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K. Nishio
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MA08 - Treatment Monitoring in Advanced NSCLC (ID 386)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:R. Perez-Soler, T. Reungwetwattana
- Coordinates: 12/06/2016, 11:00 - 12:30, Lehar 3-4
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MA08.10 - Detection of the T790M Mutation of EGFR in Plasma of Advanced NSCLC Patients with Acquired Resistance to EGFR-TKI (WJOG8014LTR) (ID 5377)
12:06 - 12:12 | Author(s): K. Nishio
- Abstract
- Presentation
Background:
NSCLC patients with activating mutations of the EGFR initially respond well to TKIs, but about half such patients develop TKI resistance through acquisition of a secondary T790M mutation. Whereas next-generation EGFR-TKIs have been developed to overcome T790M-mediated resistance, performance of a second tumor biopsy to assess T790M mutation status can be problematic.
Methods:
We developed and evaluated liquid biopsy assays for detection of TKI-sensitizing and T790M mutations of EGFR by droplet digital PCR (ddPCR) in EGFR mutation–positive patients with acquired EGFR-TKI resistance.
Results:
A total of 260 patients was enrolled between November 2014 and March 2015 at 29 centers for this West Japan Oncology Group (WJOG 8014LTR) study. Plasma specimens from all subjects as well as tumor tissue or malignant pleural effusion or ascites from 41 patients were collected after the development of EGFR-TKI resistance. All plasma samples were genotyped successfully and the results were reported to physicians within 14 days. TKI-sensitizing and T790M mutations were detected in plasma of 120 (46.2%) and 75 (28.8%) patients, respectively. T790M was detected in 56.7% of patients with plasma positive for TKI-sensitizing mutations. For the 41 patients with paired samples obtained after acquisition of EGFR-TKI resistance, the concordance for mutation detection by ddPCR in plasma compared with tumor tissue or malignant fluid specimens was 78.0% for TKI-sensitizing mutations and 65.9% for T790M.
Conclusion:
Noninvasive genotyping by ddPCR with cell-free DNA extracted from plasma is a promising approach to the detection of gene mutations during targeted treatment.
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P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.02-082 - The Feasibility of Cell-Free DNA Sequencing for Mutation Detection in Non–Small Cell Lung Cancer Was Detemined by Tumor Volume (ID 6001)
14:30 - 14:30 | Author(s): K. Nishio
- Abstract
Background:
Targeted therapeutics such as tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) have recently been introduced into clinical practice for individuals with NSCLC positive for actionable mutations of EGFR or ALK fusions, respectively. Molecular profiling that is able to predict the response to such drugs has thus become an important therapeutic strategy, allowing selection of the most appropriate treatment for individual patients.
Methods:
Matched lung cancer tissue and serum specimens were collected from 150 patients who underwent surgery at Tokyo Medical University Hospital from January 2013 to July 2014. All tissue samples were stored at –80°C until analysis. Tumor DNA and cfDNA samples were subjected to analysis with next-generation sequencing (NGS) panels for mutation detection.
Results:
All tumor DNA samples were successfully sequenced with the Ion Proton platform. The median read number per amplicon was 15,632. We identified TP53 mutations in 58 cases (38.7%); EGFR mutations in 56 (37.3%); KRAS mutations in 15 (10.0%); CTNNB1 mutations in 7 (4.7%); ERBB2, PIK3CA, BRAF, and PTEN mutations in 3 each (2.0%); and ERBB4, MET, ALK, FGFR2, NRAS, AKT1, and FBXW7 mutations in 1 each (0.7%). No mutation was detected in 22.0% (33/150) of the samples. Serum cfDNA was extracted for all 150 patients, with a median yield (copy number) of 4936 (range, 572 to 373,658). A total of 149 of the 150 (99.3%) cfDNA samples were successfully sequenced with the Ion Proton platform, with sequencing failure being due to an insufficient read number per amplicon in the one unsuccessful case. The median read number per amplicon for the 149 successfully sequenced cfDNA samples was 33,982.
Conclusion:
These results suggested that detection of mutations in cfDNA of patients with disease at stage IA or IB or at T2a or lower is difficult, and that the feasibility of mutation detection with cfDNA may depend on the T factor rather than the N factor. Tumor volume in the cfDNA mutation–positive group was significantly greater than that in the cfDNA mutation–negative group (159.1 ± 58.0 versus 52.5 ± 9.9 cm[3], p = 0.014). The maximum tumor diameter calculated at diagnosis was also larger in the cfDNA mutation–positive group than in the cfDNA mutation–negative group (5.3 ± 0.7 versus 4.1 ± 0.3 cm, p = 0.050). These results suggested that tumor volume is a determining factor for the feasibility of mutation detection with cfDNA.
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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-120 - EGFR T790M, L792F, and C797S Mutations as Mechanisms of Acquired Resistance to Afatinib (ID 4818)
14:30 - 14:30 | Author(s): K. Nishio
- Abstract
Background:
Afatinib is effective for lung cancers harboring common EGFR mutations, Del19 and L858R. We reported that tumors with exon 18 mutations are especially sensitive to afatinib compared with first generation (1G) EGFR- tyrosine kinase inhibitors (TKIs). However, data on the mechanisms of acquired resistance to afatinib are limited
Methods:
We established afatinib-resistant cells from Ba/F3 cells transfected with common or exon 18 (G719A and Del18) mutations and PC9 (del E746_A750), HCC4006 (del E746_A750), and 11_18 (L858R) cell lines by chronic exposure to increasing concentrations of afatinib. Separately, afatinib-resistant clones were established from above Ba/F3 cells by exposure to fixed concentrations of afatinib using N-ethyl-N-nitrosurea (ENU) mutagenesis. Re-biopsy samples from patients whose tumors acquired resistance to afatinib were collected. EGFR secondary mutations and bypass tracks were analyzed by Sanger sequence, western blot, and real time PCR.
Results:
Afatinib-resistant cells transfected with Del19, L858R, or G719A developed T790M, whereas those with Del18 acquired novel L792F mutation. ENU mutagenesis screening established 84 afatinib-resistant clones. All Del19 clones and most of the other clones acquired only T790M. However, C797S occurred in subsets of L858R, G719A, and Del18 clones. Additionally, subsets of Del18 clones acquired L792F. C797S-acquired cells were sensitive to erlotinib. L792F demonstrated intermediate resistance between T790M and C797S to both 1G and 3G-TKIs, whereas L792F was the least resistant to 2G-TKIs, particularly dacomitinib. Chronic exposure of Del18+L792F cells to dacomitinib induced additional T790M acquisition. T790M was detected in 1 of 4 clinical samples, whereas no EGFR secondary mutations were detected in afatinib-resistant PC9, HCC4006, or 11_18 cell lines. Regarding bypass tracks, IGF1R was over expressed in all of the three afatinib-resistant cell lines compared with parental cells, whereas expression of AXL and PTEN were not changed. Neither mutations in PIK3CA and BRAF nor amplification of MET and FGFR1 were detected in clinical samples and resistant cell lines.
Conclusion:
L792F and C797S, in addition to major T790M, can develop in afatinib-resistant cells, and these minor mutations appear to exhibit sensitivity to dacomitinib and erlotinib, respectively. These secondary mutations should be tested in clinical practice. Bypass track through IGF1R may be associated with acquired resistance to afatinib.
