Mutant allele frequency predicts the efficacy of EGFRTKIs in lung adenocarcinoma harboring L858R EGFR
mutation
Akira Ono, * Hirotsugu Kenmotsu, * Masakuni Serizawa, ‡ Hisao Imai, * Tetsuhiko Taira, * Tateaki
Naito, * Haruyasu Murakami, * Takashi Nakajima, ¶ Yasuhisa Ode, ** Masahiro Endo, † Nobuyuki
Yamamoto,∏ Yasuhiro Koh,‡Toshiaki Takahashi. *
*
Division of Thoracic Oncology, Shizuoka Cancer Center
Division of Thoracic Surgery, Shizuoka Cancer Center
¶ Division of Diagnostic Pathology, Shizuoka Cancer Center
† Division of Diagnostic Radiology, Shizuoka Cancer Center
‡ Division of Drug Discovery and Development, Shizuoka Cancer Center Research Institute
∏ Third Department of Internal Medicine, Wakayama Medical University
**
Akira Ono, MD
I/We have no real or apparent
conflicts of interest to report
Conflict of interest
• I have no conflict of interest
Background
• EGFR mutation tests on a commercial basis
– Cycleave PCR, Direct (Sanger) sequencing, PCR-Invader
– PNA-LNA PCR clump, ARMS
• Information from EGFR mutation testing is limited to a simple
binary “mutant”or“wild-type”classification in clinical practice
• Possible predictive factors for EGFR TKIs in mutant RGFR
lung adenocarcinoma
– BIM, cytokeratin 19, de novo T790M
– exon 20 incertion, and intratumor heterogeneiety
Cancer Discov 2011;1:352, J Thorac Oncol 2013;8:892
Ann Oncol 2014;25:423, Clin Cancer Res 2008;14:4877
Cancer Sci 2008;99:929
Background
• The relative abundance of EGFR mutation might be capable
of prediting benefit from EGFR-TKI treatment in patients
with advanced NSCLC
J Clin Oncol 2011;29:3316
• No previous study has evaluated the quantitative value of
the mutant allele frequency of the EGFR mutation
Workflow of Shizuoka Cancer Center Mutation
Study
Anonymous ID
Pathology Lab
Unlinkabe
Clinicians
Mutation
analysis
Plasma
separation
Peripheral
blood
Peripheral
blood
DNA
extraction
Registration
Office
Electronic Medical Chart
Pleural
effusion
Detection
of fusion
genes
Banking
Registration
Linkable
Biopsy
specimens
RNA
extraction
Data linking
Surgically
resected
specimens
Samples stored at -80°C
DNA
extraction
Research Institute
(Drug Discovery & Development Div)
Clinicians
Pyrosequencing
Mutations
Pyrosequencing
Gene Name
Position
AA mutant
AKT1
PTEN
HER2
Q56
K57
D67
E17
R233
exon 20
G719C/S
G719A
deletion
T790M
insertion
L858R
L861Q
G12C/S/R
G12V/A/D
G13C/S/R
G13D/A
Q61K
Q61R/L
Q61H
G466V
G469A
L597V
V600E
E542K
E545K/Q
H1047R
Q61K
Q61L/R
Q56P
K57N
D67N
E17K
R233*
insertion
DDR2
S768
S768R
G719
EGFR
exon 19
T790
exon 20
L858
L861
G12
KRAS
G13
Q61
BRAF
PIK3CA
NRAS
MEK1 (MAP2K1)
G466
G469
L597
V600
E542
E545
H1047
Q61
Nucleotide mutant
2155G>T/A
2156G>C
Single base
substitution-type
mutations
2369C>T
2573T>G
2582T>A
34G>T/A/C
35G>T/C/A
37G>T/A/C
38G>A/C
181C>A
182A>G/T
183A>T/C
1397G>T
1406G>C
1789C>G
1799T>A
1624G>A
1633G>A/C
3140A>G
181C>A
182A>T/G
167A>C
171G>T
199G>A
49G>A
697C>T
2304T>A
Pyrogram
Mutant EGFR
Wild-type EGFR
Objective
• To evaluate the predictive implications of the
frequency of L858R allele for EGFR-TKIs in
patients with advanced EGFR-mutant lung
adenocarcinoma
A Flow-diagram of the patients included in the
analysis
Of patients enrolled in the Shizuoka Lung Cancer Mutation Study from July 2011 to March 2013,
driver mutation analysis was performed in 705 patients.
102 lung adenocarcinoma patients were identified with L858R mutations
by pyrosequencing method using histological specimens.
In 48 patients EGFR mutation status was
assessed using cycleave method.
29 advanced lung adenocarcinoma patients had
EGFR L858R mutation assesed on commercial
basis testing before initial therapy
Characteristics of 48 pts assessed by cycleave
method
Characteristics
Median age (range)
n
68 (46-90)
Gender; male/ female (%)
19 (40)/ 29 (60)
Smoking status; yes/ no (%)
19 (40)/ 29 (60)
Stage; I/ II/ III/ IV (%)
Pathological specimen; surgical/ non-surgical
4 (8)/ 6 (12)/ 8 (17)/ 30(63)
14 (29)/ 34 (71)
Median mutant allele frequency of L858R (range)
18.5% (8-82)
Between cycleave and pyrosequencing; Concordance/
discordance (%)
45(94)/ 3 (6)
Receiver Operating Characteristic (ROC)
analysis
Mutant allele frequency of 9% AUC: 0.967
Characteristics of 29 pts assessed commercially
available EGFR testing
Charasteristics
n
Median age (range)
69 (47/84)
Gender; male/ female (%)
14 (48)/ 15(52)
Smoking status; yes/ no (%)
13 (45)/ 16 (55)
Stage IIIb/ IV (%)
4 (14)/ 25 (86)
ECOG PS 0/ 1/ 2/ 3 (%)
11 (38)/ 15 (52)/ 2 (7)/ 1 (3)
Treatment line of initial EGFR-TKIs;
first/ second/ third (%)
21 (72)/ 5 (17)/ 3 (10)
Median mutant utant allele frequency (range)
18% (8-63)
All specimen materials type was Formalin-Fixed Paraffin embedded (FFPE).
Progression-free survival
Frequency ＞ 9
mPFS (days)
Frequency ≦ 9
284
92
P=.0026
Conclusions
• Mutant allele frequency predicts the efficacy of EFGR-TKIs
therapy in patients with lung adenocarcinoma harboring the
L858R mutation
• These results should be evaluated and validated in a
prospective study of serial mutation burden monitoring
using highly sensitive techniques such as digital PCR
Thank you for your attention
Back-up silde
Discussion
• Major Limitation
– Small number
•
I think that the small number of patients could affect outcomes not by mutant
allele frequency but by the co-variable of the subset of patients
– Intratumor heterogeniety
•
•
There rare some previous reports on views of two sides.
heterogeniety rate (28%; Cancer Sci, 38%; JCO)
The shift in tumors from EGFR mutation status to wild-type status
observed
in that study(JCO) suggests that intura-tumor
heterogenieety
–
The shift of EGFR mutation status and found patients who archived PR were
more likely to shift EGFR mutation than those of SD,PD after cytotoxic
chemotherapy
–
It is noteworthy, some patients had low frequency and low abundance of EGFR
mutations , thus they had shifted the EGFR
muation from wild-type to
positive after cytotoxic chemotherapy
–
•
psudeheterogeniety (Yatabe; JCO)
Discussion
• Major Limitation
– Tumor content
• There rare some previous reports on views of two sides.
• low correlation of histopathological estimates of tumor content and
frequency of mutant alleles
• the estimated tumor density in cytologic samples and the frequency of
mutated alleles were well associated
• Tumor biopsy specimens containing 10% or more tumor content
evaluated by hematoxylin-eosin staining were used for this study.
Further evaluation of tumor content of each specimens is evaluating in
our pathologic division
Discussion
• Mutation? or amplification?
–
Mutation precedes the amplification and that EGFR
amplification may occur during the progressionto
gene
invasive cancer
Both mutation and amplification may be required for constitutive
activation of EGFR 19 deletion
– L858R mutation exhibited constitutive
phosphorylation of EGFR,
regardless of the absence or presence of EGFR amplification
– weak EGFR mutation signals area without amplification
may
not reach the threshold when a less sensitive
method→psudoheterogeneiety
– I considered that I would prefer to search the mutation
than
amplification in L858R mutation.
–
Discussion
• Cut-off value of mutant allele frequency
– In metastatic colorectal cancer, it is notable that patients with
tumor harboring KRAS mutations at ratio < 10%, as determined by
pyrosequencing, benefit less from anti-EGFR therapy than patients
with tumors harboring KRAS wild-type
– The cut–off value was arbitrarily assigned as 10% mutant allele
burden
– Our study defined the cut-off value more sceientifically
Discussion
• Is too much sensitivity of EGFR mutation detection
methods good?
–
May be identifies a sub-set of tumors which would be
classified falsepositive by high sensitivity methods, with
potentially implication for
the prescription EGFR-TKIs
–
Ultrasensitive molecular assays can be problematic.
If an
ultrasensitive molecular assay findings is positive
while
anassay finding conventional sensitivity is negative, the results is either
interprited as a possible false positive due to
mispriming or low crosscontamination, or as a ture positive reflecting a very small mutated
subclone
–
Technical artifacts may be seen with ultrasensitive
methods that
require experience and caution in
interpretation. In my opinion, I
considered that it is not
necessary to detect the“true positive”using
the
ultrasensitive methds
Discussion
•
2-tiered testing strategy, in which both standerd-sensitivity and high
sensitivity testing is performed and reported
→ If it could set the cut-off value using pyrocequencing
method, I
considered that it may be not necessary to perform the strategy
•
EGFR false-positive results of mutation detection may be serious problem
in a clinical course, leading to prescription of a deleterious treatment.
•
Potential applications of mutation burden
– patient selection of postoperative adjuvant therapy
– decision-making at acquisition of resistance (T790M)during EGFR-TKI therapy
Discussion
• Progression-free survival
– Progression-free survival was defined as the time from
commencement of EGFR-TKI therapy to disease progression
according to RECIST criteria or death resulting from any cause.
Patient withdrawals due to adverse events have analyzed as
censored cases. Censored point have defined the last day which
perform radiographic evaluation on the target lesion
– All these patients had preserved the effectiveness of EGFR-TKI
therapy at censored point. The follow-up time from initial EGFR-TKI
therapy to subsequent therapy of each patient who had withdrawn
were 316 days, 54 days, 406 days, and 648 days.
想定質問
• About biomarker study、the frequency EGFR mutation
– the overall detection rate of genetic alterations in our study was lower
(48% in the current study vs. 70% in Kohno et al.), especially in EGFR
mutations (35% vs. 53%). One of the most likely reasons for this
difference is that we had significantly more smokers in our study (68% vs.
51%, p < 0.0001), which probably reflects the characteristics of our local
patient cohort. Importantly, there was no significant difference in the
overall detection rate or frequency of EGFR mutations in never-smoker
patients between these studies, which supports our hypothesis that
differences in mutation rates were affected by the smoking status of the
study cohort.
• Turnaround time TAT (promptly)
– CAP/IASLC/AMP Lung Cancer Biomarkers Guideline