ANALYTICAL AND QUANTITATIVE CYTOPATHOLOGY AND HISTOPATHOLOGY

1. 119
BACKGROUND: Droplet digital PCR (dd-PCR) is a
recently developed method with improved reliability and
sensitivity. The present study compared RT-qPCR and
dd-PCR techniques for detecting the EGFR G719S and
T790M mutations in formalin-fixed, paraffin-embedded
(FFPE) tissue samples of lung cancer and cervical cancer.
STUDY DESIGN: We collected 54 tumor samples from
cervical cancer patients and 60 lung cancer patients
with stage. The expression of EGFR in FFPE samples
was detected through immunohistochemistry assay, and
the EGFR mutation status was determined by DNA
extraction from FFPE slides followed by droplet digital
PCR.
RESULTS: The expressions of EGFR were significant-
ly increased in FFPE samples of both lung cancer and
cervical cancer. On the other hand, the EGFR G719S
and T790M mutations were detected in lung cancer
tissue but not detected in cervical cancer by dd-PCR
method. The results of the dd-PCR analysis were con-
firmed by Scorpion amplification refractory mutation
system (ARMS) assay.
CONCLUSION: The establishment of highly sensitive
droplet digital PCR detection is a new method for stud-
ies on FFPE samples. The EGFR genotype assay results
supplemented clinical information and could facilitate
therapeutic approach design. (Anal Quant Cytopathol
Histpathol 2020;42:119–124)
Keywords: cervical cancer, droplet digital PCR,
EGFR genes, epidermal growth factor receptor,
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/20/4204-0119/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Highly Sensitive Droplet Digital PCR for the
Detection of EGFR G719S and T790M
Mutations in Formalin-Fixed Paraffin-Embedded
Cervical Cancer and Lung Cancer Patients
Hongwen Yang, M.M., Kui He, M.D., Weile Dong, M.D., Jinchuan Fang, M.M.,
Suyun Zhong, B.M., Lixia Tang, M.M., and Lihua Long, B.M.
From the Department of Gynecology, Women & Children Health Institute Futian Shenzhen, Shenzhen; the Department of Abdominal
Surgery, The Second People’s Hospital of Futian District, Shenzhen; and the Department of Gynecology, The First Affiliated Hospital of
University of South China, Hengyang, China.
Hongwen Yang is Physician, Department of Gynecology, Women & Children Health Institute Futian Shenzhen.
Kui He is Physician, Department of Abdominal Surgery, The Second People’s Hospital of Futian District, Shenzhen.
Weile Dong is Physician, Department of Gynecology, The First Affiliated Hospital of University of South China, Hengyang.
Jinchuan Fang is Physician, Department of Gynecology, Women & Children Health Institute Futian Shenzhen.
Suyun Zhong is Physician, Department of Gynecology, Women & Children Health Institute Futian Shenzhen.
Lixia Tang is Physician, Department of Gynecology, Women & Children Health Institute Futian Shenzhen.
Lihua Long is Physician, Department of Gynecology, Women & Children Health Institute Futian Shenzhen.
Hongwen Yang, M.M., and Kui He, M.D., contributed equally to this work.
This work is supported by grants from the Nonprofit Health Research of Futian District, Shenzhen, in 2016 (No. FTWS20160044).
Address correspondence to: Hongwen Yang, M.M., Department of Gynecology, Women & Children Health Institute Futian Shenzhen,
No. 2002 Jintian Road, Futian District, Shenzhen 518017, China (yhwftfy@hotmail.com).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. FFPE, gene amplification, immunohistochemistry,
lung cancer, paraffin embedding, polymerase chain
reaction, tissue embedding, tumor biomarkers.
Epidermal growth factor receptor (EGFR) was
identified as a tumor therapy target which plays
important roles in tumor cell differentiation, pro­
liferation, metastasis, and invasion.1-5 It was re-
ported that EGFR is highly expressed in tumor
tissues in 90% of cervical cancer patients.6 These
patients showed lower recurrence-free survival
and overall survival rate as compared to those
with low EGFR expression in tumors.6 Moreover,
it was demonstrated that EGFR is also associated
with radiation resistance. In tumor cells with high
EGFR expression, radiation could induce auto­
phosphorylation of EGFR, which in turn affects
downstream signal pathway activity.7,8
Currently, two distinct therapeutic approaches
were employed for targeting EGFR in various ma-
lignancies. They are monoclonal antibodies (e.g.,
cetuximab) and small molecule tyrosine kinase
inhibitors (TKIs) (e.g., gefitinib). The anti-EGFR
agents could increase the anti-tumor effect of ra-
diotherapy and chemotherapy through inhibiting
EGFR phosphorylation and blocking signal trans-
duction.9 In recent years Yamanaka et al10 dem­
onstrated that gefitinib could inhibit epidermal
growth factor–induced activities of cervical cancer
cells in vitro. Liu et al11 substantiated that gefitinib
could suppress cervical cancer cell proliferation
and induce radiosensitization of cervical carcinoma
cells.
The increasing amount of evidence demonstrated
that sensitivity of anti-EGFR agents shows correla-
tion with EGFR mutations. Several mutations exist
in the EGFR gene, mainly in exons 18–21.12,13 The
G719S in exon 18, L858R in exon 21, and exon 19
deletion mutations result in changes in ATP bind-
ing domain of EGFR and subsequently enhance
competition between TKI and ATP, which in turn
improves TKI therapeutic effects,12 whereas the
T790M in exon 20 leads to acquisition of EGFRTKI
resistance.14 Recently, the L858R in exon 18 and
exon 19 deletion mutations have been detected and
no mutations were found among all human neo-
plastic samples analyzed.15,16 However, the detec-
tion of G719S in exon 18 and T790M in exon 20
mutation remains not be reported.
Droplet digital PCR (dd-PCR) is a recently de-
veloped method that may facilitate the detection
of cDNAs. The dd-PCR method is based on the
principle of partitioning of the sample into thou-
sands of micro-reactions.17 As compared with tra­
ditional real-time qPCR methods, the dd-PCR tech-
nique has some favorable features. For example,
dd-PCR performs absolute quantification, while
RT-PCR is based on relative quantification; thus,
dd-PCR overcomes the normalization and calibra-
tor issues. Moreover, dd-PCR showed increased
precision and sensitivity. In the present study we
employed droplet digital PCR (dd-PCR) meth-
ods to screen the G719S and T790M mutations of
EGFR gene in lung cancer tissue and cervical can-
cer tissue. The result of the present study may pro-
vide a novel method for the detection of cDNAs
in FFPE samples.
Materials and Methods
Sample Collection
A total of 54 tumor samples from cervical cancer
patients and 60 non–small cell lung cancer pa-
tients were collected between August 2013 and
November 2017 at Women & Children Health Insti-
tute Futian Shenzhen. This study was approved by
the ethic committee of Women & Children Health
Institute Futian Shenzhen, and each patient signed
the informed consent form.
Immunohistochemistry Assay
The pathobiology of tumor tissues was analyzed
using immunohistochemistry-SP (streptavidin per-
oxidase) according to a previous study.17 The anti-
EGFR antibody (ab52894) was purchased from
Abcam.
DNA Extraction
DNA was extracted from FFPE samples. Briefly,
the tissue sections were cut from 5–8 FFPE slices
(10 μm). Genomic DNA was extracted using TIAN-
quick FFPE DNA Kit (TIANGEN, DP330) and
quantified using NanoDrop ND2000 (Thermo Fish-
er Scientific).
Droplet Digital PCR
Primers and probes were designed based on EGFR
sequence (NM_001346900.1) from NCBI database
using Primer Premier 6.0. Bio-Rad QX200TM dd-
PCR System (Bio-Rad) was used for droplet digi-
tal PCR according to the manufacturer’s manual.
The reactions were performed in final volumes of
20 μL. In particular, 10 μL of ddPCR Supermix,
0.4 μL of forward primer (5 μM), 0.4 μL of reverse
primer (5 μM), 0.2 μL of wild-type probe (5 μM),
120 Analytical and Quantitative Cytopathology and Histopathology®
Yang et al

3. 0.2 μL of mutation probe (5 μM), 4 μL of extracted
genomic DNA, and H2O to reach the final volume
were used. A volume of 50 μL droplet generation
oil was used to generate droplets in QX200 droplet
generator (Bio-Rad). The droplets were transferred
to a 96-well PCR plate for PCR amplification. The
program is as follows: hold at 95°C for 5 minutes,
40 cycles of 94°C for 30 seconds, 60°C for 1 minute,
1 cycle of 60°C for 30 minutes, and ending at 4°C.
After amplification, the positive droplets (HEX/
FAM) were counted using QX200 droplet reader
(Bio-Rad). The data were analyzed using Quanta-
Soft 1.7.4 software (Bio-Rad).
Amplification Refractory Mutation System (ARMS)
Technology
The ADx-ARMS kit (Amoy Diagnostics, Xiamen,
China) was used to detect the EGFR mutation at
exons 18 and 20 by the LightCycler 480 (Roche)
according to the manufacturer’s instructions. The
thermocycling profile of the PCR reaction was
95°C for 5 minutes, 15 cycles of 95°C for 20 sec-
onds, 64°C for 20 seconds, 72°C for 20 seconds,
and then 31 cycles of 95°C for 25 seconds, 60°C for
35 seconds, and 72°C for 20 seconds.
Statistical Analyses
Data analysis was performed using SPSS version
22.0 software. Results are established as mean±
95% confidence intervals.
Results
High Expression of EGFR in Cervical and Lung Cancer
Tissues
The cervical cancer patients included 16 patients
with stage IB2, 3 patients with stage IIA1, 18 pa­
tients with stage IIA2, and 20 patients with stage
IIB. The early stage lung cancer patients included
29 patients at stage I and 31 at stage II. First, we
detected EGFR expression in tumor tissues. The
tumor tissues from patients were collected dur­
ing surgery and taken for immunohistochemistry
assay. The results showed that EGFR was highly
expressed in tumor tissues among all samples of
the cervical cancer and lung cancer patients (Fig-
ures 1–2).
EGFR Genotype Detection of EGFR G719S and
T790M Mutations in Cervical Cancer dd-PCR
Furthermore, we detected EGFR genotype using
droplet digital PCR in 56 patients with cervical
cancer. Genomic DNA was extracted from FFPE
slides, followed by PCR amplification and dd-PCR
assay. FAM labeled probes were designed for de­
tecting wild-type genotype sequences, whereas
HEX labeled probes were designed for detecting
Volume 42, Number 4/August 2020 121
Highly Sensitive Droplet Digital PCR
Figure 1
Flowchart of the study
design and EGFR expression
detection in cervical tumor
tissues. ddPCR = droplet
digital PCR, FFPE = formalin-
fixed, paraffin-embedded,
IHC = immunohistochemistry.

4. for EGFR mutations (Figure 3A). The droplets read-
ing results showed that all the amplicons possess
FAM-positive fluorescence only (Figure 3B). The
G719S mutation in exon 18 and T790M mutation
in exon 20 were not detected in all samples (Fig-
ure 3B). It is indicated that G719S and T790M mu-
tations are quite rare in cervical cancer. Therefore,
the chemotherapy sensitivity in this study was not
relative to EGFR G719S and T790M mutations. The
results of the dd-PCR analysis were confirmed by
Scorpion ARMS assay.
EGFR Genotype Detection of EGFR G719S and
T790M Mutations in Lung Cancer dd-PCR
Next, EGFR G719S and T790M mutations were
analyzed by using genomic DNA extracted from
FFPE tissue specimens in 60 patients with lung
cancer (Table I). T790M was a common mutation
(21/60), while the frequency of G719S was low
(1/60) (Table I). The results of the dd-PCR analy-
sis were confirmed by Scorpion ARMS assay.
Discussion
In this study we investigated EGFR expression in
cervical cancer and lung cancer tissues with early
stage (from stage IB2 to IIB). The immunohisto-
chemistry assay showed that EGFR was highly
expressed in both cervical cancer and lung cancer
tissues analyzed. It was reported that EGFR ex­
pression level is the highest at early stage (stage
I–II) in cervical tumor tissues, median values of
which were 7.8 fmol/mg protein.18 The expression
of EGFR was highly associated with the develop-
ment of lung cancer,19 and for cervical cancer, even
though the prognostic role of EGFR is controver-
sial, the results in a previous study suggested that
EGFR expression confers prognostic information in
addition to that based on clinicopathologic param­
eters of FIGO stage and lesion size.19
The EGFR mutation is relative to sensitivity to
chemotherapy and anti-EGFR agents. Fang et al20
122 Analytical and Quantitative Cytopathology and Histopathology®
Yang et al
Figure 2 Results of immunohistochemistry staining of EGFR in
lung cancer tissue.
Figure 3
EGFR G719S and T790M
mutations detection using
droplet digital PCR.
(A) Scheme of primers and
probes design and (B) results
of ddPCR assay for the
detection of exon 18 mutation
and exon 20 mutation.
E18 = exon 18, E20 = exon
20.
Table I EGFR G719S and T790M Mutations in Cervical and
Lung Cancer Detection Using Droplet Digital PCR
G719S T790M
mutation mutation
Cervical cancer
Positive 0 0
Negative 50 50
Lung cancer
Positive 21 1
Negative 39 59

5. reported that presence of EGFR exon 19 deletion
mutation improved overall survival for patients
with advanced non–small cell lung cancer who
received platinum-based doublet first-line chemo-
therapy. For anti-EGFR therapies, the mutation
L858R, G719S, and exon 19 deletion mutations
were demonstrated to improve sensitivity to EGFR
tyrosine kinase inhibitors,21-23 whereas T790M
mutation in ATP binding pocket of EGFR kinase
domain results in acquirement of resistance to
EGFR TKIs.24,25 Therefore, detecting EGFR gene
status is critical to provide information for EGFR-
TKI therapy.21
In the current study, EGFR G719S and T790M
mutation status of collected cervical tumor sam-
ples and lung cancer tissues were detected using
dd-PCR. Droplet digital PCR technology provides
a more precise and sensitive method than tradi-
tional PCR. Moreover, it is well known that the
input of genomic DNA extracted from FFPE sam-
ples is low,22 while dd-PCR requires only a small
amount of the DNA samples. Therefore, if dd-PCR
has high accuracy, it may be an ideal method for
the detection of EGFR mutations in FFPE sam-
ples. Our data showed that no EGFR G719S and
T790M mutations were detected in cervical tu-
mor samples. This information indicated that
G719S and T790M mutations may be quite rare
in cervical cancer, and these tumors in patients
would be sensitive to EGFR-TKIs. On the other
hand, EGFR G719S and T790M mutations were
all detected in lung cancer tissue, and T790M
was a common mutation, while the frequency of
G719S was rare. These results were consistent
with the previous reports on the distribution of
the G719S and T790M mutations among patients
with lung cancer.23 Interestingly, compared with
ARMS assay, which was now the “gold stan-
dard” for detecting the EGFR mutations,23 the
sensitivity and specificity of dd-PCR were all
100%. Taken together, these results suggested that
dd-PCR may be a potential novel method for the
fast and accurate detection of EGFR mutations in
FFPE samples.
To sum up, our study provided important in-
formation in addition to clinical information and
could be taken as a guide for further therapy.
In addition, the establishment of highly sensitive
detection methods for EGFR mutations makes it
possible to analyze EGFR genotype in FFPE sam-
ples, which could combine pathobiology and ge-
netics to produce comprehensive information.
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