1. No category

Predominance of EGFR mutations responsible for resistance to tyrosine kinase inhibitors
among tumor DNAs assessed by picoliter droplet digital PCR analysis of cell-free plasma DNAs
Yoshitaka Seki1,2, Yutaka Fujiwara3, Takashi Kohno1, Erina Takai4, Kuniko Sunami3, Hidehito Horinouchi3, Shintaro Kanda3, Hiroshi Nokihara3, Shun-ichi Watanabe5, Noboru Yamamoto3, Kazuyoshi Kuwano2, Yuichiro Ohe3. 1Division
of Genome Biology and 4Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan
3Department of Thoracic Oncology and 5Department of Thoracic Surgery, National Cancer Center Hospital, Tokyo, Japan
Introduction & Objectives
Detecting mutations by two TaqMan probes Exon 19 DEL
mutations
Study cohort consisting of 8 LADC patients who received EGFR-TKI therapy
Eight patients with advanced LADCs were subjected to the cfDNA analysis(Table).
Tumor tissues from all patients were diagnosed as positive for TKI-sensitive EGFR mutations.
cfDNA was obtained after the cancers acquired resistance to EGFR-TKIs
Assessment of the predominance of T790M+ tumor cells among tumor cells. Two ddPCR assays for a cfDNA sample
Wild-type
Del
mut
L858R or T790M
mutations
M
Wild-type probe
Mutation probe
Reference probe
Wild-type probe
Fractions
obtained
RainDrop® Digital PCR System PCR mixture
C
T790M DNA
/all EGFR DNA
19DEL or L858R DNA
/all EGFR DNA
1000
Exon 19
DEL
T790M
Analysis of cell line genome DNA
Negative droplet
FAM+ droplet
VIC+ droplet
69
60
60
0
-500
1500
19DEL : 228
500
0
Wild type : 887
0
1000
2000 3000
-500
1500
Wild type : 1322
1000
500
Wild type : 599
0
1000
Wild type : 501
1000
0
T790M : 2
1000
2000
3000
0
T790M : 99
0
1000
●NSCLC with common EGFR mutation
(exon 19 deletion, L858R)
●Systemic disease progression on EGFR-TKI within 30 days
●Patients without HBV/HCV infection
cfDNAs from eight LADC patients acquiring resistance to EGFR-TKIs were subjected
to picoliter-ddPCR assay that enables us to assess fractions of EGFR DNAs with
T790M, L858R and representative exon 19 deletion mutations among all EGFR DNAs.
2000
3000
cfDNAs from 4 patients was consistent with those of their
re-biopsied tissues from the lesion acquiring resistance.
T790M mutation was deduced to have occurred in a variety of
fractions (7-97%) of tumor DNAs.
Table. Plasma cell-free DNAs subjected to picoliter-ddPCR
Patient
Age Sex
no.
cfDNA before
acquiring
resistance
cfDNA after acquiring
T790M
mutation in resistance
rebiopsied
Sensitive T790M
Sensitive T790M
T790M
tissues
mut (%) mut (%)
mut (%) mut (%) fraction
Duration
EGFR
of
Smoking
EGFR-TKI
Modes of disease
mutation Stage
(pack years)
(Best response) therapy progression
in tumor
(months)
1
62
Female Never
19DEL
IV
Erlotinib (PR)
6.6
Malignant pericarditis 5.8
Negative Positive
30.7*
15.0*
0.49
2
74
Female Never
19DEL
IV
Erlotinib (PR)
12.4
Brain metastasis
-
-
Positive
24.7*
9.5*
0.39
3
67
Female Never
L858R
IV
Gefitinib (PR)
12.0
Primary diseases and
liver metastasis
-
Positive
4.0*
0.86*
0.22
4
65
Male
Never
19DEL
IV
Erlotinib (PR)
10.0
Malignant pleuritis
3.0
Negative -
5
68
Male
Never
19DEL
IV
Erlotinib (SD)
8.6
Malignant pleuritis
-
-
-
1.1*
Negative -
6
65
Female Never
L858R
IV
Gefitinib (PR) & 6.4 &
Erlotinib (SD)
5.3
Liver and spinal
metastases
-
-
-
5.8
5.6
0.97
7
53
Female Never
19DEL
IV
Gefitinib (PR)
Bone metastases
-
-
-
16.0
1.2
0.074
8
51
Female Never
19DEL
IV
Erlotinib (SD) & 31.5 &
Gefitinib (SD)
3.2
Primary diseases and
spinal metastasis
-
-
-
10.8
Negative -
2000 3000
500
0
69
12.3
Negative Negative Negative
20
0.0
0
PC9
(n=4)
II-18
(n=4)
Exon 19
deletion
H1975
(n=4)
L858R
H1975
(n=4)
PC9
(n=9)
T790M
Analysis of cfDNA from two patients with
LADC harboring the EML4-ALK fusion
10
5.8
5
EGFR cfDNA profile of eight patients who acquired resistance Quantitative analysis using
serially diluted DNA 40
Droplet measurement
19DEL : 55
Results: All eight patients examined after acquiring resistance to
EGFR-TKIs showed positivity for TKI-sensitive mutations, and five
of them (62.5%) also for T790M mutations. The mutation status of
Patients eligibility:
FAM intensity (Arb. units)
80
ddPCR
assay
1000
500
89
No. of droplets
positive for
EGFR mutations
B
19DEL or L858R
After TKI resistance
acquisition
Before
EGFR-TKI therapy
Droplet creation
100
Thermal cycling
T790M
Fraction: T790M DNA/19DEL or L858R DNA
||
T790M tumor cells/ all tumor cells
(i.e., fraction of TKI-resistant tumor cells)
Mutant DNA (%)
A
Analysis of cfDNA from LADC patients
Validation of analysis method
Genomic DNA extracted from cell lines was used to assess the accuracy and reproducibility of
picoliter-ddPCR. The quantitative nature of the analysis was validated using serially diluted DNA
(R2 = 0.96), and the limit of detection was defined as more than 0.75% mutant alleles.
To set the threshold for calling EGFR mutations in patient cfDNA, we subjected cfDNA from two
patients with LADC harboring the EML4-ALK fusion to picoliter-ddPCR. These results revealed
that only a few droplets among millions were positive for EGFR mutations (mean = 2.5,
standard error, SE = 1.4); the threshold for a positive call was tentatively set to 10 droplets,
based on the equation: mean + 5 × SE = 9.5. Using this threshold, the rate of false-positive
droplet detection was predicted to be less than 0.0002%.
T790M DNA detected (%)
Wild-type
of Respiratory Diseases, Department of Internal Medicine, Jikei University School of Medicine, Japan
Experimental Design
VIC intensity (Arb. units)
The new generation EGFR-tyrosine kinase inhibitors (TKIs),
which suppress the kinase activity of EGFR proteins with
secondary T790M resistant mutation, are being developed
to overcome resistance to therapy using current EGFR-TKI.
Non-invasive monitoring of EGFR gene mutations
conferring sensitive (L858R and ex. 19 deletion mutations)
and T790M as resistant mutations to TKIs are inevitable for
efficient therapy of lung adenocarcinoma (LADC) using new
generation EGFR-TKIs.
We aimed to evaluate the utility of picoliter droplet digital
PCR (pdPCR)-based cell-free plasma DNA (cfDNA)
examination to assess the tumor progression status of
LADC patients against EGFR-TKI treatments.
2Division
100
Circle size:
Tumor cfDNA fraction
in total cfDNA
R² = 0.96
% of T790M
positive
% of T790M
negative
1
0.3
1
19DEL L858R T790M
all
(n=3) (n=5) (n=6) (n=14)
10
H1975 DNA mixed (%)
100
?
?
12 m
Erlotinib
7 m
#4
30.7%
#2
#5
?
Erlotinib
12 m
24.7%
Gefitinib
6m
Erlotinib
5 m
Erlotinib
1.1%
9 m
Conclusion
97%
5.8%
#7
?
0% Erlotinib 3.0%
10 m
?
0
0
5.8%
#6
22%
Erlotinib
4.0%
(-)
10
2.5
0.8
#1
#3
#8
?
Gefitinib
12 m
7.4%
16.0%
Erlotinib
32 m
Gefitinib 10.8%
3m
+ another TKI
Picoliter-ddPCR-based examination of
cfDNAs present a robust noninvasive
assessment of tumor resistant status
against EGFR-TKI treatments.
Information of the predominance of T790M
mutated DNAs among tumor DNAs might
be helpful for T790M targeted therapy.
A prospective study to validate the utility of
the picoliter-ddPCR assay is on-going on
patients receiving EGFR-TKI therapy.