Lung Cancer 117 (2018) 1–6

Contents lists available at ScienceDirect

Lung Cancer
journal homepage: www.elsevier.com/locate/lungcan

High ratio of T790M to EGFR activating mutations correlate with the T

osimertinib response in non-small-cell lung cancer
Ryo Ariyasua, Shingo Nishikawaa, Ken Uchiboria,b,c, Tomoko Oh-harab, Takahiro Yoshizawaa,
Yosuke Dotsua, Junji Koyamaa, Masafumi Saikia, Tomoaki Sonodaa, Satoru Kitazonoa,
⁎
Noriko Yanagitania, Atsushi Horiikea, Naohiko Inasec, Kazuo Kasaharad, Makoto Nishioa, ,
⁎
Ryohei Katayamab,
a
Department of Thoracic Medical Oncology, The Cancer Institute Hospital, Japanese Foundation for Cancer Research, 3-8-31, Ariake, Koto-ku, Tokyo, 135-8550, Japan
b
Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31, Ariake, Koto-ku, Tokyo, 135-8550, Japan
c
Department of Respiratory Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-
8510, Japan
d
Respiratory Medicine, Kanazawa University Faculty of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Objectives: Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor
EGFR that can overcome resistance due to the Thr790Met (T790M) mutation. However, osimertinib occasionally
T790M shows limited efficacy in a small population of patients. We investigated the correlation between the ratio of
Osimertinib T790M to EGFR activating mutation and the response to osimertinib.
ddPCR
Materials and methods: Between April 2016 and April 2017, 44 patients started osimertinib therapy at the Cancer
Institute Hospital of the Japanese Foundation for Cancer Research. We performed EGFR mutation analysis of
cytological samples from 33 patients using droplet digital PCR. We calculated the ratio of T790M to EGFR
activating mutations and correlated it with the systemic response to osimertinib.
Results: In tumors from the 33 patients, the average ratio of T790M to EGFR activating mutations was 0.420.
Twenty-one of the 33 patients had tumors with a T790M ratio of ≥0.4. The osimertinib response rate was
significantly higher (92.3%) in patients with a T790M ratio of ≥0.4 than in those with a T790M ratio of < 0.4
(52.6%; p = 0.0237). We examined the correlation between the T790M ratio and the tumor reduction rate and
obtained a coefficient of r = 0.417 (p = 0.0175). In patients with a T790M ratio of ≥0.4, the median pro-
gression-free survival was 355 days, which was longer, but not significant, than that in patients with a T790M
ratio of < 0.4 (median: 264 days). In patients with a T790M ratio of ≥0.4, the median treatment duration from
first-line therapy onward was 931 days, which was significantly longer than that in patients with a T790M ratio
of < 0.4 (median, 567.5 days) (p = 0.044).
Conclusion: The T790M ratio to EGFR activating mutation in tumor may correlate with the response to osi-
mertinib, and patients with a higher T790M ratio have a longer treatment history.

1. Introduction to EGFR and is detected in approximately 50–60% of patients with

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-
TKIs) such as gefitinib, erlotinib, and afatinib strongly suppress the
growth of EGFR-mutant non-small-cell lung cancer (NSCLC) cells [1,2]
and prolong the progression-free survival (PFS) of EGFR-mutant NSCLC
patients. However, cancer cells inevitably acquire resistance to EGFR-
TKIs, and the disease progresses within approximately 1 year in most
patients [3–6]. One of the mechanisms of resistance is the secondary
point mutation Thr790Met (T790M), which impairs the binding of TKIs
acquired EGFR-TKI resistance [7–9]. Osimertinib, a third-generation
EGFR-TKI, was developed to overcome resistance by covalently binding
to T790M-mutated EGFR [10]. Osimertinib showed greater efficacy
than platinum-based chemotherapy in T790M-positive advanced
NSCLC patients in a phase 3 clinical trial [11]. However, the response
rate in the trial was approximately 71%, indicating that not all patients
benefit from osimertinib.
Tumor heterogeneity and drug resistance have been reported to be
important in cancer treatment [12,13]. Several mechanisms of

⁎
Corresponding authors.
E-mail addresses: mnishio@jfcr.or.jp (M. Nishio), ryohei.katayama@jfcr.or.jp (R. Katayama).

https://doi.org/10.1016/j.lungcan.2017.12.018
Received 3 October 2017; Received in revised form 27 December 2017; Accepted 28 December 2017
0169-5002/ © 2017 Elsevier B.V. All rights reserved.
R. Ariyasu et al.
resistance to EGFR-TKIs, such as MET amplification, PIK3CA mutation,
transformation to small-cell lung cancer, and epithelial-to-mesench-
ymal transition, have been described, moreover, some cancer patients
have multiple resistance mechanisms functioning simultaneously
[12,13]. Past report showed that the ratio of T790M to EGFR activating
mutation in cell samples or plasma samples may serve as a predictive
biomarker of response to rociletinib, another third-generation EGFR-
TKI [14,15]. Therefore, we hypothesized that patients who did not re-
spond to osimertinib have subclones with resistance mechanism(s)
other than T790M and have a low relative ratio of T790M to EGFR
activating mutations. In such cases, a correlation between the response
to osimertinib and the T790M ratio in tumors would be expected.
Therefore, we quantified the T790M ratio using droplet digital PCR
(ddPCR) and examined the correlation between the ratio and the re-
sponse to osimertinib.
2. Materials and methods
2.1. Patient selection
Between April 2016 and April 2017, 44 NSCLC patients started
osimertinib therapy in the Department of Thoracic Medical Oncology at
the Cancer Institute Hospital of the Japanese Foundation for Cancer
Research. Of the cytological samples obtained from each patient before
osimertinib treatment, 34 yielded sufficient quantities of DNA for
cobas® EGFR Mutation Test and ddPCR analysis (one patient’s cytolo-
gical sample tested negative for the T790M mutation using the cobas®
EGFR Mutation Test; five patients tested with plasma samples and had
no cytological samples; there were insufficient quantities of DNA
available from 5 patient samples after cobas® EGFR Mutation Test, and
they were therefore excluded from the ddPCR analysis). In addition, for
comparison, we performed ddPCR analysis using DNA from 25 NSCLC
patients who underwent cytological analysis between September 2016
and May 2017 after acquiring resistance to EGFR-TKIs with no T790M
detection by the cobas® EGFR Mutation test. All patients underwent
computed tomography every two to three months with a few excep-
tions. All patients provided written informed consent, and this study
was approved by the Institutional Review Board of the Cancer Institute
Hospital of Japanese Foundation for Cancer Research.
2.2. ddPCR and T790M ratio measurement
DNA was extracted from cytological samples using the cobas® EGFR
Mutation Test kit (Roche Molecular Systems Inc. New Jersey, USA).
DNA concentration was measured using the Qubit dsDNA HS Assay kit
(Invitrogen, Life Technologies, CA, USA) on a Qubit 2.0 Fluorometer
(Invitrogen, Life Technologies, CA, USA). For PCR amplification, 10 ng
of DNA was used (if the concentration of template genomic DNA was
low, 4 μL of DNA containing a minimum of 0.8 ng was included in the
final 20 μL reaction mixture). Each ddPCR reaction mixture and 70 μL
of droplet generation oil were loaded into a droplet generator (Bio-Rad
Laboratories, Hercules, CA, USA). Primers and probes for the detection
of EGFR exon 19 deletion, L858R, and T790M were obtained from Bio-
Rad. Cycling conditions for PCR included an initial incubation at 95 °C
for 10 min, followed by 40 cycles of denaturation at 94 °C for 30 s and
extension at 55 °C for 60 s, and finally enzyme inactivation at 98 °C for
10 min. A negative control containing human reference genomic DNA
(Promega, Madison, WI, USA), positive control DNA known to harbor
EGFR activating mutations, and a non-template control were included
in every run. PCR samples were read with a QX200 Droplet Reader
(Bio-Rad) as per the manufacturer’s instructions. Analysis of ddPCR
data for allele calling was performed with QuantaSoft Analysis Pro
version 1.0.596 (Bio-Rad). Fractional abundance of the activating mu-
tations (exon 19 deletion and L858R) and T790M were measured, and
the ratio of T790M to activating mutations was calculated (Fig. 1).
Assays were considered positive if there were ≥10 copies per reaction
2
Lung Cancer 117 (2018) 1–6
(20 μL) based on false-positive droplet counts from human reference
genomic DNA (Supplemental Table 1). To validate the analysis of the
ratio of T790M to EGFR activating mutations, we performed ddPCR
using samples containing EGFR mutant DNA with known allele fre-
quencies (Riken Genesis, Tokyo, Japan) (Supplemental Figure).
2.3. Quantitative PCR (qPCR)
EGFR and MET gene copy was measured and calculated using
FastStart Essential DNA Green Master on the LightCycler 96 platform
(Roche), according to the manufacturer’s instruction. The following
PCR primers were used.
EGFR exon 18: forward primer 5′-CTTGTGGAGCCTCTTACACC-3′,
reverse primer 5′-AATTCAGTTTCCTTCAAGATCC-3′.
MET exon 2: forward primer 5′-TGGTGCAGAGGAGCAATGG-3′, re-
verse primer 5′-ACGCTGCCACAGCTAATGAG-3′.
LINE-1: forward primer 5′-AAAGCCGCTCAACTACATGG-3′, reverse
primer 5′-TGCTTTGAATGCGTCCCAGAG-3′.
The optimized annealing temperature was set at 60 °C. We defined
the fold change in EGFR to LINE-1 higher than four-fold as an “ampli-
fication” considering the past reports [16]. To validate qPCR analysis,
we performed qPCR using genomic DNA from HCC827 (human EGFR
mutant lung cancer cell line with EGFR amplification) and MKN45
(human gastric cancer cell line with MET amplification) mixed with
human reference genomic DNA (Promega).
2.4. Statistical analysis
We evaluated the systemic response to osimertinib using the
Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 retro-
spectively. And we evaluated the tumor reduction rate from baseline in
target lesions quantified by RECIST methodology. We used Fisher’s
exact and chi-square tests for categorical comparison of data as ap-
propriate, based on group number, and compared differences in con-
tinuous data using the Mann–Whitney U test. We performed Pearson’s
correlation test to examine the relationship between variables. The PFS
and time to treatment failure (TTF) in the two groups were represented
using the Kaplan–Meier method and compared using the log-rank test.
All p-values were based on a two-sided hypothesis. We performed all
statistical analyses using the JMP statistical software package 13 (SAS
Institute Inc., Cary, NC, USA).
3. Result
We first compared the validity of detecting EGFR mutations using
ddPCR and the cobas® EGFR Mutation Test. All DNA samples testing
positive for the T790M mutation using the cobas® method also showed a
positive result using ddPCR. Three out of 26 samples testing negative
for T790M using the cobas method showed a positive result using
ddPCR (Supplemental Table 2). These results suggest that ddPCR is not
inferior to the cobas method. To validate the analysis of the ratio of
T790M to EGFR activating mutations, we performed ddPCR using
samples containing EGFR mutant DNA with known allele frequencies.
The results showed that even at 1% allele frequencies, the ratio of
T790M to EGFR activating mutation was constant (Supplemental
Table 3 and Supplemental Fig. 1).
Next, we evaluated the validity of qPCR for EGFR and MET ampli-
fication. We could detect EGFR and MET amplification with 3:7 mixture
of positive control DNA and human reference genomic DNA which
mimic 30% tumor cell contents in the tissue samples (Supplemental
Fig. 2). Therefore, we evaluated EGFR and MET amplification with
qPCR using genomic DNA from 18 out of 34 cases (we were not able to
perform qPCR with the remaining 16 cases because of the limited
amount of the remaining samples). One case had EGFR amplification
(approximately 14-fold) and no cases had MET amplification (MET
amplification levels were under two-fold in all cases) (Supplemental
R. Ariyasu et al. Lung Cancer 117 (2018) 1–6

Fig. 1. The calculation of T790M ratio. Fractional abundance of activating mutation and T790M were measured with droplet counts by ddPCR. And the ratio of T790M to activating
mutation was calculated.

Fig. 3). EGFR amplification might skew the ratio of T790M to EGFR Table 1
activating mutation, so we exclude the case with EGFR amplification Patients characteristics.
from subsequent analysis.
N (%)
Results of ddPCR analysis using DNA from 33 EGFR-TKI-resistant
patient biopsy specimens showed that the average ratio of T790M to Total 33 (100%)
EGFR activating mutation was 0.420 (median: 0.350, range: Age (median, range) 67 (41–85)
Sex
0.099–1.0). We divided the patients based on a threshold T790M ratio
Females 24 (72.7%)
of 0.4 considering the average T790M ratio. Twenty-one of the 33 pa- Males 9 (27.3%)
tients (63.6%) had tumors with a T790M ratio of ≥0.4. (In one patient, Smoking history
the fractional abundance of EGFR activating mutations was 8.49% and Yes 10 (30.3%)
that of the T790M mutation was 10.6%, resulting in a T790M ratio of No 23 (69.7%)
Performance Status
over 1.0, but we considered the T790M ratio to be 1.0. In another pa-
0 10 (30.3%)
tient, the fractional abundance of activating mutations was un- 1 21 (63.6%)
detectable due to the L861Q mutation and that of the T790M mutation 2 2 (6.1%)
was 80.0%, but we assumed the T790M ratio to be 0.8 on the pre- EGFR activating mutation
Exon 19 deletion 15 (45.5%)
sumption that the activating mutation abundance was 100%).
L858R 17 (51.5%)
Patient characteristics are shown in Table 1. The median age was 67 L861Q 1 (3.0%)
years old, 27.3% of patients were male, 30.3% had a history of
smoking, 93.9% were performance status 0 or 1, 45.5% had EGFR exon

3
R. Ariyasu et al. Lung Cancer 117 (2018) 1–6

Table 2
The systemic response to osimertinib assessed using RECIST v1.1. (SD, stable disease; PD,
progressive disease; PR, partial response).

The ratio of T790M < 0.4 The ratio of T790M P value

(N = 19) ≥0.4 (N = 13)

ORR 52.6% 92.3%

PR (N) 10 12 0.024
SD or PD 9 1
(N)

Fig. 3. Progression free survival (PFS) analysis. In patients with a T790M ratio of ≥0.4,
the median PFS was 355 days, and in patients with a T790M ratio of < 0.4, the median
PFS was 264 days.

Table 3
The difference of patient characteristics and prior treatment between the two groups.

The ratio of The ratio of P value

T790M < 0.4 T790M ≥0.4
Fig. 2. Pearson correlation analysis of the relationship between the T790M ratio and the
(N = 20) (N = 13)
tumor reduction rate from baseline. The analysis yielded a correlation coefficient of
r = 0.417 (p = 0.0175). Age (median, range) 67.5 (43–85) 66 (41–83) 1.00
Sex (Females/Males) 12/8 12/1 0.056
Smoking history (Yes/No) 6/14 4/9 1.00
19 deletion, 51.5% had EGFR L858R.
Performance Status (0/1/2) 5/15/0 5/6/2 0.101
The systemic response rate to osimertinib was evaluated in 32 pa- EGFR activating mutation 9/11/0 6/6/1 0.436
tients (one patient had no measurable target lesion) and it was 68.8%. (exon 19 deletion/
In patients with a T790M ratio of ≥0.4, the systemic response rate was L858R/L861Q)
92.3% (12/13). On the other hand, in patients with a T790M ratio History of treatment
Number of treatment 8/6/6 2/3/8 0.168
of < 0.4, the response rate was 52.6% (10/19). The response rate was lines (1/2/≥3)
significantly higher in patients with a T790M ratio of ≥0.4 (p = 0.024) gefitinib 13 9
(Table 2). Pearson correlation analysis was performed to examine the erlotinib 7 9
relationship between the T790M ratio and the tumor reduction rate afatinib 3 5
cytotoxic chemotherapy 9 9
from baseline. The analysis yielded a correlation coefficient of
Duration of treatment from 567.5 (256–1922) 931 (139–2887) 0.043
r = 0.417 (p = 0.0175) (Fig. 2). In patients with a T790M ratio of 1st line therapy (median
≥0.4, the median PFS was 355 days, which was longer, but not sig- days, range)
nificant, than that in patients with a T790M ratio of < 0.4 (median:
264 days) (p = 0.273) (Fig. 3). Patients with a T790M ratio of ≥0.4
showed a trend toward a longer median TTF, which was not statistically 4. Discussion
significant compared to that in patients with a T790M ratio of < 0.4
(not reached vs. 287 days; p = 0.389). In this study, we found that the detection sensitivity of T790M using
We examined differences in patient characteristics and prior treat- ddPCR was not inferior to that using the cobas® EGFR Mutation Test (we
ment between the two groups, which are shown in Table 3. The number found the T790M mutation using ddPCR in three patient samples; the
of males was higher in patients with a T790M ratio of < 0.4, and all mutation was negative using the cobas test; this might have been due to
patients with a T790M ratio of < 0.4 had a performance status of 0 or 1, the cut-off value used). Therefore, we inferred that ddPCR is also useful
but there was no statistically significant difference between the two for detecting the EGFR T790M resistance mutation.
groups including other factors. Tumors with a T790M ratio of ≥0.4 The objective response rate to osimertinib in all of the T790M po-
were found in 40.9% (9/22) of gefitinib-treated patients, 56.3% (9/16) sitive patients was 68.8%, which is equivalent to the results in phase 3
of erlotinib-treated patients, 62.5% (5/8) of afatinib-treated patients, clinical trial. And, in this study, we found that EGFR-TKI resistant pa-
and 50.0% (9/18) of cytotoxic chemotherapy-treated patients. In pa- tients having tumors with a T790M ratio of ≥0.4 had better responses
tients with a T790M ratio of ≥0.4, the median duration of treatment to osimertinib than those having tumors with a T790M ratio of < 0.4.
from first-line therapy onward was 931 days, which was significantly Tumor heterogeneity has been reported, and resistant tumors with
longer than that in patients with a T790M ratio of < 0.4 (median: multiple different acquired resistance mechanisms are often detected in
567.5 days; p = 0.044). individual patients [12,13]. In fact, we showed that the T790M ratio to
EGFR activating mutation varied in each NSCLC patient. Clones lacking
the T790M mutation are likely to have other resistance mechanisms
such as MET amplification, cMET or HGF overexpression, ERBB2

4
R. Ariyasu et al.
amplification, PIK3CA mutation, transformation to small-cell lung
cancer, and epithelial-to-mesenchymal transition [12,13,17–19], and
such clones would not respond to osimertinib. Therefore, the response
rate to osimertinib treatment is likely to correlate with the T790M ratio
in tumor cells. Currently, osimertinib is administered based on the ex-
istence of T790M but not its ratio to EGFR activating mutation. How-
ever, if tumors with a low T790M ratio often show limited responses to
osimertinib, it would be preferable to consider this fact in advance and
formulate future treatment strategies including cytotoxic chemotherapy
while patients are still on osimertinib.
We sought to identify patients likely to have a higher T790M ratio in
their tumors. In our study, 62.5% patients with a history of afatinib
treatment had tumors with a T790M ratio of ≥0.4. This percentage was
slightly higher than that in patients with other treatment histories, but
the difference was not significant. On the other hand, we found that
patients with long treatment durations from first-line therapy onward
had a high T790M ratio. It has been reported that some EGFR-mutant
lung cancer cells have intrinsic resistance mechanisms [13,20]. Patients
with short treatment histories may have subclones with intrinsic EGFR-
TKI resistance mechanisms other than T790M. Therefore, after patients
acquire the T790M mutation, subclones with other resistance me-
chanisms might also proliferate, resulting in a relatively low T790M
ratio.
Because of tumor heterogeneity, individual cytological samples may
represent a part of the tumor different from other parts in the entire
tumor [19,21]. Recent developments allow the evaluation of EGFR
mutations by analyzing tumor cell-free DNA in plasma, which might be
a better reflection of cells from the entire tumor and help predict
treatment response and prognosis [22,23]. However, the low sensitivity
of plasma cell-free DNA is a problem, and analyzing DNA derived di-
rectly from tumor cells is sometimes necessary [24]. Therefore, the
quantification of the T790M ratio in tumor biopsy samples would be
still important in some clinical situations.
This study was limited by a few factors. First, the number of patients
was small and this was a retrospective study, which may have influ-
enced the results. Second, we used cytological samples to analyze DNA.
Histological samples are considered more appropriate for the cobas®
EGFR Mutation Test, but it has also been reported that the test can
detect mutations from cytology samples [25]. Third, to validate the
mutation ratio analysis, it would have been ideal to examine various
concentration combinations of mutant DNA mixed with wild-type DNA.
Instead, we analyzed the fractional abundance of T790M twice to
confirm our results and support the reliability of our data. Forth, if
patients have co-existent resistant mechanisms other than T790M, they
may not respond to osimertinib regardless of T790M ratio. Moreover,
EGFR amplification might influence the calculation of the ration of
T790M to EGFR activating mutations. Therefore, we evaluated two
major resistant mechanisms other than T790M (MET amplification and
SCLC transformation) and EGFR amplification in this analysis [26,27].
Among 18 cases, one patient had EGFR amplification, and no cases had
MET amplification, all patients diagnosed as adenocarcinoma cytolo-
gically before osimertinib administration. We excluded one patient with
EGFR amplification from the analysis of the T790M ratio. However, we
could not evaluate EGFR and MET amplification in all cases due to the
limited amount of the remaining samples and we could not fully eval-
uate the mechanism which might reduce the response of osimertinib in
case with high T790M ratio. It would be preferable to evaluate the
detailed resistance mechanisms including possible co-existent resistant
mechanism in future research.
5. Conclusion
A positive correlation between the T790M ratio to EGFR activating
mutation in tumor cells and the response to osimertinib was observed in
EGFR-T790M resistance mutation-positive NSCLC patients using
ddPCR, and patients with a longer treatment history were more likely to
5
Lung Cancer 117 (2018) 1–6
have a higher T790M ratio.
Conflict of interest
M. Nishio received research funding from Novartis, ONO
Pharmaceutical, Chugai Pharmaceutical, Bristol-Myers Squibb, TAIHO
Pharmaceutical, Eli Lilly, Pfizer, Astellas Pharma, AstraZeneca, and
received honorarium from Pfizer, Bristol-Myers Squibb, ONO
Pharmaceutical, Chugai Pharmaceutical, Eli Lilly, TAIHO
Pharmaceutical, and AstraZeneca. A. Horiike received research funding
from Chugai Pharma, Quintiles, MSD, Daiichi Sankyo and received
honorarium from Pfizer, Chugai Pharma, Eli Lilly, AstraZeneca. N.
Yanagitani is a consultant of Chugai Pharmaceutical. R. Katayama re-
ceived research funding from TAIHO Pharmaceutical, Fujifilm. All
other authors declare no conflict of interest.
Funding source
This study was supported in part by MEXT/JSPS KAKENHI grant
number JP17H06327 (to R. Katayama), JP16H04715 (to R. Katayama),
the grant from the MHLW/AMED grant number 17cm0106203h0002
and 17ck0106231h0002 (to R. Katayama), and the grant from the
Vehicle Racing Commemorative Foundation (to R. Katayama).
Acknowledgements
The authors thank Dr. Siew-Kee Low (Cancer Precision Medicine
Center, Japanese Foundation for Cancer Research, Japan) and Dr.
Yukinori Yatsuda (Bio-Rad Laboratories) for technical advice.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.lungcan.2017.12.018.
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