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K.R. Jakobsen
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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 - 17:00 | Author(s): K.R. Jakobsen
- 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: 2
- 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 - 17:00 | Author(s): K.R. Jakobsen
- 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 - 17:00 | Author(s): K.R. Jakobsen
- 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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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-023 - Exosomes and Their Potential for Detection of Lung Cancer (ID 831)
09:30 - 17:00 | Author(s): K.R. Jakobsen
- Abstract
Background:
A recent study showed that advanced lung adenocarcinoma patients have a distinct exosomal protein-profile compared to a matched group without cancer (Jakobsen et al., 2015, JEV). To improve the overall survival, it is however crucial to develop tools capable of detecting early stages of lung cancer as well. In addition, it is unsettled if different histologic subclasses result in distinct exosomal protein profiles. The aim of this study is to explore the potential of using exosomal proteins as biomarkers in lung cancer patients of all stages and of different histology histology.
Methods:
Plasma was isolated from patients suspected of having lung cancer. Patients diagnosed to be cancer free were defined as controls. Based on previous experiments a panel of 47 antibodies were selected for exosome-capture using a highly sensitive extracellular vesicle protein array (EV Array). 10 µl unpurified plasma was applied to the EV Array and captured exosomes were visualised by binding of biotin-conjugated CD9, CD63 and CD81 antibodies. The information from all 47 markers was investigated by multivariate analysis by partial least squares discriminant analysis (PLS-DA).
Results:
The study included 504 patients; 153 control patients and 351 patients with NSCLC (adenocarcinoma 70%, squamous cell 24%, other 6%). 51% had locally advanced or advanced disease and 49% had local disease. Multivariate analysis produced a combined marker model separating cancer patients from controls regardless of stage and histology. Area under the curve (AUC) was for each stage: I: 0.74 (0.68-0.82), II: 0.68 (0.57-0.79), III: 0.77 (0.62-0.91) and IV 0.79 (0.73-0.83). For all stages AUC was 0.755, CI (0.72-0.81) with sensitivity 0.70 and specificity 0.66. The accuracy of the test was 0.69.
Conclusion:
We demonstrate that the EV array is able to lung cancer in advanced as well as low stages regardless of histology.
