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K. Nishio
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MINI 10 - ALK and EGFR (ID 105)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:T. Yap, T. Li
- Coordinates: 9/07/2015, 16:45 - 18:15, Mile High Ballroom 1a-1f
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MINI10.10 - A Multicenter Prospective Biomarker Study in Afatinib-Treated Patients with EGFR-Mutation Positive Non-Small Cell Lung Cancer (ID 472)
16:45 - 18:15 | Author(s): K. Nishio
- Abstract
- Presentation
Background:
Afatinib is an oral, irreversible ErbB family blocker and one of the key drugs for patients with EGFR mutation positive advanced non-small cell lung cancer (NSCLC). Although treatment with afatinib has a clinical benefit for these patients, such individuals inevitably develop drug resistance as with other TKIs. This is a multicenter prospective biomarker study to inform the usefulness of noninvasive liquid biopsy in the treatment of EGFR-tyrosine kinase inhibitors (EGFR-TKIs) and explore the molecular mechanism of acquired-resistance against afatinib.
Methods:
Eligible patients were EGFR-TKIs naïve and had histologically and cytologically confirmed stage IIIB/IV adenocarcinoma of the lung with activating EGFR mutations. Patients remained on afatinib treatment until disease progression or unacceptable toxicity. Tumor samples were collected upon before afatinib treatment and after disease progression. Plasma samples were collected upon before and during afatinib treatment (4 and 24 weeks after initiation) and after disease progression. DNA derived both from tumors and plasma was analyzed using Scorpion-ARMS (ARMS), digital PCR (dPCR) and next generation sequencing (NGS). We used a nanofluidic dPCR system (BioMark HD System; Fluidigm) with a digital chip to detect activating or resistance mutations of EGFR in a quantitative and highly sensitive manner. NGS on an Ion Torrent PGM device (Thermo Fisher Scientific) was applied to detect target molecules which contribute to the survival and growth of lung cancer cells. We compared the sensitivity of these methods in detection of EGFR activating mutations in plasma DNA.
Results:
A total of 35 EGFR mutation positive NSCLC patients were enrolled. Twenty one patients harbored a deletion in exon 19 and fourteen patients had an L858R missense mutation in exon 21. Twenty seven (77.1%) patients had an objective response. In plasma DNA obtained before afatinib treatment, dPCR and NGS detected EGFR activating mutations more sensitively compared with ARMS (83.9% v 58.1%; p <0.005, 74.2% v 58.1%; p =0.059, respectively). Concordance of EGFR activating mutations detected by dPCR and NGS was 26/31 (84%) (kappa value: 0.52). All of the mutation type detected by NGS on plasma DNA completely corresponded to that found in matching tumor tissue by NGS. As of March 2015, serial plasma DNA was analyzed in 9 patients. The copy number of activating mutation was markedly decreased in 5 of 9 patients.
Conclusion:
EGFR activating mutations in plasma DNA were frequently detected by dPCR or NGS. We will present the detailed data for monitoring mutation load in plasma DNA during the afatinib treatment.
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ORAL 03 - New Kinase Targets (ID 89)
- Event: WCLC 2015
- Type: Oral Session
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:F. Blackhall, R. Juergens
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 4a-4f
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ORAL03.03 - EGFR Exon 18 Mutations in Lung Cancer: Molecular Predictors of Sensitivity to Afatinib or Neratinib but Not to Other EGFR-TKIs (ID 1748)
10:45 - 12:15 | Author(s): K. Nishio
- Abstract
- Presentation
Background:
Lung cancers harboring common EGFR mutations respond to EGFR tyrosine kinase inhibitors (TKIs),whereas exon 20 insertions (Ins20) are known to be resistant to these drugs. However, little is known about the role of mutations in exon 18. Inspired by clinical observation that a patient with adenocarcinoma harboring exon 18 deletion (Del18: delE709_T710insD) responded to afatinib, this study aimed to establish a rational therapeutic strategy for lung cancers harboring exon 18 mutations.
Methods:
The mutational status of lung cancers registered in Aichi Cancer Center (ACC) database between 2001 and 2015 was reviewed. Three representative mutations in exon 18, Del18, E709K, and G719A, were introduced into Ba/F3, NIH3T3, and HEK293 cells using retroviral vector. The 90% inhibitory concentrations (IC90s) of first generation (1G) (gefitinib and erlotinib), second generation (2G) (afatinib, dacomitinib, and neratinib), and third generation (3G) TKIs (AZD9291 and CO1686) in these cells were determined and compared with the corresponding IC90s in cells expressing exon 19 deletion (Del 19) and with the trough concentration (C~trough~) at the recommended doses for each drug. Clinical data on the treatment response of tumors harboring exon 18 mutations were collected from the ACC and Catalogue of Somatic Mutations in Cancer (COSMIC) databases.
Results:
Among the 1355 EGFR mutations registered in the ACC database, Del19, L858R, and Ins20 were detected in 40%, 47%, and 4%, respectively. Of note, exon 18 mutations including G719X, E709X, and Del18 were present in 3.2% (n=43), accounting for 38% of the remaining. According to the COSMIC database, exon 18 mutations accounted for 4.1% (654/16,138) of all EGFR mutations present from exons 18-21. Mutations at codons 709 and 719 accounted for 84% of all exon 18 mutations. Ba/F3 cells expressing Del18, E709K, or G719A grew in the absence of interleukin 3, and NIH3T3 cells transfected with these mutations formed foci with marked pile-up, indicating that these mutations act as oncogenic drivers. IC90s of 1G and 3G TKIs in cells transfected with Del18, E709K and G719A were much higher than those in cells transfected with Del19 (by >50-, >25-, and >11-fold, respectively). In contrast, IC90 of afatinib in these three mutations ranged from only 2- to 6-fold greater than that in Del19 and was <1/40 of its C~trough~. Notably, cells transfected with exon 18 mutations exhibited higher sensitivity to neratinib (by 25-fold for E709K, by 5-fold for G719A, and by a comparable extent for Del 18) than those expressing Del19. Western blot analyses showed that these differential sensitivities corresponded to different degrees of suppression of EGFR phosphorylation in HEK293 cells. Furthermore, analyses of the ACC and COSMIC databases clearly indicated that patients with lung cancers harboring G719X exhibited higher response rate to afatinib or neratinib (~80%) than to 1G TKIs (35-56%).
Conclusion:
Our data indicated that lung cancers harboring exon 18 mutations, although rare, should not be overlooked in clinical practice and that these cases are best treated with afatinib or neratinib, although the currently available in vitro diagnostic kits do not detect all exon 18 mutations.
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ORAL 42 - Drug Resistance (ID 160)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:R.C. Doebele, J.V. DeGregori
- Coordinates: 9/09/2015, 18:30 - 20:00, Mile High Ballroom 4a-4f
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ORAL42.02 - Qualitative and Quantitative Heterogeniety in Acquiring Resistance to EGFR Kinase Inhibitors in Lung Cancer (ID 572)
18:30 - 20:00 | Author(s): K. Nishio
- Abstract
- Presentation
Background:
Acquisition of resistance to EGFR- tyrosine kinase inhibitors (TKIs) is one of important issues in lung cancer researches. Several resistance mechanisms have been identified. However, inter-tumor heterogeneity in acquisition of resistance to EGFR-TKIs is currently unclear.
Methods:
Eleven autopsied patients who developed acquired resistance to EGFR-TKI monotherapy were included in this study. All patients harbored activating EGFR mutations (exon 19 deletion or L858R mutation), and developed acquired resistance to EGFR-TKI after initial response to the drug. Details of patient characteristics are summarized in Table 1. The resistance mechanisms of seven patients have been reported in our previous analyses (Suda K, et al. Clin Cancer Res 2010, and Suda K, et al. APLCC 2014). In this study, we analyzed acquired resistance mechanisms in twenty-eight tumor samples obtained from the four additional patients using target sequencing technique by next-generation sequencer.
Results:
Among eleven patients, four developed T790M EGFR secondary mutation in all TKI-refractory lesions. One patient developed MET amplification in all TKI-refractory lesions. Three patients harbored both TKI-refractory lesions with T790M mutation and those with MET amplification. The other three patients showed respective resistance mechanisms (Table 1).Table 1. Summary of resistant mechanisms in eleven patients.
In the target sequence analysis, allele count data were further analyzed in tumor samples with T790M mutation, and we observed diverse T790M/activating EGFR mutation allele ratio ranging from 2 – 51%. In the analysis for time to treatment failure (TTF), we observed longer TTF in patients who developed single resistance mechanism compared with those who developed multiple resistance mechanisms (Fig. 1; p = 0.055). Figure 1Pt. ID Age/Sex Pack-Year Resistant Mechanisms TTF (m) C1 57/F 0 T790M or MET 13.8 C2 48/F 0 T790M or MET 11.0 C3 58/M 34 MET 14.5 C4 75/M 0 T790M 43.9 C5 93/F 0 T790M 14.8 C6 62/M 26 T790M 9.1 P1 86/F 0 T790M 10.8 P2 72/M 27 T790M or MET 3.8 P3 89/F 0 EGFR loss with MET or Unknown 9.0 P4 84/F 0 Unknown 22.6 A1 76/F 0 SCLC transformation or T790M 5.0
Conclusion:
In this study, we observed qualitative heterogeneity and quantitative heterogeneity of T790M allele ratio in acquisition of resistance to EGFR-TKIs in lung cancers. Qualitative heterogeneity in resistance mechanisms would have a correlation with TTF of EGFR-TKIs.
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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: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-091 - Digital PCR Analysis of Plasma Cell-Free DNA as a Noninvasive Detection of the Drug Resistance Mechanisms in EGFR Mutant NSCLC (ID 2613)
09:30 - 17:00 | Author(s): K. Nishio
- Abstract
Background:
Although the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have shown dramatic effects against EGFR mutant non-small-cell lung cancer (NSCLC), patients demonstrate resistant by various mechanisms, such as second-site point mutation that substitutes methionine for threonine at position 790 (T790M) in EGFR, and amplification of mesenchymal-epithelial transition (MET) proto-oncogene and human epidermal growth factor receptor 2 (HER2). With the development of overcoming resistance to EGFR-TKIs, identification of the mechanisms of drug resistance is urgently needed. However, tumor samples for detecting the resistant mechanisms were not easily available in patients with EGFR mutation-positive NSCLC relapsed after EGFR-TKIs treatment. Here, we examined the correlation of T790M mutation, activating EGFR mutations, HER2 amplification, and MET amplification in relapsed NSCLC patients between plasma and tumor samples using digital PCR assay as an alternative and noninvasive method.
Methods:
A total of 18 patients obtained pairs of tumor and blood samples after resistance to EGFR-TKI treatment were enrolled in this study. T790M mutation, activating EGFR mutations, MET amplification, and HER2 amplification in relapsed NSCLC patients after EGFR-TKIs treatment were analyzed by digital PCR.
Results:
Digital PCR analysis of T790M mutation in plasma had a sensitivity of 81.8% and specificity of 85.7%, with the overall concordance between plasma and tissue samples 83.3%. Analysis of primary active mutation in plasma showed inconsistent results with lower sensitivity of 66.7% and concordance of 70.6% compared to those of T790M mutation. MET gene copy number gain of tumor DNA by digital PCR was observed in three patients. Of these patients, one patient exhibited positive for MET amplification by FISH, whereas no patient demonstrated MET and HER2 copy number gain in plasma DNA.
Conclusion:
Digital PCR analysis in plasma is feasible and accurate method for detecting the T790M mutation in NSCLC resistance to EGFR-TKIs treatment.
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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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P3.04-032 - Clinical Applications of Next Generation Sequencing on Therapeutic Decision-Making in Lung Cancer (ID 1007)
09:30 - 17:00 | Author(s): K. Nishio
- Abstract
Background:
The identification of driver mutations, such as epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), have already been successfully translated into clinical practice. The clinical implementation of genomic profiling for NSCLC with high-throughput and multiplex genotyping tests is thus warranted in order to prioritize appropriate therapies for individual patients.
Methods:
The present study has recruited lung cancer patients at Kinki University Hospital from June 2013. To screen patients with lung cancer for genetic alterations relevant to novel molecular-targeted therapeutics, we have applied a Ion AmpliSeq RNA Fusion Lung Cancer Research Panel to detect known fusion transcripts such as ALK, ROS1, RET, and NTRK1 rearrangements simultaneously in a RNA sample obtained from FFPE lung cancer tissues. Deep sequencing was also performed using the Ion AmpliSeq Colon and Lung Cancer Panel. There were two co-primary endpoints for this study. First, we assessed the percentage of patients with additional therapy options uncovered by detecting potentially actionable genetic alterations. Second, we evaluated the percentage of patients who actually received genotype-directed therapy.
Results:
From June 2013, one hundred ten patient tumor samples were sequenced with these assays, and 104 (95%) patients received the results of Ion AmpliSeq Colon and Lung Cancer Panel and 106 (96%) patients received the results of the Ion AmpliSeq RNA Fusion Lung Cancer Research Panel with a >90% success rate for genotyping. An actionable driver alteration was detected in 43 (39%) of tumors from patients, leading to use of a targeted therapy in 23 (21%).
Conclusion:
Multiplexed genomic testing can aid physicians in matching patients with targeted treatments and appropriate clinical trials.
