ORIGINAL ARTICLE

Distribution and Detectability of EGFR Exon 20

Insertion Variants in NSCLC
Sai-Hong Ignatius Ou, MD, PhD,a,* Jin-Liern Hong, PhD,b
Petros Christopoulos, MD, PhD,c,d Huamao M. Lin, PhD,b Sylvie Vincent, PhD,e
Eric N. Churchill, PhD,f Junpei Soeda, MD, PhD,g Daniel Kazdal, PhD,d,h
Albrecht Stenzinger, MD,d,h Michael Thomas, MDd,i
a
Department of Internal Medicine, Division of Hematology-Oncology, University of California Irvine School of Medicine,
Orange, California
b
Global Evidence and Outcomes Oncology, Takeda Development Center Americas, Inc., Lexington, Massachusetts
c
Department of Thoracic Oncology, Thoraxklinik and National Center for Tumor Diseases at Heidelberg University Hospital,
Heidelberg, Germany
d
Translational Lung Research Center Heidelberg (TLRC-H), Member of the German Center for Lung Research (DZL),
Heidelberg, Germany
e
Oncology Therapeutic Area Unit, Takeda Development Center Americas, Inc., Lexington, Massachusetts
f
Global Medical Affairs Oncology, Takeda Pharmaceuticals U.S.A., Inc., Lexington, Massachusetts
g
Japan Medical Affairs, Japan Oncology Business Unit, Takeda Pharmaceutical Company Ltd., Tokyo, Japan
h
Center for Molecular Pathology (CMP), Institute of Pathology Heidelberg (IPH), University Hospital Heidelberg,
Heidelberg, Germany
i
Department of Internal Oncology of Thoracic Tumors, Thoraxklinik and National Center for Tumor Diseases at Heidelberg
University Hospital, Heidelberg, Germany

Received 29 September 2022; revised 13 December 2022; accepted 22 January 2023

Available online - 3 February 2023

ABSTRACT
Introduction: EGFR exon 20 insertion (ex20ins) mutations
represent 5% to 10% of EGFR mutations in NSCLC. Identi-
fying patients with EGFR ex20ins is challenging owing to the
limited coverage of polymerase chain reaction (PCR) assays
and the relatively recent use of next-generation sequencing
(NGS). This study analyzes the spectrum of EGFR ex20ins
variants in a large patient population from a global clinical
trial and several real-world cohorts and the ability of PCR
kits to identify these alterations.
Methods: We conducted this retrospective analysis in pa-
tients with NSCLC who underwent NGS or other sequencing
testing and had a known EGFR ex20ins mutation. Patients
were gathered from a clinical trial (NCT02716116), a chart
review study in Germany, and the LC-SCRUM-Japan, GENIE,
and U.S. COTA databases. Proportions of patients with

*Corresponding author. BeiGene, Bristol-Myers Squibb, Boehringer Ingelheim, Celgene, Chugai,

Drs. Stenzinger and Thomas contributed equally to this work as co-
senior authors and as last authorship.
Disclosure: Dr. Ou reports receiving consulting and honoraria for
AstraZeneca, BeiGene, Caris Life Sciences, Elevation Oncology, John-
son & Johnson/Janssen, Eli Lilly, and Pfizer; and having stock owner-
ship with Turning Point Therapeutics and Elevation Oncology. Dr.
Christopoulos reports receiving consulting and honoraria for Roche,
Takeda, Chugai, Boehringer Ingelheim, Gilead, AstraZeneca, Janssen,
Daiichi Sankyo, Novartis, and Eli Lilly; and research funding from
Roche, Takeda, Amgen, Merck, AstraZeneca, and Novartis. Drs. Hong,
Lin, Vincent, Churchill, and Soeda report having employment with
Takeda. Dr. Kazdal reports receiving personal fees from AstraZeneca,
Bristol-Myers Squibb, Pfizer, Eli Lilly, Agilent, and Takeda, outside of
the submitted work. Dr. Stenzinger reports having consulting and
advisory roles for Aignostics, Amgen, AstraZeneca, Bayer, Bristol-
Myers Squibb, Eli Lilly, Illumina, Incyte, Janssen, Merck Sharp &
Dohme, Novartis, Pfizer, Roche, Seattle Genetics, Takeda, and Thermo
Fisher Scientific; and receiving research funding (to the institution)
from Bayer, Bristol-Myers Squibb, Chugai, and Incyte. Dr. Thomas re-
ports receiving honoraria for advisory board with AstraZeneca,
Daiichi Sankyo, GlaxoSmithKline, Janssen Oncology, Eli Lilly, Merck,
Merck Sharp & Dohme, Novartis, Pfizer, Roche, Sanofi, and Takeda;
research funding (all to institution) from AstraZeneca, Bristol-Myers
Squibb, Merck, Roche, and Takeda; and nonfinancial support (travel
costs) from AstraZeneca, Bristol-Myers Squibb, Janssen Oncology,
Merck Sharp & Dohme, Pfizer, Roche, and Takeda.
Previous presentations: Previously presented at the 2021 World Con-
ference on Lung Cancer, September 8–14, 2021. Virtual.
Address for correspondence: Sai-Hong Ignatius Ou, MD, PhD, Division of
Hematology-Oncology, Department of Internal Medicine, Chao Family
Comprehensive Cancer Center, University of California Irvine School of
Medicine, 200 South Manchester Avenue, Suite 200, Orange, CA 92602.
E-mail: siou@hs.uci.edu or ignatiusou@gmail.com.
ª 2023 International Association for the Study of Lung Cancer.
Published by Elsevier Inc. This is an open access article under the
CC BY license (http://creativecommons.org/licenses/by/4.0/).
ISSN: 1556-0864
https://doi.org/10.1016/j.jtho.2023.01.086

Journal of Thoracic Oncology Vol. 18 No. 6: 744–754

June 2023
ex20ins variants that could have been detected by six
commercially available and widely used PCR kits were
calculated in each data set.
Results: Overall, 636 patients with NSCLC harboring EGFR
ex20ins mutations were included in this analysis and 104
unique EGFR ex20ins variants were identified across the
data sources. The proportion of patients whose ex20ins
could have been detected by any PCR test alone ranged
from 11.8% to 58.9% across the data sources.
Conclusions: Our findings suggest that the PCR tests eval-
uated would have missed more than 40% of patients with
NSCLC harboring EGFR ex20ins mutations. NGS-based ge-
netic testing is preferable than standard PCR assays and can
substantially improve the identification of the diverse pro-
file of EGFR ex20ins variants in NSCLC.
 2023 International Association for the Study of Lung
Cancer. Published by Elsevier Inc. This is an open access
article under the CC BY license (http://creativecommons.
org/licenses/by/4.0/).
Keywords: Epidermal growth factor receptor; Exon 20
insertion mutation; Polymerase chain reaction; Next-gen-
eration sequencing; Non–small cell lung cancer
Introduction
EGFR is a receptor tyrosine kinase-signaling protein.1
EGFR mutations typically occur in exons 18 to 21, which
encode the intracellular tyrosine kinase portion of the
protein.2,3 Mutations in the EGFR tyrosine kinase domain
are associated with overexpression of the protein and
have been found to drive NSCLC development.1 EGFR
exon 20 insertions (ex20ins) are atypical EGFR muta-
tions that represent 5% to 10% of EGFR mutations in
NSCLC.3 In patients with NSCLC, atypical EGFR mutations
have been associated with worse outcomes than com-
mon EGFR mutations, such as exon 19 deletions or
L858R mutations.4 EGFR ex20ins mutations are hetero-
geneous insertions with many variants identified to
date.5–7 EGFR ex20ins mutations have different sub-
types, and most confer primary resistance to first- and
second-generation tyrosine kinase inhibitors (TKIs).2,5
Previous studies using three-dimensional modeling
have revealed that the resistance of far-end exon 20
mutations to first- and second-generation TKIs is a result
of alterations in the drug-binding pocket which cause a
shift of the phosphate-binding loop that then reduces
drug-binding affinity.5,8 Amivantamab, an EGFR and
mesenchymal–epithelial transition (MET) bispecific
antibody, and mobocertinib, an oral TKI targeting EGFR
ex20ins mutations, have been approved by the U.S. Food
and Drug Administration for the treatment of adult
EGFR Ex20ins Detectability in NSCLC 745
patients with locally advanced or metastatic NSCLC with
EGFR ex20ins mutations whose disease has progressed
on or after platinum-based chemotherapy.9,10 Positive
outcomes are associated with correct identification of
EGFR mutation types to guide treatment, but reliable and
standardized detection methods for identifying all mu-
tations remain elusive.1,2,11 Although amivantamab and
mobocertinib were found to have efficacy in clinical
trials, both studies were limited by a mix of diagnostic
methods used to detect ex20ins mutations.9,10
Two molecular tests widely used to detect EGFR
mutations are polymerase chain reaction (PCR) and
next-generation sequencing (NGS).7 PCR-based assays
are widely available and sensitive, and they yield results
quickly.12 Nevertheless, NGS has increased the incidence
of observed ex20ins mutations and improved the
detection of unique ex20ins variants because of its
ability to sequence the entire gene.2 The availability of
accurate and efficient diagnostic tools will become
increasingly important as targeted therapies for patients
with EGFR ex20ins mutations continue to be
developed.13
The purpose of this study was to evaluate the capa-
bility of PCR versus NGS to identify EGFR ex20ins in
patients with NSCLC and to identify specific EGFR
ex20ins variants in patients with NSCLC by analyzing a
global clinical trial and real-world data.
Methods
Study Design
This retrospective analysis was conducted in patients
with NSCLC by using one global clinical trial and four
real-world data sources that followed patients who un-
derwent NGS or other sequencing testing and had
confirmed EGFR ex20ins. NCT02716116 is a global
phase 1/2 trial conducted to evaluate the safety, phar-
macokinetics, and antitumor activity of mobocertinib in
patients with NSCLC (data cutoff date: November 1,
2020).10 The German Chart Review Study is a retro-
spective medical chart review study of patients with
NSCLC harboring EGFR ex20ins treated in 12 German
academic centers.14 The LC-SCRUM-Japan study is an
industry-academia collaborative cancer genome
screening project to construct a large-scale patient reg-
istry of rare subtypes of lung cancer; this study in-
tegrates clinical and genomic information in Japan (data
cutoff date: December 31, 2019).15,16 The American As-
sociation for Cancer Research Project Genomics Evi-
dence Neoplasia Information Exchange (GENIE)
database is a registry of real-world cancer genomics data
from leading cancer centers around the world (version
11.0).17 The U.S. COTA database18 comprises longitudi-
nal data abstracted from electronic health records of
746 Ou et al Journal of Thoracic Oncology Vol. 18 No. 6

health care provider sites in the routine practice in the
United States (data cutoff date: November 1, 2020).
Patients were included in the analysis if they had
NSCLC harboring EGFR in-frame insertions in the exon 20
coding region (amino acids 763–775) and if the ex20ins
mutations were detected by NGS or conventional
sequencing. Patients were excluded if their specific EGFR
insertions were not reported or if the reported mutation
did not result in the addition of amino acids in the loop
(i.e., p.S768_V769delinsIL and p.H773_V774delinsLM).
Statistical Analysis
Descriptive analyses of demographics and distribu-
tions of ex20ins variants were used for this study. De-
mographics, including age, sex, and race, were
summarized with descriptive statistics. Ex20ins variants
were analyzed by amino acid position change. The dis-
tribution of ex20ins variants was summarized separately
in five data sets using frequencies and percentages. The
ability of a PCR kit to identify patients with ex20ins was
quantified as the proportion of patients with ex20ins
variants that could have been detected by PCR testing on
the basis of manufacturer-provided coverage informa-
tion in each data set. The six PCR kits evaluated for this
analysis were as follows: Roche Cobas EGFR mutation
test version 2 (Roche Diagnostics; Indianapolis, IN);
Qiagen therascreen EGFR RGQ PCR kit version 2 (Qiagen;
Hilden, Germany); AmoyDx EGFR 29 mutation detection
kit (AmoyDx; Xiamen, People’s Republic of China);
PANAGENE clamp EGFR mutation detection kit version 2
(Panagene; Daejeon, South Korea); Diacarta QClamp
EGFR mutation detection test (Diacarta; Pleasanton, CA);
and BIOCARTIS Idylla EGFR mutation assay (Biocartis
U.S.; Jersey City, NJ). According to the manufacturer-
provided coverage information, collectively, these kits
cover the following EGFR ex20ins variants: A767_V769dup
(ASV), S768_D770dup (SVD), D770_N771insG, H773dup (H),
P772_H773insTTP, P772_H773insGNP, H773_V774insPH,
and V774_C775insHV. Across the six PCR kits, five
could detect A767_V769dup (ASV), two could detect
S768_D770dup (SVD), four could detect D770_N771insG,
five could detect H773dup (H), and one could detect
P772_H773insTTP, P772_H773insGNP, H773_V774insPH,
and V774_C775insHV. The analyses were conducted using
SAS version 9.4 (Cary, NC). The same six PCR kits were
used in each analysis.
An additional analysis was conducted to understand
the ability of PCR kits to identify patients with EGFR
ex20ins who can benefit from mobocertinib, using data
from the mobocertinib phase 1/2 trial (data cutoff date:
November 1, 2020).10 Among patients treated with
mobocertinib 160 mg once daily who were previously
treated with platinum-based chemotherapy, patients
were stratified according to whether their ex20ins var-
iants were detectable by PCR testing. For each subgroup,
confirmed objective response rates (ORRs) assessed by
an independent review committee (IRC) were calculated.
Results
Patient Demographics
Overall, 636 patients with NSCLC harboring EGFR
ex20ins variants from across the world were included in

Table 1. Patient Demographics

Mobocertinib German Chart GENIE U.S. COTA
Characteristic Phase 1/2 Study Review Study LC-SCRUM-Japan Database Database
No. of patients 157 109 95 245 30
Age,a y
Mean (SD) 60.4 (11.7) 64.1 (11.4) 61.5 (11.7) 62.6 (12.6)b 59.6 (10.0)
Median (min, max) 62 (28, 84) 66 (34, 83) 63 (34, 90) 63 (18, 89)b 60 (41, 80)
Category, n (%)
<65 94 (59.9) 50 (45.9) 51 (53.7) 137 (55.9) 21 (70.0)

65 63 (40.1) 59 (54.1) 44 (46.3) 108 (44.1) 9 (30.0)
Sex, n (%)
Female 107 (68.2) 71 (65.1) 56 (58.9) 165 (67.3) 21 (70.0)
Male 50 (31.8) 38 (34.9) 39 (41.1) 80 (32.7) 9 (30.0)
Race, n (%)
White 89 (56.7) 104 (95.4) 0 (0.0) 153 (62.4) 16 (53.3)
Black 12 (7.6) 0 (0) 0 (0.0) 23 (9.4) 5 (16.7)
Asian 49 (31.2) 4 (3.7) 95 (100) 39 (15.9) 5 (16.7)
Other/unknown 7 (4.5) 1 (0.9) 0 (0.0) 30 (12.2) 4 (13.3)
a
Ages were collected at different time points: at trial enrollment or registration (Mobocertinib phase 1/2 study and LC-SCRUM-Japan), at sequencing (GENIE),
or at diagnosis (German chart review study and U.S. COTA).
b
In GENIE, any patients younger than 18 years of age were treated as aged 18 years (n ¼ 1) and those older than 89 years of age were treated as aged 89 years
(n ¼ 4).
GENIE, American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange; Max, maximum; Min, minimum; Y, year.
June 2023 EGFR Ex20ins Detectability in NSCLC 747

Table 2. Identification of EGFR ex20ins Variants Across Data Sources

Data Source

Detected Variants and Mobocertinib German Chart LC-SCRUM- GENIE U.S. COTA All Data
Detectability by PCR Phase 1/2 Study Review Study Japan Database Database Sources
Testing (n ¼ 157) (n ¼ 109) (n ¼ 95) (n ¼ 245) (n ¼ 30) (N ¼ 636)
No. of unique EGFR ex20ins variants
Detected by sequencing 39 33 20 66 12 104
Detectable by 1 of PCR 7 7 7 7 5 7
tests
% of patients with EGFR ex20ins as detected by sequencing
Detectable by a PCR 14.0–48.4 15.6–45.9 21.1–58.9 11.8–37.1 13.3–46.7 15.3–45.1
test alone (range), %
Missed by a PCR kit, % >51.6 >54.1 >41.1 >62.9 >53.3 >54.9
EGFR ex20ins, EGFR exon 20 insertion; GENIE, American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange; PCR,
polymerase chain reaction.

this analysis: 157 patients from the mobocertinib phase
1/2 study, 109 patients from the German chart review
study, 95 patients from LC-SCRUM-Japan, 245 patients
from the GENIE database (235 of whom were from the
United States), and 30 patients from the U.S. COTA
database (Table 1). The median age of patients from each
data source was above or equal to 60 years. Most pa-
tients (58.9%–70.0%) from each data source were
female.
Identification of EGFR ex20ins Variants Across
Data Sources
A total of 104 unique EGFR ex20ins variants were
identified across the five data sources using NGS or other
sequencing testing (Table 2). There were 39 unique
variants from the mobocertinib phase 1/2 study, 33
from the German chart review study, 20 from LC-
SCRUM-Japan, 66 from the GENIE database, and 12
from the U.S. COTA database. The percentage of patients

EGFR Tyrosine Kinase Domain

Exon 18 Exon 19 Exon 20 Exon 21 Exon 22 Exon 23

C-Helix Loop After C-Helix

EGFR in-frame insertions in the exon 20 coding region included in this analysis
761 762 763 764 765 766 767 768 769 770 771 772 773 774 775
D E A Y V M A S V D N P H V C

Possible to be detected by PCR kits

Not possible to be detected by PCR kits

Figure 1. Schematic diagram of the EGFR gene tyrosine kinase domain and the ex20ins mutations detectable and unde-
tectable by PCR. Top portion represents the EGFR gene. Bottom portion represents exon 20 of EGFR and the individual
mutations in each amino acid detected by PCR (orange font) versus mutations undetectable by PCR (blue font). ex20ins, exon
20 insertion; PCR, polymerase chain reaction.
748 Ou et al Journal of Thoracic Oncology Vol. 18 No. 6

Mobocertinib Phase 1/2 Study

50 Possible to be detected by PCR
Not possible to be detected by PCR

40 37 Identified in 1 patient and not detected by PCR:

D770_N771delinsAGH N771delinsGF P772_H773insT
D770_N771delinsEGN N771delinsGY P772dup (P)
D770_N771insGD N771delinsKH S768_V769insIL
Number of Patients

29 D770_N771insSTN N771delinsKPP V769_D770insATP

30
D770delinsMASV N771delinsPH V769_D770insEASV
H773_V774insA P772_H773delinsHNPY V769_D770insGW
H773delinsTPHPT P772_H773insDNP V769_D770insV
N771_P772insH P772_H773insGT
20 18 N771delinsFH P772_H773insPNP

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Proportions of patients with EGFR ex20ins detectable by a PCR test

PCR 1 PCR 2 PCR 3 PCR 4 PCR 5 PCR 6

76 (48.4%) 47 (29.9%) 47 (29.9%) 22 (14.0%) 37 (23.6%) 76 (48.4%)

Figure 2. Identification of EGFR ex20ins variants and the proportion of patients whose EGFR ex20ins mutations were
detectable by a PCR test from the mobocertinib phase 1/2 study. Bar graph represents patients with EGFR ex20ins variants
from the mobocertinib phase 1/2 study. Orange bars are mutations that were detectable by PCR, and blue bars are mutations
that were not detectable by PCR. EGFR ex20ins, EGFR exon 20 insertion; PCR, polymerase chain reaction.

with ex20ins variants that were detectable by PCR German Chart Review Study
testing ranged from 11.8% to 58.9% across all data From the 109 patients in the German chart review
sources (15.3%–45.1% in all data sources combined). study, 33 unique variants were identified using NGS.
The most common variants were A767_V769dup (ASV) Nevertheless, only seven variants could have been
and S768_D770dup (SVD). The number of unique identified by PCR tests (Fig. 3). The most common
ex20ins variants able to be detected by PCR ranged from ex20ins variants detectable by PCR were the
five to seven across all data sources (Fig. 1). A767_V769dup (ASV) insertion (n ¼ 23) and the
S768_D770dup (SVD) insertion (n ¼ 16), represent-
ing 36% of the patients in the German chart review
Mobocertinib Phase 1/2 Study
study. The most common ex20ins variants not
From the 157 patients in the global mobocertinib
detectable by PCR were the N771_H773dup (NPH)
phase 1/2 study, 39 unique variants were identified
insertion (n ¼ 12), representing 11% of the patients
using NGS or other sequencing testing. Nevertheless,
in the German chart review study. Detectability of
only seven variants could have been identified by PCR
ex20ins by the PCR tests ranged from 15.6% to 45.9%
tests (Fig. 2). The most common ex20ins variants
(Fig. 3).
detectable by PCR were the A767_V769dup (ASV)
insertion (n ¼ 37) and the S768_D770dup (SVD) inser-
tion (n ¼ 29), representing 42% of patients in the LC-SCRUM-Japan
mobocertinib study. The most common ex20ins variants From the 95 patients in LC-SCRUM-Japan, 20 unique
not detectable by PCR were the N771_H773 dup (NPH) variants were identified using NGS. Nevertheless, only
insertion (n ¼ 18) and the D770delinsGY insertion (n ¼ seven variants could have been identified by PCR tests
8), representing 17% of the patients. Detectability of (Fig. 4A). The most common ex20ins variants detectable
ex20ins by the PCR tests ranged from 14.0% to 48.4% by PCR were the A767_V769dup (ASV) insertion (n ¼
(Fig. 2). 27) and the S768_D770dup (SVD) insertion (n ¼ 17),
June 2023 EGFR Ex20ins Detectability in NSCLC 749

40 German Chart Review Study Possible to be detected by PCR

Not possible to be detected by PCR
35

30 Identified in 1 patient and not detected by PCR:

A763_Y764insFQEA H773_V774insGNPH N771delinsTH
Number of Patients
D770_N771insGV N771_P772insT N771dup (N)
25 23 D770_V774dup (DNPHV) N771_P772insV P772delinsPGG
D770delinsGY N771delinsKG V769_D770insQ
D770delinsNNN N771delinsNPHH V769_D770insTSV
20
H773_V774insCPH N771delinsSH
16
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Proportions of patients with EGFR ex20ins detectable by a PCR test

PCR 1 PCR 2 PCR 3 PCR 4 PCR 5 PCR 6
50 (45.9%) 34 (31.2%) 34 (31.2%) 17 (15.6%) 23 (21.1%) 50 (45.9%)

Figure 3. Identification of EGFR ex20ins variants and the proportion of patients whose EGFR ex20ins mutations were
detectable by a PCR test from the German chart review study. Bar graph represents patients with EGFR exon 20 insertion
variants from the German chart review study. Orange bars are mutations that were detectable by PCR, and blue bars are
mutations that were not detectable by PCR. EGFR ex20ins, EGFR exon 20 insertion; PCR, polymerase chain reaction.

representing 46% of the patients in LC-SCRUM-Japan. only five unique variants could have been identified by
The most common ex20ins variants not detectable by PCR tests (Fig. 4C). The most common ex20ins variants
PCR were the N771_H773dup (NPH) insertion (n ¼ 8) detectable by PCR were the S768_D770dup (SVD)
and the H773_V774insAH insertion (n ¼ 5), representing insertion (n ¼ 6), the A767_V769dup (ASV) insertion
14% of the patients in LC-SCRUM-Japan. Detectability of (n ¼ 4), and the H773dup insertion (n ¼ 4), representing
ex20ins by the PCR tests ranged from 21.1% to 58.9% 47% of the patients in the U.S. COTA database. The most
(Fig. 4A). common ex20ins variants not detectable by PCR were
the H773_V774insAH insertion (n ¼ 3) and the
GENIE Database N771_P772insH insertion (n ¼ 3), representing 20% of
From the 245 patients in the GENIE database, 66 the U.S. COTA study. Detectability of ex20ins by the PCR
unique variants were identified using NGS. Nevertheless, tests ranged from 13.3% to 46.7% (Fig. 4C).
only seven variants could have been identified by PCR
tests (Fig. 4B). The most common ex20ins variants Mobocertinib-Indicated Patients and Treatment
detectable by PCR were the A767_V769dup (ASV) Outcomes
insertion (n ¼ 37) and the S768_D770dup (SVD) inser- Among 114 platinum-pretreated patients who
tion (n ¼ 37), representing 30% of the patients in the received mobocertinib in the phase 1/2 clinical trial, 95
GENIE database. The most common ex20ins variants not had known ex20ins variants. An independent review
detectable by PCR were the N771_H773dup (NPH) committee (IRC)–assessed confirmed responses by
insertion (n ¼ 19) and the M766delinsIGQR insertion presence of ex20ins variants and by ex20ins variants
(n ¼ 16), representing 14% of the patients in the GENIE detectable by one of the PCR tests are found in Table 3. A
database. Detectability of ex20ins by the PCR tests total of 55 patients had variants that were detectable by
ranged from 11.8% to 37.1% (Fig. 4B). PCR testing; 13 of these patients had a confirmed
response to mobocertinib (confirmed ORR per IRC: 24%
U.S. COTA Database [95% confidence interval: 13%–37%]). Of 40 patients
From the 30 patients in the U.S. COTA database, 12 with variants that were not detectable by PCR testing, 14
unique variants were identified using NGS. Nevertheless, patients had a confirmed response to mobocertinib
750 Ou et al Journal of Thoracic Oncology Vol. 18 No. 6

A LC-SCRUM-Japan Study Possible to be detected by PCR

Not possible to be detected by PCR
30
27

25 Identified in 1 patient and not detected by PCR:

N771_P772insVDN V769_D770insDST
Number of Patients
P772_H773insDNP V769_D770insGSV
20
17 P772_H773insGDP V769_D770insGT
P772_H773insYNP
15
10
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Proportions of patients with EGFR ex20ins detectable by a PCR test

PCR 1 PCR 2 PCR 3 PCR 4 PCR 5 PCR 6
56 (58.9%) 39 (41.1%) 39 (41.1%) 20 (21.1%) 27 (28.4%) 56 (58.9%)

B 50 GENIE Version 11.0 Study Possible to be detected by PCR

Not possible to be detected by PCR

40 Identified in 1 patient and not detected by PCR:

37 37 A767_S768insTVA D770delinsGTH N771_P772insHN P772_H773insNAH
A767delinsGHS H773_V774insGNPH N771_P772insTP P772_H773insPH
D770_N771insGF H773_V774insHH N771delinsKHKH P772_H773insQ
Number of Patients

D770_N771insKD H773_V774insY N771delinsKP P772_H773insYNP

30 D770_N771insKRR H773delinsPNPY N771delinsKPP S768_V769insMDS
D770_N771insQPT H773delinsPRD N771delinsSTH V769_D770insATP
D770_N771insSMD M766_A767insWPA N771delinsTH V769_D770insCV
D770delinsEL M766delinsIGHA P772_C775dup (PHVC) V769_D770insGG
20 19 D770delinsEQPA N771_P772insG P772_H773insGDP V769_D770insGQ
16 D770delinsEQPL N771_P772insHH P772_H773insIC V774_C775insQPP

12 12
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Proportions of patients with EGFR ex20ins detectable by a PCR test

PCR 1 PCR 2 PCR 3 PCR 4 PCR 5 PCR 6

91 (37.1%) 54 (22.0%) 54 (22.0%) 29 (11.8%) 37 (15.1%) 91 (37.1%)

Figure 4. Identification of EGFR ex20ins variants and the proportion of patients whose EGFR ex20ins mutations were
detectable by a PCR test from LC-SCRUM-Japan, GENIE version 11.0, and U.S. COTA databases. Bar graphs represent patients
with EGFR ex20ins variants from the (A) LC-SCRUM-Japan database, (B) GENIE version 11.0 database, and (C) U.S. COTA
database. Orange bars are mutations that were detectable by PCR, and blue bars are mutations that were not detectable by
PCR. EGFR ex20ins, EGFR exon 20 insertion; GENIE, American Association for Cancer Research Project Genomics Evidence
Neoplasia Information Exchange; PCR, polymerase chain reaction.
June 2023 EGFR Ex20ins Detectability in NSCLC 751

C 10
U.S. COTA Database Possible to be detected by PCR
9 Not possible to be detected by PCR
8

Number of Patients
7
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Proportions of patients with EGFR ex20ins detectable by a PCR test
PCR 1 PCR 2 PCR 3 PCR 4 PCR 5 PCR 6

14 (46.7%) 8 (26.7%) 8 (26.7%) 9 (30.0%) 4 (13.3%) 14 (46.7%)

Two patients were reported to have 2 EGFR ex20ins mutations.

Figure 4. Continued.

(confirmed ORR per IRC: 35% [95% confidence interval: these PCR tests were designed to detect one to five
21%–52%]). variants as determined by the manufacturer. Thus, there
was considerable variability in the ability of the six PCR
Discussion kits to detect ex20ins mutations; the proportion of pa-
We identified a diverse spectrum of EGFR ex20ins tients whose ex20ins variants could have been detected
variants in patients with NSCLC detected by NGS or by any PCR test alone ranged from 11.8% to 58.9%
other sequencing testing, with a total of 104 unique across the data sources. The lowest proportion of
ex20ins variants across the five data sources. PCR detected ex20ins variants with a PCR assay was in pa-
detected fewer variants, and a maximum of only seven tients in the GENIE database, which was the largest data
variants were detectable by one of the PCR tests evalu- set analyzed (n ¼ 245). The findings of our analysis
ated in this study. The most common ex20ins variants suggest that the PCR tests evaluated would have missed
that could be detected by PCR, A767_V769dup (ASV) and more than 40% of patients with NSCLC harboring EGFR
S768_D770dup (SVD), were consistent across the data ex20ins variants that could be detected by NGS. Standard
sources, and the ex20ins variants not detectable by PCR PCR kits are designed to detect prevalent EGFR muta-
were variable, revealing the high number of mutations tions only, which likely contributes to the low detection
that can be missed by PCR testing alone. In addition, rate of ex20ins variants.

Table 3. IRC-Assessed Confirmed Response in PPP Cohort From Mobocertinib Phase 1/2 Study (N ¼ 114; NCT02716116)
Stratified by Detectability of EGFR ex20ins by PCR Tests
Patients With EGFR
ex20ins Variant Detectable
by One of the PCR Tests
PPP Cohort With Known
PPP Cohorta EGFR ex20ins Variants Yes No
Confirmed Response (N ¼ 114) (n ¼ 95) (n ¼ 55) (n ¼ 40)
Confirmed ORR per IRC, n (%) [95% CI] 32 (28) 27 (28) 13 (24) 14 (35)
[20–37] [20–39] [13–37] [21–52]
a
19 patients had unknown or unconfirmed ex20ins.
CI, confidence interval; EGFR ex20ins, EGFR exon 20 insertion; IRC, independent review committee; ORR, objective response; PCR, polymerase chain reaction;
PPP, platinum-pretreated patients.
752 Ou et al
Patients with NSCLC harboring EGFR ex20ins muta-
tions that are missed by PCR testing may not receive
effective treatment, resulting in poor outcome. It has
been found that frequently used treatment options, such
as chemotherapy, EGFR TKIs, and immunotherapy, are
not effective in patients with ex20ins, particularly in
second- or later-line settings.19–21 Nevertheless, novel
therapies, such as mobocertinib and amivantamab, were
found to have improved outcomes in patients with
NSCLC harboring EGFR ex20ins mutations.9,10 The ben-
efits of mobocertinib and amivantamab were observed
across EGFR ex20ins mutation subtypes, regardless of
mutation frequency or position.9,10 We analyzed the
ability of PCR kits to identify patients with EGFR ex20ins
among 95 platinum-pretreated patients in the mobo-
certinib phase 1/2 trial10 with known ex20ins variants.
PCR testing was unable to detect ex20ins variants in 40
patients—14 (35%) of whom had a confirmed response
to mobocertinib. Thus, the EGFR ex20ins variant would
have been missed by PCR testing in 14 patients who
responded to mobocertinib in the phase 1/2 trial.
Single-gene PCR assays are limited in their ability to
detect EGFR mutations; multiplex PCR can only capture a
set of insertions on the basis of primers used in the
assay, and recent studies have revealed that PCR
methods miss at least 50% of ex20ins mutations.7 In
addition, a study comparing allele-specific PCR to NGS in
identifying EGFR mutations found that ex20ins muta-
tions were the most overlooked EGFR mutations.22 These
results further support that NGS-based genetic testing
can substantially enhance the identification of the
diverse profile of EGFR ex20ins variants in NSCLC and is
preferred compared with standard PCR assays. One
additional advantage of NGS testing is its ability to
characterize the co-mutations present in these tumors;
emerging data suggest that mutated TP53 is associated
with shorter survival for patients with EGFR ex20ins-
positive tumors treated with chemotherapy and EGFR
inhibitors.14
A retrospective analysis conducted at a single center
in Denmark compared quantitative PCR testing (n ¼
1169 samples) with NGS (n ¼ 670 samples) to detect
EGFR mutations in patients with lung cancer.23 Results
revealed that NGS detected a significantly greater num-
ber of EGFR ex20ins mutations than PCR (10% [10/98]
versus 3% [5/143]; p ¼ 0.015); it was uncertain
whether PCR would have identified these additional
mutations. Furthermore, NGS also detected rare EGFR
mutations, uncommon combinations of EGFR mutations,
and mutations in other clinically relevant genes, such as
KRAS and BRAF. A retrospective analysis in the People’s
Republic of China compared Sanger sequencing (n ¼
5244 samples), real-time PCR (n ¼ 13,329 samples), and
NGS (n ¼ 2751 samples) for EGFR mutations in patients
Journal of Thoracic Oncology Vol. 18 No. 6
with NSCLC.24 NGS identified 11 novel EGFR mutations
in exons 18 to 21 versus two novel EGFR mutations
identified with Sanger sequencing and three identified
with real-time PCR. NGS also identified mutations in non-
EGFR genes in 65% of patients with EGFR mutations
(854 of 1308), most often TP53; approximately one-third
of these non-EGFR mutations identified by NGS were
potentially treatable by targeted therapies. In a retro-
spective study conducted in the United Kingdom evalu-
ating detectability of uncommon EGFR mutations
(defined as mutations in exons 18–21, excluding exon 19
deletion, L858R, T790M, and ex20ins), uncommon mu-
tations were detected by NGS in 43 of 303 patients
(14%); however, these mutations were not detectable by
PCR in 30% (13 of 43) of the cases.25 Fortunately, in the
United States, use of PCR testing in patients with NSCLC
has decreased over time whereas use of NGS has
increased2; however, the use of PCR and NGS can vary by
global region, and PCR is more popular in some regions.26
With the recent approval of therapies targeting EGFR
ex20ins mutations, health care practitioners can use NGS
to identify and properly treat this patient population.
An important consideration in the adoption of a
particular testing method is the cost effectiveness of
performing the technique. Recent studies have revealed
that NGS has a cost advantage over common single-
sequence methods in the detection of mutations in pa-
tients with metastatic NSCLC because NGS has greater
efficiency in detecting mutations, requires a shorter time
to process test results (leading to quicker initiation of
treatment), and is associated with a reduced need for
rebiopsy.27,28 One of these studies found that cost sav-
ings increased when the proportion of NGS-tested pa-
tients increased, further supporting wider adoption of
this technique.27 The value of NGS is expected to
continue to improve with increased availability of NGS
companion diagnostics that will refine treatment plans
for patients with unique genomic profiles.29 Further-
more, NGS companion diagnostics, which use circulating
tumor DNA from plasma such as the Guardant360 and
FoundationOne Liquid CDx, will reduce the need for bi-
opsy and potentially capture mutations in patients with
insufficient tumor tissue available for testing.30
This study had some limitations. This study evaluated
patients with EGFR ex20ins mutations as detected by
NGS or other sequencing testing, but these patients
might still be subject to selection bias. Furthermore, in
this study, the frequencies of ex20ins mutations were
summarized on the basis of amino acid change. The PCR
kits, however, may not be able to identify all possible
nucleotide sequences. For example, both EGFR nucleo-
tide sequences, c.2307_2308insGCCAGCGTG31 and
c.2309_2310ACdelinsCCAGCGTGGAT,32 can result in
amino acid change, A767_V769dup (ASV). Cobas PCR kit
June 2023
can capture both nucleotide sequences, but some PCR
kits only identify one sequence. Last, this study used
different data sources, and detailed information on NGS
methods, such as platform and annotation tools, was not
available for all data sources.
Our analysis reinforces the importance for molecular
diagnostic groups to be knowledgeable about the type of
assay used to detect EGFR mutations and for clinicians to
be aware of the potential limitations of standard PCR
assays.
CRediT Authorship Contribution
Statement
Sai-Hong Ignatius Ou: Conceptualization, Formal
analysis, Investigation, Methodology, Validation, Visuali-
zation, Writing—original draft preparation, Writing—
review and editing.
Jin-Liern Hong: Methodology, Analysis, Writing—
review and editing.
Petros Christopoulos: Data curation, Resources,
Funding acquisition, Writing—review and editing.
Huamao M. Lin: Supervision, Investigation,
Writing—review and editing.
Sylvie Vincent: Data curation, Formal analysis,
Methodology, Validation, Visualization, Writing—review
and editing.
Eric N. Churchill: Conceptualization, Data curation,
Methodology, Visualization, Writing—review and
editing.
Junpei Soeda: Data curation, Formal analysis,
Writing—review and editing.
Daniel Kazdal: Data curation, Investigation, Meth-
odology, Writing—review and editing.
Albrecht Stenzinger: Conceptualization, Supervi-
sion, Data curation, Investigation, Methodology,
Writing—review and editing.
Michael Thomas: Data curation, Resources, Funding
acquisition, Writing—review and editing, Supervision.
Acknowledgments
The study was sponsored by Takeda Development Center
Americas, Inc. (Lexington, MA). The authors thank the
National Cancer Center East (Japan) and LC-SCRUM for
providing the data. The authors thank the Center for Mo-
lecular Pathology, Institute of Pathology, University Hos-
pital Heidelberg, Germany, for providing molecular data.
The authors thank the patients in the global mobocertinib
phase 1/2 study, their families, and their caregivers. The
authors thank the phase 1/2 study investigators and their
team members at each study site; and colleagues from
Millennium Pharmaceuticals, Inc. Medical writing support
for the development of this manuscript, under the direc-
tion of the authors, was provided by Corey Burgin, PhD,
EGFR Ex20ins Detectability in NSCLC 753
and Amy Zannikos, PharmD, of Peloton Advantage, LLC, an
OPEN Health company, Parsippany, NJ, and funded by
Takeda Development Center Americas, Inc., Lexington, MA,
and complied with the Good Publication Practice guide-
lines (DeTora LM, et al. Ann Intern Med. 2022;175:1298–
1304). Teodor G. Paunescu, PhD (Takeda Pharmaceuticals
USA, Inc., Lexington, MA) is acknowledged for editorial
assistance.
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