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L. Heasley
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MA15 - Immunotherapy Prediction (ID 400)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Chemotherapy/Targeted Therapy/Immunotherapy
- Presentations: 1
- Moderators:O. Arrieta
- Coordinates: 12/07/2016, 14:20 - 15:50, Schubert 1
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MA15.11 - Acquired Resistance Mechanisms to EGFR Kinase Inhibitors Alter PD-L1 Expression Status in Lung Cancer (ID 4652)
15:32 - 15:38 | Author(s): L. Heasley
- Abstract
Background:
Immunotherapies that target PD-1/PD-L1 exploit the primary roles of cytotoxic agents in lung cancers. However, tyrosine kinase inhibitors (TKIs) are still considered to be the first choice in lung cancer patients with EGFR mutations. Although immunotherapies may be applied as second line or later therapeutic approaches in these patients, after acquisition of resistance to EGFR-TKIs, it is unclear if acquired resistance mechanisms alter PD-L1 expression status that is employed as an important predictive biomarker for PD-1/PD-L1 targeting agents.
Methods:
Lung cancer cell lines with EGFR mutations (HCC827, HCC4006, PC9, and H1975) and their isogenic descendants with acquired resistance to various EGFR-TKIs were examined in this study. The resistance mechanisms of descendants include T790M secondary mutation, MET gene amplification, epithelial to mesenchymal transition (EMT), and loss of amplified EGFR mutant allele. PD-L1 expression status was analyzed by immunohistochemistry (IHC) and immunoblotting. Effects of acquired resistance mechanisms on PD-L1 expression were also evaluated by shRNA mediated knockdown of candidate molecules, and co-localization analysis using fluorescent imaging. IFN-gamma was used to mimic immune cell attack. Published microarray data of cells with acquired resistance to EGFR-TKIs were also employed to evaluate our findings.
Results:
PD-L1 expression was upregulated in several resistant cells and correlated with EGFR activation. In addition, we found that the phosphorylation of EGFR tyrosine (Y) 992 site, but not Y845, Y1068, or Y1173, was correlated with increased expression of PD-L1. We also observed that TKI-resistant cells with marked E-cadherin downregulation (HCC4006 erlotinib resistant cells and H1975 osimertinib resistant cells), one of hallmarks of EMT, showed decreased expression of PD-L1. However, one cell line (853#10), displaying EMT-like phenotype but only slight E-cadherin downregulation, showed PD-L1 upregulation. Published microarray data from three TKI-resistant lines with EMT-like features also support the correlation of low E-cadherin and reduced PD-L1 expression. ShRNA mediated knockdown of E-cadherin decreased the expression of PD-L1 in parental cell lines. IFN-gamma treatment upregulated PD-L1 expression in both parental and in resistant cells with E-cadherin downregulation, however PD-L1 expression in resistant cells was still lower and localized mainly in the cytoplasm rather than the cell membrane.
Conclusion:
We observed a dramatic change of PD-L1 expression status in lung cancers with EGFR mutation after acquisition of resistance to EGFR-TKIs, depending on the resistance mechanisms. These results support the importance of re-biopsy after acquisition of resistance to EGFR-TKIs, not only for the resistance mechanisms but also for the evaluation of PD-L1 expression status.
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P3.02b - Poster Session with Presenters Present (ID 494)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.02b-084 - Rational Combinations to Improve Outcome for EGFR-TKIs in NSCLCs with EGFR Mutations (ID 6002)
14:30 - 14:30 | Author(s): L. Heasley
- Abstract
Background:
Background: EGFR-TKIs produce high response rates in tumors with EGFR activating mutations as first line therapy and after development of T790M resistance. But complete responses are rare and all patients progress. We conducted serial RNAseq analyses of EGFR mutant cell lines exposed to gefitinib and osimertib. We found a rapid induction of gene reprogramming indicative of WNT signaling and EMT signaling that developed within 72 hours of drug exposure and lasted through at least 57 days. Important genes involved included E-cadherin, vimentin, ZEB1&2, axin2, IL8 and others. We therefore elected to determine if inhibitors of Wnt or EMT reprograming would produce synergistic growth inhibition in EGFR mutant cell lines exposed to osimertib. Prior clinical studies indicated that the HDAC inhibitors can safely be combined with EGFR-TKIs.
Methods:
Methods: Growth inhibition in the HCC4006 line by osimertib (10-100nM) in combination with the WNT/Bcatenin inhibitors AZD1366, ICG01, E7449 (30-270nM), WntC59, IWP2-V2, LGK974 (90-810nM), and with the HDAC inhibitors etinostat (300-1000nM), panobinostat (10-50nM) and romidepsin (1-6nM) was assessed by 5 day MTT assays. Growth inhibition by osimertib (2.5-20nM) + etinostat (60-300nM) was also evaluated in the PC9, HCC827, H3255 and PC9T790M EGFR mutant lines. Analysis of the combined drug effects was by the median-drug effect method using the CalcuSyn program to determine the combination indices (CI). CI values of < 1 are indicative of drug synergy.
Results:
Results: The CI values from combining 30nM osimertib with the midrange concentration of the WNT pathway inhibitors and HDAC inhibitors in the HCC4006 line varied from 0.18 to 1.2 and most were ≤ 0.75. The tankyrase inhibitor AZD1366 produced the most synergistic effect with CI = 0.18. CI values of ≤ 0.5 were observed with WntC59 and ICG01. The HDAC inhibitors all produced CI values of ≤ 0.75 with the lowest value of 0.23 for etinostat. CI values of osimertib combined with panobinostat and romdidepsin were 0.51 and 0.73 respectively. CI values for 30 nM osimertib combined with etinostat in the other EGFR mutant cell lines were also synergistic ranging from 0.37 in PC9 to 0.96 in the H3255 line. Synergy was also observed with romidepsin and panobinostat in these lines.
Conclusion:
Conclusions: The combination of WNT pathway inhibitors or HDAC inhibitors with EGFR-TKIs produce synergistic growth inhibition and may prevent EMT and survival pathways in EGFR mutant lung cancers.
