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ORIGINAL PAPER
Mutation abundance affects the efficacy of EGFR tyrosine kinase
inhibitor readministration in non-small-cell lung cancer
with acquired resistance
Ze-Rui Zhao • Jin-Feng Wang • Yong-Bin Lin • Fang Wang •
Sha Fu • Shu-Lin Zhang • Xiao-Dong Su • Long Jiang •
Yi-Gong Zhang • Jian-Yong Shao • Hao Long
Received: 3 November 2013 / Accepted: 4 December 2013
Springer Science+Business Media New York 2013
Abstract There is no consensus in the salvage treatment
for non-small-cell lung cancer (NSCLC) with acquired
resistance to primary epidermal growth factor receptor–
tyrosine kinase inhibitors (EGFR-TKIs). Fifty-one consecutive
EGFR-mutated NSCLC patients with TKI retreatment
after acquired resistance were enrolled in this study. The
quantitation of mutation abundance was performed by realtime
fluorescent quantitative PCR. The correlation between
mutation abundance and outcomes of readministrated TKI
was analyzed by survival analysis. Patients with high
(H) mutation abundance (24/51) had a significantly (logrank,
P\0.05) longer (5.27–2.53 months) median progression-
free survival (PFS), compared with the low
(L) abundance group (27/51), whereas the median overall
survival showed no difference (21.00–18.20 months, logrank
P = .403) between the two groups. Objective response
and disease control rates in group H and group L regarding
the second round TKI treatment were 8.3, 70.8 and 0,
48.1 %, respectively. Groupings with different mutation
abundances were significantly associated with PFS under
multivariate Cox proportional hazards regression model
[hazard ratio (HR) for group H vs. L, 0.527; P = .036].
Mutation abundance affects the efficacy of EGFR-TKIs readministration
in NSCLC with acquired resistance. The
quantitative mutation abundance ofEGFRmay be a potential
predictor for selecting optimal patients to readministrate
EGFR-TKIs after acquired resistance to primary TKI.
Keywords Epidermal growth factor receptor Tyrosine
kinase inhibitor Non-small-cell lung cancer Drug
resistance Target therapy
Introduction
Recently, several trials had confirmed the utility of epidermal
growth factor receptor–tyrosine kinase inhibitors
(EGFR-TKIs) in metastatic non-small-cell lung cancer
(NSCLC) [1, 2]. Unfortunately, most patients who initially
responded to EGFR-TKIs would show a progression disease
(PD) within 12 months [3]. Successful strategies for
conquering such acquired resistance [4] have yet to be
Ze-Rui Zhao, Jin-Feng Wang and Yong-Bin Lin have contributed
equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s12032-013-0810-6) contains supplementary
material, which is available to authorized users.
Z.-R. Zhao Y.-B. Lin X.-D. Su L. Jiang Y.-G. Zhang
H. Long (&)
State Key Laboratory of Oncology in Southern China,
Department of Thoracic Surgery, Sun Yat-Sen University Cancer
Center, Guangzhou, China
e-mail: longhao@sysucc.org.cn
Z.-R. Zhao Y.-B. Lin F. Wang S. Fu X.-D. Su
L. Jiang Y.-G. Zhang J.-Y. Shao H. Long
Lung Cancer Research Center, Sun Yat-Sen University,
Guangzhou, China
e-mail: shaojy@sysucc.org.cn
J.-F. Wang
Department of Cardiothoracic Surgery, The Third Affiliated
Hospital of Guangzhou Medical University, Guangzhou, China
F. Wang S. Fu J.-Y. Shao
State Key Laboratory of Oncology in Southern China, and
Department of Molecular Diagnostics, Sun Yat-Sen University
Cancer Center, Guangzhou, China
S.-L. Zhang
Department of Surgical Oncology, The Third Affiliated Hospital
of Guangzhou Medical University, Guangzhou, China
123
Med Oncol (2014) 31:810
DOI 10.1007/s12032-013-0810-6
established, albeit several resistant mechanisms like secondarily
EGFR mutations (e.g., T790 M) or other pathways
to EGFR genotypes were reported [5].
Previous studies have demonstrated the coexistence of
EGFR-mutated as well as wild-type cells in one specimen
[6, 7]. Another study also suggested that TKI-sensitive
cells may still exist even after a certain proportion of original
mutated cells had transformed into resistant cells,
thus, readministration of TKI could still yield clinical
benefit [8]. In current practice, TKI rechallenge is one of
the most common therapeutic approaches though the progression-
free survival (PFS) varies among studies, and
most of the results are barely beyond 2 months [9]. Since
there is currently no standard therapy for TKI failure, we
designed this study to find optimal patients that will benefit
from TKI readministration.
In this study, we detected the copy numbers of mutated
(mt)-EGFR gene and wild-type (wt)-EGFR gene to calculate
mt-EGFR abundance by real-time fluorescent quantitative
polymerase chain reaction (qPCR). Consequently,
we sought to explore the relationship between mutation
abundance and the efficacy of readministrated TKI after
resistance.
Materials and methods
Patient selection
Three hundred and thirty five consecutive patients with
histologically or cytologically confirmed recurrent or
metastatic NSCLC in Sun Yat-Sen University Cancer
Center were reviewed retrospectively. Patients with
acquired resistance to EGFR-TKIs, according to Jackman
et al.’s [4] criteria were enrolled: (1) previously received
treatment with a single-agent EGFR-TKI; (2) harbored a
drug sensitivity associated mutation site(e.g., G719X, exon
19 deletion, L858R, L861Q); (3) responded (C6 months)
to initial gefitinib or erlotinib treatment [complete response
(CR), partial response (PR), or stable disease (SD)
according to the response evaluation criteria in solid
tumors (RECIST) [10]]; (4) PD while on continuous
EGFR-TKI within the last 30 days. Patients were categorized
as non-smokers (B100 lifetime cigarettes) or smokers
([100 lifetime cigarettes).
Patients who had retreated gefitinib (250 mg/d) or erlotinib
(150 mg/d) after the first PD were eligible for final
analysis (Fig. 1); cessation of the 2nd EGFR-TKIs therapy
was permitted only when the patients incurred unbearable
toxicity or disease progression according to version 1.1 of
the guidelines set out by RECIST Committee [10]. Evaluation
of treatment response by computer tomography was
performed after the first 4 weeks and repeated every
8 weeks. Study protocol was approved by the institutional
review boards of Sun Yat-Sen University Cancer Center.
Written informed consent was obtained from each patient.
DNA isolation
Genomic DNA was isolated using conventional techniques
with QIAamp DNA kit (Qiagen, Courtaboeuf, France).
Before genomic DNA isolation, paraffin-embedded tumor
blocks were reviewed for tumor quality and content. If the
formalin-fixed and paraffin-embedded (FFPE) tumor
Fig. 1 Enrollment and
outcomes. EFGR epidermal
growth factor receptor(EFGR),
tyrosine kinase inhibitor(TKI),
cycle threshold(Ct), real-time
fluorescent quantitative
PCR(qPCR), progression
disease(PD), glyceraldehyde
3-phosphate
dehydrogenase(GAPDH)
810 Page 2 of 8 Med Oncol (2014) 31:810
123
blocks were large, then the removal of as much non-tumor
cells and necrotic cells as possible was done to enrich
tumor cell percentages. By using a hematoxylin and eosinstained
slide, every micro-sampled specimen was crosschecked
to confirm that micro-dissected areas were not
contaminated by non-cancerous cells. If the FFPE tumor
blocks were small, like those obtained from needle biopsy,
this procedure was omitted in order to guarantee sufficient
genomic DNA isolated for qPCR.
EGFR mutational status detection
The methodology of mutation detection has been previously
described in detail [11]. After the extraction of DNA
from tumor tissues, qualitative detection of mt-EGFR was
done using a fluorescence-based, real-time detection
method [ABI PRISM 7500 Sequence Detection System
(Taqman); Perkin-Elmer Applied Biosystems, Foster City,
CA.]. The primers and probe sequences are shown in
Table 1.
Quantitative detection of mt-EGFR, wt-EGFR
After the extraction of DNA from tumor tissues, quantitative
detection of mt-EGFR, wt-EGFR and an internal reference
gene [glyceraldehyde 3-phosphate dehydrogenase
(GAPDH)] was done using qPCR (by using the ABI
PRISM 7500 Sequence Detection System Applied Biosystems,
Foster City, CA). mt-EGFR, wt-EGFR and
GAPDH standards were diluted into a series gradient
concentration for standards template: 107, 106 , 105 , 104 ,
103 and 102 copies/ll. The primers and probe sequences
used are shown in Table 1.
The standard curve method was used to determine the
number of copies of mt-EGFR, wt-EGFR and GAPDH
relative to corresponding gene standards, and quantitation
was based on standard curves established from a series
gradient concentration of mt-EGFR, wt-EGFR and
GAPDH standards. Corresponding number of copies of mt-
EGFR, wt-EGFR and GAPDH could be ascertained against
a standard curve, soon after the values of Ct for mt-EGFR,
wt-EGFR and GAPDH were measured. All calculations
were performed using the ABI PRISM 7500 System
Sequence Detection Software Version 1.4 (Applied Biosystems,
Foster City, CA).
mt-EGFR abundance calculation
Here, mt-EGFR abundance was defined as the percentage
of mt-EGFR gene in the whole EGFR gene, which was
borrowed from a concept in chemistry: abundance of elements.
Theoretically, the calculation equation of mt-EGFR
abundance is shown as the following:
Due to non-tumor cells and necrotic cells of which
EGFR genes were wt-EGFR in the samples, the mt-EGFR
abundance calculated using the original number was not
accurate. Therefore, wt-EGFR dosage in tumor cells should
not be included in non-tumor cells.
Tumor wt-EGFR = Whole wt-EGFR dosage
Non-tumor wt-EGFR dosage
In this study, the reference GAPDH gene was subtracted
from the GAPDH in tumor used to estimate the number of
copies of EGFR gene dosage of non-tumor cells and
necrotic cells in the sample. Additionally, the GAPDH in
mt-EGFR cells can be substituted by mt-EGFR dosage.
Thus, an approximate non-tumor wt-EGFR dosage justified
by subtracting the incalculable GAPDH-wt-EGFR can be
calculated as follows:
Non-tumor wt-EGFR ¼ Whole GAPDH
GAPDH mt-EGFR GAPDH-wt-EGFR
Whole GAPDH mt-EGFR dosage
Consequently, the final calculation equation of mt-
EGFR abundance can be shown as follows:
EGFR mutation abundance ¼
mt-EGFR dosage
mt-EGFR dosage t wt-EGFR dosage
EGFR mutation abundance
mt-EGFR dosage
2mt-EGFR dosage t wt-EGFR dosage whole GAPDH dosage
Med Oncol (2014) 31:810 Page 3 of 8 810
123
Statistical analysis
The initial progression-free survival (1st PFS) was defined as
the interval between the beginning of EGFR-TKI and the
progression time. Likewise, 2nd PFS was defined as the
period from the start of TKI retreatment to the date at which
disease progression or death was noted. Overall survival
(OS) was defined as the period from the start of TKI retreatment
to the date of death. Objective response rates
(ORRs) were defined as (CR ? PR)/whole patients; disease
control rates (DCRs) were defined as (CR ? PR ? SD)/
whole patients. PFS and OS were analyzed by the Kaplan–
Meier method, and the log-rank test was used to compare the
difference within groups.The comparison in different groups
was performed using the v2 test or Fisher’s exact test as
needed. Multivariate Cox proportional hazards regression
model was used to evaluate independent predictive factors
associated with 2nd PFS.Atwo-sided P value of less than .05
was considered statistically significant. All analyses were
conducted using PASW statistical software, version 18.0
(SPSS Inc., Chicago, IL).
Results
Patients’ characteristics
From May 2003 to September 2011, 51 patients were
recruited into this study. Baseline characteristics were
showed in Table 2. Only one patient (2.0 %) had a
rebiopsy after resistance. All patients finished the second
round of EGFR-TKIs therapy until a PD was documented.
EGFR mutation abundance distribution and grouping
In consideration of the fold changes of gradient concentration
for standards template, we organized the raw data using
a logarithmic transformation. Patients were classified into
two abundance groups according to previous experience
[12]: those abundanceC0.5015 as the high EGFR abundance
group (group H: with higher number of copies of the mutated
allele or fraction, as opposed to low abundance population)
and inversely, those abundance\0.5015 as the low abundance
group (group L), given that the corresponding median
abundance in this study was 0.5015 and the variable were
normally distributed (P = .006). Consequently, objects
were divided into two groups as 24 patients (47.1 %) in
group H and 27 patients (52.9 %) in group L.
Mutation status and treatment details
In total, 28 patients (54.9 %) harbored deletion mutation in
exon 19 (19 in group H vs. 9 in group L) and 23 patients
(45.1 %) had a point mutation of L858R in exon 21 (5 in
group H vs. 18 in group L), respectively (P = .002). As for
the 2nd EGFR-TKI therapy, 7 of 51 patients (13.7 %) had
switched TKI regimen (all switch from gefitinib to erlotinib:
3 in group L, 3 in H, and one patient participated in the
afatinib clinical trial in group H, respectively).
Table 1 Primers and probe sequences used for detection
Gene Content Sequences
EGFR exon 19 Primer 1 F: 50-CTGGATCCCAGAAGGTGAGAAA-30ab
R: 50-AGCAGAAACTCACATCGAGGATTT-30ab
Probe 1 (wild type) 50-HEX-AAGCAACATCTCCGAAAGCCAACAAGG-BHQ1-30a
Probe 2 (15 bp del) 50-FAM-TCCCGTCGCTATCAAGACATCTCCGAAA-BHQ1-30ab
Probe 3 (18 bp del) 50-HEX-TCGCTATCAAGGAATCGAAAGCCAAC-BHQ1-30ab
EGFR exon 21 Primer 2 F: 50-AACACCGCAGCATGTCAAGA-30ab
R: 50-CCTTACTTTGCCTCCTTCTGCAT-30ab
Probe 4 (wild type) 50-HEX-CAGTTTGGCCAGCCCAAAATCTGTG-BHQ1-30a
Probe 5 (L858R) 50-FAM-TTTGGCCCGCCCAAAATCTGT-BHQ1-30ab
Probe 6 (L861Q) 50-HEX-CACCCAGCTGTTTGGCC-BHQ1-30ab
GAPDH Primer 3 F: 50-GGTGGTGAATACCATGTACAAAGCT-30ab
R: 50-CCAACACCCCCAGTCATACG-30ab
Probe 3 50-HEX-AGTGCCCCACATGGCCGCTTC-TAMRA-30ab
FAM and HEX were reporter group of Taqman probe
BHQ1 and TAMRA were quencher group of Taqman probe
a Primer and probe used for qualitative detection of mt-EGFR
b Primer and probe used for quantitative detection of mt-EGFR
810 Page 4 of 8 Med Oncol (2014) 31:810
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No significant association between the length of time a
patient responded to the initial TKI and the likelihood that
they benefited from the readministration (independent of
high/low abundance) was found (P = .643). Additionally,
we did not find that such switching would have impacted the
outcomes [as the 2nd PFS of switched arm vs. continuation
arm in H: 3.93 months (95 % CI 2.85–5.01 months) vs.
4.40 months (95 % CI 2.73–6.07 months), log-rank
P = .824; and in L: 4.50 months (95 %CI 1.19–7.81 months)
vs. 2.53 months (95 % CI 0–6.04 months), log-rank
P = .865, respectively]. Besides the 3 patients (12.5 %, one
received three lines and two received one line) in group H
and 6 (22.2 %, two received two lines and four received one
line) in group L who had received documented post 2nd TKI
chemotherapies (P = .473), the PFS for the rest of them did
not differ significantly (H: 6.10 months 95 % CI
0–13.14 months; L: 3.93 months 95 % CI 0–9.54 months,
log-rank P = .372).
A chart illustrating the length of treatment on TKI prior
to PD, other chemo post-PD, and TKI post-PD for each
patient was shown (Supplementary Fig. 1).
Efficacy of the second round TKI
The last follow-up time was May 2013 and median followup
duration was 42.50 months from the initial TKI therapy
(range 19.77–101.13 months). Forty-seven patients
(92.2 %) exhibited PD and thirty-two patients (62.7 %,
Table 2 Patients characteristics
TKI tyrosine kinase inhibitor, CI
confidence interval, CNS central
nervous system, ECOG Eastern
cooperative Oncology Group,
PR partial response, SD stable
disease, PD progression disease
* Comparison between the two
groups, Fisher’s exact test
used, log-rank test used
Characteristic mt-EGFR high
abundance
mt-EGFR low
abundance
P*
N % N %
Age, years .229
Median 63.50 61
Range 43–79 29–76
Gender .095
Female 9 37.5 17 63.0
Male 15 62.5 10 37.0
Pathology .671
Adenocarcinoma 22 91.7 23 85.2
Non-adenocarcinoma 2 8.3 4 14.8
Smoking status .336
Smoker 8 33.3 5 18.5
Never-smoker 16 66.7 22 81.5
ECOG .195
B1 23 95.8 22 81.5
[1 1 4.2 5 18.5
Initial TKI response .546
PR 9 37.5 7 25.9
SD 15 62.5 20 74.1
Progression on initial TKI, months .616
Median 15.79 12.92
95 % CI 13.06–19.13 10.51–24.25
Types of progression .554
CNS progression 6 25.0 9 33.3
Local or non-CNS progression 18 75.0 18 66.7
Time to readministration of TKI after PD, months .757
Mean 1.21 1.45
95 % CI 0–2.53 0.49–2.41
Chemotherapy before readministration of TKI .508
At least 1 cycle 4 16.7 7 25.9
PFS median 6.97 1.53 .667
95 % CI 0.11–13.83 0.61–2.45
None 20 83.3 20 74.1
Med Oncol (2014) 31:810 Page 5 of 8 810
123
17/24 in H vs. 15/27 in L, P = .385) evidenced death in the
last date. The median 2nd PFS and OS were 3.93 months
(95 % CI 3.87–6.36 months) and 10.17 months (95 % CI
11.54–17.90 months), respectively. The difference of
median 2nd PFS in group H and L was significant
(H: 5.27 months, 95 % CI 2.36–8.18 months vs. L:
2.53 months, 95 % CI 0–5.07 months, log-rank P = .033).
However, no significant results were observed in OS
(H: 16.00 months, 95 % CI 0.55–31.45 months vs. L:
14.70 months, 95 % CI 4.80–24.60 months, log-rank
P = .033) (Table 3, Fig. 2).
Using grouping based on mutation abundance, mutation
site, smoking history, pathological differentiation, physical
status score, brain metastasis in the initial therapy, rash
condition and PD site for the failure of the 1st TKI as
variables, the multivariate Cox proportional hazards
regression model showed that only the grouping variable
was significantly associated with 2nd PFS [hazard ratio
(HR), 0.527; 95 % CI .290–.959; P = .036].
ORRs and DCRs tend to be higher in group H than in
group L (8.3 and 70.8 % vs. 0 and 48.1 %); though, the
differences were not statistically significant (P = .216 and
.154, respectively).
The most common adverse event was grade 1 or 2 rash,
which affected 10 patients (19.6 %), whereas no grade 3
skin rash was observed. Besides, no dose reduction or
discontinuation of TKI due to unbearable TKI-associated
toxicity was required.
Discussion
To the best of our knowledge, this article represents the
first demonstration of how EGFR mutation abundance
might indicate the extent of efficacy for TKI readministration
after acquired resistance of EGFR-TKIs. Our results
Table 3 Efficacy of readministrated TKI
Efficacy mt-EGFR high
abundance
mt-EGFR low
abundance
P*
N % N %
PFS (months) .033
Median 5.27 2.53
95 % CI 2.36–8.18 0–5.07
OS (months) .403
Median 21.00 18.20
95 % CI 5.97–36.03 14.70–21.70
Best response .098
PR 2 8.3 0 0
SD 15 62.5 13 48.1
PD 7 29.2 14 51.9
ORRs 2 8.3 0 0 .216
DCRs 17 70.8 13 48.1 .154
TKI tyrosine kinase inhibitor, CI confidence interval, PFS progression
free survival, OS overall survival, PR partial response, SD stable
disease, PD progression disease, ORR objective response rate, DCR
disease control rate
a Comparison between the two groups, b Fisher’s exact test used
Fig. 2 a. Progression-free survival (PFS) and b overall survival (OS) of patients in the two groups. Group H high EGFR abundance group,
Group L low EGFR abundance group, CI confidence interval
810 Page 6 of 8 Med Oncol (2014) 31:810
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show that a potential method in genomic level could be
used to select patients with higher mt-EGFR abundance as
candidates for receiving EGFR-TKI retreatment.
Salvage treatment for acquired resistance to EGFR-TKI
in patients harboring EGFR mutation with NSCLC remains
controversial even though a set of plausible mechanism to
resistance has been reported [13]. Theoretically, several
options to overcome EGFR-TKI resistance are exist (readministration
of TKIs; second-generation TKIs, e.g.,
afatinib or dacomitinib; anti-EGFR combinations, e.g.,
EGFR-TKI combined with anti-EGFR antibody or anti-
PD1 immunotherapy). Recent retrospective report showed
that retreatment of TKI might be useful for ex-responders
following a drug holiday [14]. Therefore, it is postulated
that a certain proportion of ‘‘oncogene-addicted’’ cells
might still remain even when a resistance was notified.
Several studies [8, 15, 16] reported the clinical outcomes
of a readministrated EGFR-TKIs after the primary resistance,
and the PFS and OS of these trials varied from 2.0 to
3.4 months and 11.4 to 12.0 months, respectively. Though
these differences may be partly explained by the various
enrolled criteria among trials (e.g., patients with clinical
benefit[6 months of initial EGFR-TKIs were enrolled in
Koizumi’s [15] study but[3 months in Oh’s [8] trial), a
significant and better response to TKI retreatment was
observed in those who had a PFS more than 6 months
during the initial TKI treatment [9]. Therefore, we
acknowledge the acquired resistance criteria introduced by
Jackman [4] as a reasonable rational to select patients for
the readministration of TKI in the current study.
Formation of tumor cells is supposed to generate monoclone
to multi-clones as a result of clonal evolution and
genetic/epigenetic instability [17]. As for the EGFR heterogeneity
in lung cancer, recent reports by microdissection
methods have indicated that tumors are composed of
mixed populations of mt-EGFR and wt-EGFR cells, and
patients with greater EGFR mutation frequency showed
longer PFS [6, 7], suggesting that the intratumoral heterogeneity
does indeed exist. Besides, Zhou et al. [18] found
that the 1st PFS of patients in low abundance group
(mutation negative by DNA sequencing, positive by
ARMS) was significantly shorter than that in high abundance
(mutation positive by both sequencing and ARMS).
Giving that the sensitivities to detect EGFR mutation for
sequencing and ARMS are 10 and 1 % [19], respectively,
this relative abundance may help in indicating the best
responders to TKI therapy. In our study, the sensitivity of
qPCR is about 10 % [19], which may partly explain why
no significant difference was found in 1st PFS between
groups despite a relatively longer 1st PFS was noticed in
group H. Based on all this evidence, it is speculated that
during the process of acquired resistance, a certain proportion
of mutated cells would transform into refractory
clones through various mechanisms, whereas, some other
tumor cells might remain molecularly dependent on the
EGFR pathway, which indicates that a TKI retreatment
could yield benefit. Consequently, after resistance, tumors
with higher mutation abundance may have more remaining
sensitive cells than those with lower abundance.
The mutation distribution difference in this study could
be of trivial influence to the results since no evidence has
indicated these two sites were associated with significantly
different outcomes of EGFR-TKI. Therefore, baseline
characteristics between group H and L were well balanced
in this study. Although a better outcome was reported for
patients receiving 2nd round TKI after a EGFR-TKI-free
holiday [20], we did not observe this difference as another
pilot study [15] had indicated. A similar insignificant result
was noticed regarding PD sites with 2nd PFS in this study.
Besides, we did not find a prolonged OS in group H, and
this discrepancy may be partly explained by patients that
might still display high response after switching to other
treatments as other prospective trials had indicated [21].
By removing non-tumor cells and using qPCR as an
accurate and reproducible quantization of gene copy
method, we minimized the confounding factors. However,
as only one patient had a rebiopsy after resistance, the
frequency of EGFR T790 M mutations as well as other
resistant mechanisms (mainly, activation of EGFR signaling
pathways via other aberrant molecules, e.g., c-MET
amplification or PIK3CA mutations) is unclear in the current
study. Therefore, some limitations remained as
patients harboring EGFR T790 M mutations are more
likely to benefit from prolonged TKI administration [22].
Future studies may be designed to testify the utility of a
readministrated EGFR-TKIs in TKI resistant cases with
higher EGFR mutation abundance.
Acknowledgments This work was supported by National High
Technology Research and Development Program of China (863
Program) (2012AA02A502).
Conflict of interest The authors declare that there is no conflict of
interest.
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置顶卡
沉默卡
喧嚣卡
变色卡
千斤顶
显身卡
