oncogenic driver has been identified in the 45% of initial lung adenocarcinoma specimens. Supported by Free to Breathe
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MINI 13 - Genetic Alterations and Testing (ID 120)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:Y. Koh, R.K. Thomas
- Coordinates: 9/08/2015, 10:45 - 12:15, 205+207
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MINI13.03 - Characterization of MET Gene and MET Protein Expression in Lung Cancer (ID 2155)
10:55 - 11:00 | Author(s): P.A. Bunn, Jr
- Abstract
- Presentation
Background:
Activation of the MET signaling pathway can propel the growth of cancer cells in non-small cell lung cancer (NSCLC). Increased MET gene by amplification and/or polysomy can cause MET protein overexpression; less common causes include mutations, translocations, and alternative RNA splicing. Clinical trials using MET as a biomarker for selection of lung cancer patients who might most benefit from targeted therapy have experienced variable outcomes. We aimed to characterize the relationship between MET protein overexpression and MET amplification or mean copy number alterations in patients with NSCLC.
Methods:
The Lung Cancer Mutation Consortium (LCMC) is performing an ongoing study of biomarkers with patients with NSCLC from 16 cancer center sites across the United States. For this analysis, 403 cases had complete data for MET protein expression by immunohistochemistry (IHC, monoclonal antibody SP44, Ventana) and MET gene amplification by fluorescence in-situ hybridization (FISH, MET/CEP7 ratio). Pathologists evaluated MET expression using the H-score, a semi-quantitative assessment of the percentage of tumor cells with no, faint, moderate, and/or strong staining, ranging from 0-300. Spearman's correlation was used to analyze the correlation between MET protein expression (H-scores) and FISH results (MET/CEP7 ratio (N=403) and MET copy number (N=341). Protein overexpression using 5 different cut-offs was compared with amplification defined as MET/CEP7 ≥ 2.2 and high mean copy number defined as ≥ 5 MET gene copies per cell using the Fisher’s exact test. Cox Proportional Hazards models were built to examine the associations of these different definitions of positivity with prognosis, adjusting for stage of disease.
Results:
MET protein expression was significantly correlated with MET copy numbers (r=0.17, p=0.0025), but not MET/CEP7 ratio (r=-0.013, p=0.80). No significant association was observed between protein overexpression using a commonly used definition for MET positivity (“at least moderate staining in ≥ 50% tumor cells”) and MET amplification (p=0.47) or high mean copy number (p=0.09). A definition for MET protein overexpression as “≥ 30% tumor cells with strong staining” was significantly associated with both MET amplification (p=0.03) and high mean copy number (p=0.007), but a definition of “≥ 10% tumor cells with strong staining” was not significantly associated with either. Definitions of protein overexpression based on high H-scores (≥200 or ≥250) were associated with high MET mean copy numbers (p=0.03 and 0.0008, respectively), but not amplification (p=0.46 and 0.12, respectively). All 5 definitions of MET protein overexpression demonstrated a significant association with worse prognosis by survival analyses (p-values ranged from 0.001 to 0.03). High MET copy number (p=0.045) was associated with worse prognosis, but MET amplification was not (p=0.07).
Conclusion:
Evaluation of NSCLC specimens from LCMC sites confirms that MET protein expression is correlated with high MET copy number and protein overexpression is associated with worse prognosis. Definitions of MET protein overexpression as “an H-score ≥250” and “≥30% tumor cells with strong staining” were significantly associated with high mean MET copy number. It may be worth reevaluating the performance of MET as a biomarker by different definitions of positivity to predict response to MET-targeted therapies.
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MINI 27 - Biology and Other Issues in SCLC (ID 152)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:P.A. Bunn, Jr, J. Sage
- Coordinates: 9/09/2015, 16:45 - 18:15, 605+607
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MINI27.14 - The Aurora Kinase B Inhibitor AZD1152-HQPA Inhibitor in Small Cell Lung Cancer (SCLC) (ID 2161)
18:00 - 18:05 | Author(s): P.A. Bunn, Jr
- Abstract
- Presentation
Background:
Aurora kinase expression has been associated with a poor prognosis in non-small cell lung cancer (NSCLC) and aurora kinase inhibitors have activity in preclinical lung models. Aurora kinases are required for mitosis and cell division. Small cell lung cancer cells have rapid proliferation and higher rates of MYC family amplification, which makes aurora kinase inhibition a natural target.
Methods:
23-SCLC lines with known MYC family amplification and MYC family gene expression were exposed to varying concentrations of the specific aurora kinase B inhibitor AZD1152-HQPA. The percentage growth inhibition compared to control was determined in MTS assays at 120 hours. Cell lines were classified as “sensitive” if the GI50 concentration was < 50 nM and with ≥ 80% growth inhibition at 100 nM. Fisher’s exact test was used to determine the correlation between amplification of MYC family members and sensitivity of the cell lines to growth inhibition by AZD1152-HQPA. A two-group t-test (gene expression as a continuous variable) and an odds ratio estimate (dichotomized gene expression level) were used to determine a correlation between MYC family gene expression and growth inhibition by AZD1152-HQPA. To determine whether growth inhibition correlated with the published MYC-signature gene expression, we used Fisher’s exact test. In vivo growth inhibition by AZD1152 (prodrug) was evaluated on SCLC xenografts in nude mice.
Results:
Nine (39%) of the 23 cell lines were sensitive to AZD1152-HQPA with IC50 values < 50 nM. There was a significant association between sensitivity to growth inhibition by AZD1152-HQPA and cMYC amplification (p = 0.018). The odds of being sensitive is 16 (95% CI, 1.4, 183) times higher for cMYC amplified compared to non-cMYC amplified cell lines. By a two-group t-test, the mean cMYC gene expression of 10.9 (std 4) in sensitive lines compared to 7.2 (std 3.3) in resistant lines was also significant (p = 0.026). Cell lines were separated into two groups based on cMYC gene expression > 12.9 vs < 12.9. The odds of being sensitive is 11 (95% CI, 1.2, 103) time higher for cell lines with cMYC gene expression > 12.9 compared to cell lines with cMYC gene expression < 12.9. Sensitive cell lines were enriched in a published MYC-signature of gene expression (p = 0.042). AZD1152 (prodrug) caused significant growth delay in vivo in two of these lines. The doses of AZD1152-HQPA used in this study are within the range reported to be clinically achievable.
Conclusion:
Aurora kinase inhibitors have promise in SCLC therapy. Questions that currently need answering in translating aurora kinase inhibitors in the clinical setting are: (1) the dosing schedule to avoid myelosupression, (2) should aurora kinase inhibitors be used in maintenance therapy and (3) should the aurora kinase inhibitors be evaluating in combination with chemotherapy.
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ORAL 23 - Prevention and Cancer Risk (ID 121)
- Event: WCLC 2015
- Type: Oral Session
- Track: Prevention and Tobacco Control
- Presentations: 2
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ORAL23.02 - Pioglitazone for the Chemoprevention of Lung Cancer (ID 2419)
10:56 - 11:07 | Author(s): P.A. Bunn, Jr
- Abstract
- Presentation
Background:
Prior clinical studies have shown that the oral prostacyclin agonist iloprost improves bronchial dysplasia in former smokers. Prostacyclin is a PPAR gamma agonist, and epidemiologic and pre-clinical studies suggest PPAR gamma agonists like pioglitazone may chemoprevent lung cancer. Based on these promising results, a double-blind, placebo controlled, phase II trial of pioglitazone in subjects at increased risk for lung cancer was sponsored by the Department of Veterans Affairs.
Methods:
Subjects were selected for the trial if they met one the following criteria: current or former smoker (> 10 pack years); biopsy proven endobronchial dysplasia; airflow obstruction (FEV1/FVC < 0.70); or at least mild sputum cytologic atypia. Fluorescent bronchoscopy was performed with biopsy of 6 standard endobronchial sites and all other abnormally appearing areas. Subjects also had pulmonary function testings and quantitative high resolution CT scans at the start and completion of the trial. Subjects were then randomized to oral pioglitazone or placebo for 6 months and then a second fluorescent bronchoscopy with repeat biopsy of all the central airway areas sampled on the first bronchoscopy. The endobronchial biopsies were scored on a 1-8 scale based on WHO criteria. The primary endpoint for the study is change in maximum (worst) endobronchial histology.
Results:
A total of 90 subjects (46 pioglitazone, 44 placebo) have been enrolled in the trial, with 76 completing both bronchoscopies. Subjects are well matched in terms of age, gender, tobacco exposure, and sputum cytology. No significant differences in lung function were observed between the treatment groups. While the investigators remain blinded in regards to treatment group, aggregate data is available. Overall, mild dysplasia or worse was seen in 26% of the initial biopsies. Similar to prior studies, current smokers exhibited more dysplasia at baseline compared to former smokers (32.4% vs. 16.6%) and also had more angiogenic squamous dysplasia (11.7% vs. 3.2%). Our primary endpoint is change in maximum histology, and histologic scores from matched biopsies in all participants showed a change of at least 1 grade in 50.2% (25.9% improved, 24.3% progressed). More histologic changes were observed in current smokers (59.2%) than former smokers (41.7%). Summary data for the non-normal biopsy pairs (ie those with a histologic score of at least 2 on baseline biopsy) showed that the majority of pairs (73.7%) changed by at least one grade. Current smokers exhibited more progression (29.3%) compared to former smokers (14.6%).
Conclusion:
The pioglitazone lung cancer chemoprevention trial is currently in progress. The treatment has been well tolerated and histologic changes were observed in many of the subjects.
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ORAL23.03 - Role of Inflammatory Infiltrates in Promoting Persistence or Regression of Bronchial Dysplasia (ID 3026)
11:07 - 11:18 | Author(s): P.A. Bunn, Jr
- Abstract
- Presentation
Background:
Inflammatory infiltrates show differing capacities to eliminate malignant cells. This capacity is related to the polarization of key inflammatory cells in tumor infiltrates. A pathway analysis of genes that are differentially expressed between persistent and regressive bronchial dysplasia (BD) identified 13 pathways associated with persistence of which 8 were related to inflammation. We have hypothesized that differences in inflammatory infiltrate polarization may contribute to lung carcinogenesis and have employed gene expression and in situ analyses to characterize differences in inflammatory infiltrates related to persistence and regression of pre-malignant BD.
Methods:
Normalized gene expression levels (Affymetrix Hu 1.0) of selected genes related to inflammatory cell polarization features were analyzed to find differences associated with follow-up histology for BD. Validational analyses of these relationships were undertaken in studies of baseline biopsies selected to represent persistent (n=43) and regressive BD (n=39). These biopsies were analyzed by quantitative immunohistochemistry and dual immunofluorescence studies to characterize the overall proportion of subsets of T-lymphocytes and macrophages in each of the groups. Image analysis tools (Aperio) were used to characterize the density of inflammatory cell subsets in the stromal and epithelial compartments of biopsy tissue within defined areas.
Results:
Analysis of expression levels for a subset of inflammatory cell related genes assessed in a global gene expression analysis indicated significantly higher levels of expression of macrophage M1 markers HLA-DRA (p=0.01) and inducible nitric oxide synthetase (iNOS; p=0.02) and T-helper lymphocyte marker CD4 (p=0.04) in regressive BD compared to persistent BD. There was also a trend toward higher expression of cytotoxic T-lymphocyte marker CD8 in regressive BD (p=0.25). Expression of B-lymphocyte and neutrophil markers were not different between regressive and persistent BD. CD68 immunohistochemical stains (IHC) demonstrated a trend toward an increase in macrophages per area of combined dysplastic epithelium and underlying stroma with a mean increase in IHC positivity of 1.75-fold in regressive versus persistent BD (p=0.08). CD4 and CD8 IHC showed 1.36- and 1.19-fold increases, respectively, in regressive BD but these changes were not statistically significant (p=0.36 and p=0.43 respectively). Dual immunofluorescence was undertaken to determine if polarization specific subsets of macrophages correlated with regression or persistence of BD. Analysis of a preliminary subset of regressive (n=3) and persistent (n=3) BD demonstrates a wide range of M1 to M2 ratios (range = 0.84 – 4.82 for ratio of HLA-DRA-CD68 dual positive M1 to CD206-CD68 dual positive M2 macrophages per high power field, 400X). Additional analyses of macrophages are ongoing to determine if the polarization status is related to regression or persistence of BD, and analysis of markers of T-helper lymphocyte subsets are planned.
Conclusion:
Gene expression analyses indicate that increased expression of markers of M1 macrophages and T-helper lymphocytes are associated with regression, and in situ analyses suggest that differences in the amount of inflammatory cell subsets may be related to outcome in BD. These studies could have implications for predicting the behavior of premalignant disease and manipulating inflammatory activity in preventing progression of BD to invasive lung cancer.
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ORAL 37 - Novel Targets (ID 146)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.S. Ramalingam, E. Thunnissen
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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ORAL37.07 - Lung Cancer Mutation Consortium Pathologist Panel Evaluation of MET Protein (ID 2129)
17:50 - 18:01 | Author(s): P.A. Bunn, Jr
- Abstract
- Presentation
Background:
MET is a receptor tyrosine kinase with frequently activated signaling in lung cancers. Multiple studies indicate that MET overexpression correlates with poor clinical prognosis. Tumors with MET amplification and overexpression may respond better to MET inhibitors than tumors with low expression. The prevalence of MET overexpression in lung cancer cohorts has varied from 20%-80%, as has the proportion of patient’s testing positive for prospective clinical trials with entry based on MET overexpression. The Lung Cancer Mutation Consortium (LCMC) Pathologist Panel endeavored to standardize evaluation of MET protein expression with “Round Robin” conferences.
Methods:
508 FFPE non-small cell lung cancer specimens were stained by immunohistochemistry for MET protein expression (SP44 antibody, Ventana). Seven pathologists from LCMC sites with specialized training in MET scoring evaluated 78 Aperio-scanned images of MET-stained slides in two successive rounds of 39 different cases per round. The percentage of tumor cells with membranous and/or cytoplasmic staining at different intensities were evaluated with H-scores ranging from 0 to 300. Overall group and individual pathologist’s scores were compared with intraclass correlation coefficients (ICCs). Between rounds, a “Round Robin” teleconference was conducted to review discordant cases and improve consistency of scoring. Steps to improve scoring included: review of a Roche MET training document, sharing pictures of cases with concordant scores (Figure 1), and provision of H&E images for the second round to facilitate identification of tumor areas. Figure 1
Results:
The overall average MET H-score for the 78 cases was 165.3 (H-score range: 42.5-279.7). The average H-score was <125 for 14 specimens, 125-175 for 35 specimens, and >175 for 29 specimens. The overall group ICC comparing the consistency of H-scores from all 7 pathologists improved from 0.50 (95% confidence interval: 0.37-0.64, “fair” correlation) for the first scoring round to 0.74 (95% confidence interval: 0.64-0.83, “good” correlation) for the second round. A comparison of the individual pathologist’s ICCs demonstrated improved individual scoring consistency for all seven pathologists between rounds with an average of 0.64 (“moderate” correlation, range 0.43-0.76) for the first round and 0.82 (“almost perfect” correlation, range 0.75-0.93) for the second round.
Conclusion:
Development of standardized, reproducible strategies for evaluation of complex biomarkers, such as MET, are critical to clinical trial design. The consistency of scoring for MET protein expression and other biomarkers may be improved by continuous training and communication between pathologists with easy access to H&E images and other visual aids.
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ORAL 41 - Immune Biology, Microenvironment and Novel Targets (ID 159)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.K. Padda, R. Nemenoff
- Coordinates: 9/09/2015, 18:30 - 20:00, Four Seasons Ballroom F1+F2
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ORAL41.02 - Novel Mechanism of Immune-Tolerance and Cancer Metastasis Due to Aberrant Expression of Natural Killer Immunoglobulin-Like Receptors (KIRs) (ID 2199)
18:41 - 18:52 | Author(s): P.A. Bunn, Jr
- Abstract
- Presentation
Background:
Natural Killer (NK) cells are a major defense to eliminate cancer cells. Cancer cells and metastases may have aberrantly expressed KIRs to prevent killing by NK cells. In addition, platelets may inhibit NK killing of cancer cells. Metastatic cancer cells spread through blood vessels where they constantly interact with platelets by forming tumor microemboli and thereby protected from otherwise rapid elimination from host immune defense cells such as NK cells. Here, using an in vivo model of cancer metastasis in athymic nude mice by directly injecting cancer cells into the blood stream, we study the ability of platelets and KIRs in helping cancer cells to escape from immune surveillance and promote metastasis.
Methods:
GFP-luciferase tagged human lung adenocarcinoma cell line, H2122-GL, was further transfected with KIR2DL1 (LL454) plasmids. Stable transformants were enriched by cell sorting. In vivo experimental metastasis were performed in both athymic nude mice and in Nbeal2 knockout and wild type C57 black mice, by tail vein injections of H2122 parental and KIR expressing cells, with and without pre-infusion of human platelets. Levels of tumor cells detected in the lung and other sites were closely monitored by bioluminescence imaging at various time intervals, using an IVIS200 imager.
Results:
24 hours after tail vein injection of a million parental H2122-GL, as low as 0.4 million photons were detectable in the lungs of nude mice (n=5), while those mice injected with a same number of H2122-GL-KIR2DL1 cells, they produced 1.85 million photons in the lungs, showing a 4.6 fold increase in accumulation of KIR-expressed cancer cells than those parental cells in the lung. When the nude mice were pre-infused with iv injection of human platelets followed by tail vein injection of parental or KIR-expressed H2122 cells, enhancement up to 7 fold of lung metastases of KIR expressed H2122 were detected relative to the parental cells as early as 24 hours. 5 weeks post injection, an enhancement up to 190 fold in bioluminescence intensity was found with KIR expressed cells relative to the parental cells. Interestingly, the enhancement of lung metastases was abrogated when similar experiments were repeated in the NBeal2 knockout mice, whose platelets were nonfunctional due to defective alpha-granules and deficiency in their cargo, including von Willebrand factor, thrombospondin-1, and platelet factor 4. One hour after tail vein injection, both parental and KIR expressed H2122 cells produced same but low number of lung metastases, indicating that the defective platelets in the ko mice had failed to promote lung metastases. In the wild type mice, significantly more KIR expressed H2122 cells were detected in the lung relative to parental cells. However, as expected, these early lung metastases were rejected later by the host intact immune cells.
Conclusion:
Our studies demonstrated that metastatic cancer cells acquire immune-resistance by aberrantly express Natural Killer-Cell Immunoglobulin-like Receptors (KIRs) on their surface and that KIR-expressing cancer cells interact more strongly with platelets leading to significantly increase in NK tolerance and enhancing cancer metastases in pre-clinical models.
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P1.01 - Poster Session/ Treatment of Advanced Diseases – NSCLC (ID 206)
- Event: WCLC 2015
- Type: Poster
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.01-071 - Long-Term Tolerability Among IRESSA Clinical Access Program (ICAP) Participants in the United States (US) (ID 780)
09:30 - 09:30 | Author(s): P.A. Bunn, Jr
- Abstract
Background:
Following the gefitinib (IRESSA[®]) NDA voluntary withdrawal, which previously had allowed for limited commercial distribution of IRESSA, gefitinib became available under the IRESSA Clinical Access Program (ICAP) in June 2011. The ICAP continued to provide drug access to patients who were benefiting or had benefited from treatment with gefitinib through restricted distribution (2005-2011) or through a clinical trial that was IRB approved prior to June 2005. ICAP participants constitute a unique subset of cancer patients in whom long-term use of gefitinib can be studied. Consequently, this is the first study to describe long-term safety and tolerability data for an EGFR TKI in cancer patients outside of the clinical trial setting.
Methods:
This study utilizes 2 data sources: (1) retrospective patient medical chart review of demographics, including safety and tolerability of prolonged treatment with gefitinib as part of the ICAP; and (2) retrospective review of serious adverse event (SAE) reports in the AstraZeneca safety database, as all ICAP investigators are responsible, per protocol, for reporting all SAEs observed for ICAP participants.
Results:
A total of 188 patients were enrolled in the ICAP from 134 sites across the US; 94 patients (50.0%) remain on active treatment. This study aims to include as many sites and patients as feasible. Currently, 46 sites representing 77 patients have agreed to participate; site enrollment and data collection are ongoing. As of July 16, 2015, chart abstractions of 16 patients were completed. These patients have a median age of 68.0 years, are predominantly female (75.0%), non-Hispanic white (87.5%), with a confirmed NSCLC diagnosis (93.8%). More patients received gefitinib in second line (43.8%) followed by first line (37.5%) and third line (18.8%). Median gefitinib duration prior to ICAP initiation was 11.3 years (range 9.1-13.9 years), with median gefitinib duration as part of the ICAP being an additional 3.5 years (range: 0.3-3.8 years). During the ICAP, 93.8% of patients (95% CI: 87.9-99.6) did not experience any dose reductions, interruptions, or discontinuation due to gefitinib-related adverse events. The AstraZeneca Safety Database showed 123 SAEs reported from 54 ICAP patients as of February 26, 2015. The majority of SAEs were consistent with underlying disease conditions and were considered unrelated to gefitinib therapy by investigators. 5.6% of patients (3/54) had potentially causally related SAEs as determined by investigators: one patient had procedure-related bronchitis, lung infection, and exacerbation of preexisting COPD with a fatal outcome; one patient experienced interstitial lung disease, pulmonary alveolar hemorrhage, and acute respiratory and renal failure (patient recovered with sequelae); and one patient developed dermatitis acneiform and pruritus. Of the remaining 51 patients, 20 had fatal outcomes (39.2%). The majority of fatalities (12) had insufficient information to assess cause of death and may have had other alternative causes, including underlying or concurrent diseases (6) and possible disease progression (2).
Conclusion:
: Characterization of long-term gefitinib use among this subset of NSCLC patients indicates acceptable long-term tolerability and indicates that some patients have long-term (>10 year) benefit. Clinical and genetic features associated with long-term benefit need further study.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 2
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-006 - MiRNA Signature to Assess Sensitivity to FGFR Tyrosine Kinase Inhibitors (ID 1717)
09:30 - 09:30 | Author(s): P.A. Bunn, Jr
- Abstract
Background:
Increased signaling through the FGF/FGFR signaling pathway has been implicated as a driver in a number of different malignancies including lymphomas, prostate cancer, breast cancer, and lung cancer. This pathway also appears to play a role in conferring de novo and acquired resistance to cancers driven by EGFR mutations. Consequently, drugs that inhibit FGFRs are being investigated as potential therapeutics for cancer. Here we screened a large panel of miRNAs as potential predictors of sensitivity to FGFR tyrosine kinase inhibitors (TKIs).
Methods:
A panel of 377 miRNAs (Megaplex Card A, Life Technologies) was screened for expression level differences between four lung cancer cell lines that are sensitive (IC~50~< 50 nM) and four lines that are resistant (IC~50~ > 100 nM) to ponatinib (non-specific FGFR TKI) and AZD4547 (FGFR-specific TKI). Expression levels were assayed by RT-qPCR and analyzed using the Statistical Analysis of Microarrays (SAM) method. Thirty-nine miRNAs having an estimated false discover rate (FDR) of zero and large median fold differences (> 8) between the sensitive and resistant lines were selected for signature development. RT-qPCR assays were incorporated into a custom microfluidics card (Life Technologies), which was used to profile the original 8 cell lines and 10 additional sensitive lines and 16 additional resistant lines (34 lines total). Logistic regression was then used to identify the best signature panel for distinguishing sensitive cell lines from resistant.
Results:
Univariate analysis indicated three miRNAs (let-7c, miR-338, and miR-218) that differed between the sensitive and resistant lines at p < .05. The best signature panel consisted of let-7c, miR-200a and miR-200b, which gave an area under the receiver operator characteristic (AUROC) curve of 0.90 (95% CI = 0.8 to 1). This performance was nearly as good as using FGFR1 mRNA alone (AUROC = 0.94). The predominant miRNA in our 3-miRNA signature was let-7c, which also exhibited a suggestive additive effect to using FGFR1 as a biomarker (p = 0.09). We also tested whether cell lines with high sensitivity to ponatinib can be made resistant by reducing the high level of let-7c in these lines. We have found that transient transfection of let-7c silencing RNA (Life Technologies) produces a decrease in FGFR1 mRNA levels for some cell lines but not others.
Conclusion:
It appears possible to predict sensitivity to an FGFR1 inhibitor using miRNA expression signatures. More studies, however, are needed to confirm the 3-marker signature developed in this study. Modulating let-7c, the predominant predictor within the signature, appears to modulate FGFR1 levels in a manner consistent with altering ponatinib sensitivity. This effect is most likely indirect as the mRNA of FGFR1 does not contain predicted binding sites for let-7c.
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P2.04-007 - In Vitro and in Vivo Efficacy of AZD9291 Is Enhanced by Combination with AZD4547 in EGFR Mutant Lung Cancer Cells (ID 2456)
09:30 - 09:30 | Author(s): P.A. Bunn, Jr
- Abstract
Background:
EGFR-specific tyrosine kinase inhibitors (TKIs) provide marked clinical responses in patients bearing EGFR mutated lung tumors, although acquired resistance limits the durability of the response. In light of the frequent emergence of erlotinib and gefitinib-resistant EGFR T790M mutations upon tumor progression, 3[rd] generation EGFR-specific TKIs have been developed that specifically inhibit gain-of-function EGFR mutants irrespective of T790M status. Recently, we reported a distinct mechanism of acquired resistance whereby specific EGFR mutant lung cancer cell lines including H1650 and HCC4006 cells, but not PC9 cells, undergo an epithelial-mesenchymal transition (EMT) upon chronic in vitro treatment with gefitinib. As a result, the adapted cells acquire vulnerability to FGFR inhibitors by virtue of EMT-mediated FGF2 and FGFR1 induction. Herein, we have tested the hypothesis that combination of the FGFR inhibitor, AZD4547, with the 3[rd] generation EGFR TKI, AZD9291 will yield superior anti-tumor activity relative to AZD9291 alone.
Methods:
Lung cancer cell lines bearing gain-of-function EGFR mutations (HCC4006, H1650 and PC9) were submitted to in vitro clonogenic growth assays in the presence of AZD9291 and/or AZD4547 over concentration ranges for 1 to 300 nM for each drug. For in vivo measurement of the activity of these drugs, flank xenografts were established in Nu/Nu mice with the 3 lung cancer cell lines and treated by daily oral gavage (5 days on, 2 days off) with diluent, AZD9291 (5 mg/kg), AZD4547 (12.5 mg/kg) and the combination of the two drugs at these doses. Tumor size was measured with calipers and volume was calculated using the formula, Volume=3.14(short diameter)[2](long diameter)/6.
Results:
HCC4006, H1650 and PC9 cells were highly sensitive to ZD9291 in vitro with IC~50~ values of 1.6, 7.4 and 3.3 nM, respectively. In a 2 week clonogenic growth assay, AZD9291 reduced growth of all cell lines by >95%, although viable drug resistant persisters clearly remained. While none of these cell lines exhibited significant growth inhibition in response to AZD4547 alone, combination of AZD9291 and AZD4547 further reduced clonogenic growth of HCC4006 and H1650 cells, but not PC9 cells. In flank xenograft studies, AZD9291 monotherapy induced marked tumor shrinkage (H1650, ~80% at day 10; HCC4006, ~90% at day 30; PC9, 89% at day 25), although regrowth of the tumors occurred with all three xenografts. AZD4547 yielded little or no growth inhibition as a monotherapy, but significantly enhanced the degree of tumor shrinkage and delayed the time to tumor progression in H1650 and HCC4006 tumors, but not PC9 tumors.
Conclusion:
Combination of the FGFR inhibitor AZD4547 with AZD9291 affords greater growth suppression relative to AZD9291 alone in HCC4006 and H1650 cells that undergo EMT and induction of an FGF2-FGFR1 pathway. Predictably, this combination was not more effective compared to AZD9291 alone in PC9 cells that fail to undergo EMT in response to EGFR TKI treatment. The studies support the efficacy of combined AZD9291 and AZD4547 treatment of a subset of lung tumors driven by mutated EGFR, although the features of these particular lung tumors that predict this response is unknown at this time.
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P2.06 - Poster Session/ Screening and Early Detection (ID 219)
- Event: WCLC 2015
- Type: Poster
- Track: Screening and Early Detection
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.06-007 - A miRNA Signature Derived From Independently Replicated Biomarkers of Non-Small Cell Lung Cancer (ID 1728)
09:30 - 09:30 | Author(s): P.A. Bunn, Jr
- Abstract
Background:
miRNAs have shown exceptional promise as biomarkers of lung cancer; however, no miRNA signatures have yet reached the clinic. Towards developing a signature with a high likelihood of being validated externally for clinical use, we screened a panel of 50 miRNAs shown to be effective biomarkers in at least two previous studies for distinguishing human lung cancer samples from non-cancer samples.
Methods:
Sixty tumor-normal pairs (33 adenocarcinoma, 27 squamous cell carcinoma) were used to identify the best-performing combination of 4 miRNAs for distinguishing tumor samples from normal. The miRNA levels were measured by RT-qPCR using Taqman custom-made microfluidics cards and primer pools purchased from Life Technologies. All possible combinations of 4 miRNAs were tested, and best performance was defined as the highest median area-under the receiver operating curve (AUC) obtained from 1000 bootstrap replicates. A second, independent set of 68 tumor-normal samples (half adenocarcinoma, half squamous) was used as a test set, and bootstrapping was used to determine the 95% confidence interval for the AUC.
Results:
The median AUC for the top-performing panel of 4 miRNAs in our training set was 0.96. Several other miRNA combinations exhibited AUCs > 0.95 as well. In our test set, the top-performing panel (and only panel tested) exhibited an AUC of 0.97 (0.93, 0.99). This panel consisted of miRs 26a, 145, 183 and 486. miRs 145 and183 have previously been shown, when used individually, to be significant lung tumor biomarkers in at least 4 previous studies; miR-486 has been replicated 8 times.Figure 1
Conclusion:
Consistent with previous studies, we’ve identified a panel of 4 miRNAs that shows excellent potential for diagnosing lung tumors. Each of these miRNAs has been replicated as a biomarker of lung cancer in at least two previous studies, suggesting a high likelihood of achieving clinical validation. Several previous studies have also shown that these four miRNAs are potentially useful as biomarkers for diagnosing lung cancer using blood samples, and we are currently pursuing such validation studies.
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P2.07 - Poster Session/ Small Cell Lung Cancer (ID 222)
- Event: WCLC 2015
- Type: Poster
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.07-002 - Effects of Eribulin and Radiation on a Panel of Small Cell Lung Cancer (SCLC) Cell Lines (ID 2154)
09:30 - 09:30 | Author(s): P.A. Bunn, Jr
- Abstract
Background:
Background: Chemotherapy produces high response rates in extensive stage SCLC and a modest improvement in 5-year survival rates when combined with chest radiation in limited stage SCLC. Chemotherapeutic agents for SCLC have not changed in 20 years. Eribulin is a microtubule inhibitor that arrests cells in the G2/M fraction of the cell cycle with established activity in breast cancer. Radiobiological studies demonstrated that cells in the G2/M phase of the cell cycle are less efficient at repairing radiation induced DNA damage. Thus, we investigated the effects of eribulin, radiation and the combination on growth and cell cycle distribution in a panel of SCLC cell lines.
Methods:
Methods: Growth inhibition (GI) by varying concentrations of eribulin alone, radiation alone and the combination was assessed by MTS assay at 5 days post-treatment. Growth inhibition or fraction affected (FA) was determined by 1-(x/y) where x is the MTS signal for the experimental condition and y is the MTS signal for the untreated control cells. Our goal was to use a dose of radiation that alone induced a FA of about 0.5 allowing determination of the combination effects with eribulin. Changes in the G2/M distribution of cells treated with eribulin alone, radiation alone and the combination were evaluated at 24 and 48 hours post treatment by propidium iodine staining and analysis by FACS.
Results:
Results: Four of the eight SCLC cell lines were very sensitive to eribulin with half maximal growth inhibitory concentration (FA<0.5) of < 2nM. Four lines had 0.5 FA values > 2nM. 2 Gy radiation produced 32% to 58% growth inhibition in all 8 lines irrespective of their eribulin sensitivity. Low eribulin concentrations (≤ 1.25nM) and 2Gy radiation produced >70% growth inhibition in the 4 sensitive lines, which was significantly more growth inhibition than either alone. Eribulin concentrations of >2.5nM were required to increase growth inhibition over either alone in the 4 more resistant lines and the maximal GI was less in these lines (48%-70%) even at higher concentrations. With respect to G2/M, in the 4 most sensitive eribulin lines, there was a significant increase in the G2/M fraction following eribulin alone (0.625-1.25nM), radiation alone (2 or 3Gy) and a further increase occurred with the combination treatment. In these eribulin sensitive lines, 59% to 93% of the cells were in the G2/M phase by 48 hours. In 3 of the 4 less sensitive eribulin lines, higher concentrations of eribulin (>2.5nM) were required to increase the G2/M fraction to >50% and 2 or 3 Gy irradiation increased the G2/M fraction to 59% to 64%. The combination produced maximal G2/M fractions of 64%-79%. The one most eribulin resistance line never had more than 50% GI or > 40% of cells in G2/M at any concentration or radiation dose up to 4 Gy.
Conclusion:
Conclusions: SCLC cell lines are sensitive to eribulin and radiation and the combination produced significantly more growth inhibition and cell cycle arrest then either alone. The combination warrants further evaluation in in vivo models and potentially clinical trial study in patients with SCLC.
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PLEN 04 - Presidential Symposium Including Top 4 Abstracts (ID 86)
- Event: WCLC 2015
- Type: Plenary
- Track: Plenary
- Presentations: 1
- Moderators:T. Mok, F.R. Hirsch
- Coordinates: 9/09/2015, 10:45 - 12:15, Plenary Hall (Bellco Theatre)
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PLEN04.04 - Discussant for PLEN04.03 (ID 3450)
11:19 - 11:27 | Author(s): P.A. Bunn, Jr
- Abstract
- Presentation
Abstract not provided
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YIS - Young Investigator Session incl. Q & A with Longstanding IASLC Members (ID 238)
- Event: WCLC 2015
- Type: Young Investigator Session
- Track: Other
- Presentations: 1
- Moderators:L. Carr, J.R. Jett
- Coordinates: 9/06/2015, 07:30 - 11:00, Mile High Ballroom 4a-4f
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YIS.07 - Q & A with Longstanding IASLC Members (ID 3517)
10:30 - 11:00 | Author(s): P.A. Bunn, Jr
- Abstract
Abstract not provided
