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C. Demuth
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P2.01 - Poster Session/ Treatment of Advanced Diseases – NSCLC (ID 207)
- Event: WCLC 2015
- Type: Poster
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.01-017 - Genetic Variations in the EGFR Gene Predicts Outcome in Advanced NSCLC Patients Treated with Erlotinib (ID 812)
09:30 - 09:30 | Author(s): C. Demuth
- Abstract
Background:
Genetic variations in the epidermal growth factor receptor (EGFR) gene may alter protein expression or function and influence response to tyrosine kinase inhibitors. This study evaluates the role of genetic polymorphisms in the EGFR gene in advanced non-small cell lung cancer (NSCLC) patients treated with erlotinib. EGFR mutation status was known for all patients.
Methods:
Genotypes for -216G>T, -191C>A and 181946C>T in the EGFR gene were retrospectively evaluated by DNA sequencing and polymerase chain reaction in 354 Caucasian patients with advanced NSCLC. Hundred and seven of the patients had a somatic EGFR mutation, and all patients had been treated with erlotinib. Genotypes were correlated with clinical characteristics and outcome. A multivariate analysis was conducted adjusting for clinical relevant factors, including EGFR mutation status, using Cox proportional hazards model. A subgroup analysis was performed based on the EGFR mutation status.
Results:
Patients harboring at least one variant T allele (CT or TT) at position 181946 had a significantly longer median progression-free survival (PFS) (5.6 versus (vs.) 2.9 months; p =0.032) and overall survival (OS) (8.3 vs. 6.7 months; p=0.032) compared to patients with the CC genotype. The result remained significant in a multivariate analysis; PFS, adjusted hazard ratio (AHR)=0.73 (95% confidence interval (CI): 0.55-0.98); OS, AHR=0.72 (95%CI: 0.54-0.97). Patients carrying -216GT or TT genotypes showed a trend to a better clinical outcome compared to those with the GG genotype. The -216GT or TT and 181946CT or TT combined genotypes showed an even more pronounced association with clinical outcome compared to patients with the -216GG and 181946CC genotype (PFS, AHR=0.66 (95%CI: 0.44-0.98); OS, AHR=0.58 (95%CI: 0.38-0.87)). A subgroup analysis demonstrated that the association might be most relevant in EGFR mutation-positive patients; PFS, AHR=0.27 (95% CI: 0.11-0.68); OS, AHR=0.33 (95% CI: 0.13-0.83).
Conclusion:
A combination of 181946C>T and -216G>T polymorphisms in the EGFR gene seems to be a potential predictor of longer PFS and OS in advanced NSCLC patients treated with erlotinib; especially in EGFR mutation-positive patients. A prospective randomized study is wanted to confirm our data.
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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: 3
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-003 - Two Methods for Developing in Vitro Erlotinib-Resistant Cell Lines Lead to Distinct RTK Shifts, but Both Result in EMT (ID 928)
09:30 - 09:30 | Author(s): C. Demuth
- Abstract
Background:
Several studies have investigated resistance mechanisms underlying acquired erlotinib-resistance in vitro. To mimic the in vivo distribution of the drugs, different approaches such as applying gradually increasing doses of erlotinib to the cells or exposing them to a high fixed concentration of the drug have been used. We demonstrate that two different approaches of developing erlotinib-resistant HCC827 cells results in activation of two distinct RTK bypass-signalling pathways. However, despite these differences both cell lines undergo EMT. Our finding suggests that EMT is a common marker of erlotinib-resistance.
Methods:
Two HCC827 erlotinib-resistant cell lines were established using either gradually increasing doses of erlotinib (0.01 μM – 5 μM) resulting in erlotinib-resistant HCC827ER cells. Alternatively a fixed concentration of 5 μM generated HCC827HD with erlotinib resistance. Growth of the resistant cell lines was investigated using MTS assay in combination with erlotinib, linsitinib and crizotinib. Phospho-RTK arrays (R&D Systems), qPCR and immunofluorescence were used to characterize the cells.
Results:
Phospho-RTK array analysis revealed that the erlotinib-resistant HCC827ER cells had an increased activation of MET, and copy number analysis demonstrated the activation to be caused by a MET amplification. Furthermore, HCC827ER showed growth inhibition when treated with the MET-inhibitor crizotinib. The other type of erlotinib-resistant cells, HCC827HD, had increased activation of IGF1R and also responded to the IGF1R-inhibitor linsitinib. However, a common feature is that both HCC827ER and HCC827HD gained EMT features. HCC827ER showed increased expression SLUG, SNAIL and ZEB1, whereas HCC827HD showed increased SLUG and TWIST expression. To detect the relevance of MET and IGF1R signalling in accordance to EMT in the two cell lines, we treated the HCC827ER cells with the tyrosine kinase inhibitor crizotinib (MET) and the HCC827HD cells with linsitinib (IGF1R). In both cases, we saw a decrease in EMT-marker transcription after the treatment.
Conclusion:
Our study demonstrates that different approaches to developing erlotinib-resistant cell lines can lead to distinct activation of bypass receptor tyrosine kinase signalling pathways. EMT, however, is induced in both types of erlotinib-resistance. This finding indicates that EMT is a common trait of the phenotype of erlotinib-resistant cells. More research needs to be done to establish the functional role of EMT in erlotinib resistance.
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P2.04-073 - PD-L1 Expression Is Induced by MET in an Erlotinib-Resistant Cell Line with MET Amplification (ID 833)
09:30 - 09:30 | Author(s): C. Demuth
- Abstract
Background:
The programmed cell death receptor 1 (PD-1) and its ligand PD-L1 have proved to be of significant importance in lung cancer. Production of PD-L1 helps the cancer cells evade the immune system by inactivating T-cells. Clinical trials investigating the effect of treating lung cancer patients with monoclonal antibodies targeting the PD-L1 and PD-1 shows promising results. Expression of PD-L1 is associated with epidermal growth factor receptor (EGFR) mutational status. Further, expression can be significantly decreased by targeting EGFR with tyrosine kinase inhibitors (TKIs). In vitro studies suggest that this initial regulation of PD-L1 expression by EGFR occurs through the Erk pathway. Though, currently not much is known about expression of PD-L1 when TKI-resistance develops. We have developed erlotinib-resistant cell lines. The resistant cell line gained a MET amplification. We demonstrate that PD-L1 is increases in the resistant cells and that this increment is induced by MET signalling.
Methods:
The lung cancer cell line HCC827 with a deletion in exon 19 in the EGFR gene, was treated with increasing concentrations of erlotinib over 5 months until resistance developed. MET gene amplification in the resistant cells was confirmed by PCR. The resistant cell line was used for studying the effect of EGFR and MET inhibitors on PD-L1 expression.
Results:
The HCC827 erlotinib-resistant (ER) cell line gained a MET gene amplification, as seen in previous studies. In the initial phase of erlotinib treatment the expression of PD-L1 decreases. As the dose increases and resistance starts to develop the expression of PD-L1 increases. Activation of Erk is intact in HCC827ER as compared to the parental HCC827 cell line; most likely due to the activation of MET. When HCC827ER cells are treated with the MET inhibitor crizotinib, expression of PD-L1 decreases. When erlotinib is combined with crizotinib an additional effect on PD-L1 expression is observed. These results indicate that increased PD-L1 expression in erlotinib-resistant cell lines may be caused by activation of Erk through MET signalling.
Conclusion:
Our data demonstrates that Erk-dependent PD-L1 expression is increased in cells with erlotinib resistance caused by MET gene amplification. This mechanism might even be general and include several by-pass resistance mechanisms. Our findings suggest that the role of the PD-L1/PD-1 system should also be studied in erlotinib resistant tumors.
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P2.04-076 - Dynamics of Soluble PD-1 during Treatment with EGFR-TKI in Advanced NSCLC Patients (ID 1186)
09:30 - 09:30 | Author(s): C. Demuth
- Abstract
Background:
The programmed cell death receptor-ligand pathway (PD-1/PD-L1) is hijacked by tumors in order to evade immune response. Therapy with monoclonal antibodies against PD-1 or PD-L1 in patients with advanced NSCLC has shown promising results in recent clinical trials. Preclinical studies indicate that the PD-L1 expression on tumor cells, and subsequently the PD-L1/PD-1 interaction, is increased when resistance to EGFR-TKI treatment in EGFR mutated NSCLC tumors occur. A soluble form of PD-1 receptor is present in the blood plasma, and can be detected in healthy individuals and in increased amounts in patients with autoimmune diseases and cancer. The dynamics of soluble PD-1 (sPD-1) during treatment and at the point of resistance to EGFR-TKI treatment is unknown. The aim of the present study is to assess the dynamics in plasma of EGFR mutated circulating tumor DNA and sPD-1 longitudinally during treatment with EGFR-TKI, and to study if the level of sPD-1 will increase at the time of resistance to EGFR-TKI treatment.
Methods:
Consecutive blood samples taken before initiation of treatment with erlotinib, during treatment, and at disease progression while on erlotinib from 20 patients with EGFR wildtype and 20 patients with EGFR mutated advanced NSCLC, has been collected, and will be analyzed. The amount of EGFR mutated circulating tumor DNA (in the EGFR mutated patients) and sPD-1 (all patients) will be detected by use of the Cobas® instrument (RMD) and ELISA (R&D systems), respectively. These results will be described and correlated to the clinico-pathological characteristics of the patients.
Results:
Preliminary results show that sPD-1 can be detected in plasma from lung cancer patients. The final results of the analysis will be presented at the conference.
Conclusion:
The present study is to our knowledge the first to describe the dynamics of soluble PD-1 during EGFR-TKI therapy in both EGFR mutated and EGFR wildtype patients treated with erlotinib. The results will elucidate on the role of sPD-1 as a potential biomarker of resistance to EGFR-TKI therapy. The clinical time point of increased sPD-1 in plasma could indicate a “window of opportunity”, in which these patients could be highly responsive to anti-PD-1 or anti-PD-L1 immunotherapy. Such findings have to be further investigated in prospective clinical trials.
