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Non-classic EGFR mutations in a cohort of
Dutch EGFR-mutated NSCLC patients and
outcomes following EGFR-TKI treatment
JL Kuiper
1
, SMS Hashemi
1
, E Thunnissen
2
, PJF Snijders
2
, K Gru
¨nberg
3
, E Bloemena
2
, D Sie
2
, PE Postmus
4
,
DAM Heideman
2
and EF Smit*
,1,5
1
Department of Pulmonary Diseases, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands;
2
Department of Pathology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands;
3
Department of
Pathology, Radboud University Medical Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands;
4
Clatterbridge Cancer Centre
and Liverpool Heart & Chest Hospital, Thomas Drive, Liverpool, Merseyside L14 3PE, UK and
5
Department of Pulmonary Diseases,
The Netherlands Cancer Institute, PO Box 90203, 1006 BE Amsterdam, The Netherlands
Background: Data on non-small-cell lung cancer (NSCLC) patients with non-classic epidermal growth factor receptor (EGFR)
mutations are scarce, especially in non-Asian populations. The purpose of this study was to evaluate prevalence, clinical
characteristics and outcome on EGFR-TKI treatment according to type of EGFR mutation in a Dutch cohort of NSCLC patients.
Methods: We retrospectively evaluated a cohort of 240 EGFR-mutated NSCLC patients. Data on demographics, clinical and
tumour-related features, EGFR-TKI treatment and clinical outcome were collected and compared between patients with classic
EGFR mutations, EGFR exon 20 insertions and other uncommon EGFR mutations.
Results: Classic EGFR mutations were detected in 186 patients (77.5%) and non-classic EGFR mutations in 54 patients (22.5%); 23
patients with an exon 20 insertion (9.6%) and 31 patients with an uncommon EGFR mutation (12.9%). Median progression-free
survival (PFS) and overall survival (OS) on EGFR-TKI treatment were 2.9 and 9.7 months, respectively, for patients with an EGFR
exon 20 insertion, and 6.4 and 20.2 months, respectively, for patients with an uncommon EGFR mutation. Patients with a double
uncommon EGFR mutation that included G719X/L861Q/S768I had longer PFS and OS on EGFR-TKI treatment compared with
patients with a single G719X/L861Q/S768I EGFR mutation (both P¼0.02).
Conclusions: In our Dutch cohort, prevalence and genotype distribution of non-classic EGFR mutations were in accordance with
previously reported data. The PFS and OS on EGFR-TKI treatment in patients with an uncommon EGFR mutation were shorter
compared with patients with classic EGFR mutations, but varied among different uncommon EGFR mutations.
Classic EGFR mutations. The discovery of mutations in the
epidermal growth factor receptor (EGFR) gene as oncogenic driver
in lung cancer patients has changed both the diagnostic process
and treatment of such patients. The EGFR mutations are detected
in B10% of Caucasian patients with non-squamous non-small-cell
lung cancer (NSCLC) and in up to 50% of Asian NSCLC patients
(Dearden et al, 2013). In addition to the higher prevalence in
people from Asian descent, there is a higher prevalence of EGFR
mutations in women, nonsmokers and adenocarcinoma patients
(Barlesi et al, 2016). The vast majority of EGFR mutations
comprise microdeletions in exon 19 (45–50%) and the Leu858Arg
(L858R) substitution, resulting from a point mutation in exon 21
(40–45%; Murray et al, 2008). These mutations are so-called
sensitising EGFR mutations and hereafter referred to as ‘classic
EGFR mutations’ (Supplementary Figure 1). The beneficial effect of
treatment with EGFR tyrosine kinase inhibitors (TKIs) in NSCLC
*Correspondence: Dr Professor EF Smit; E-mail: ef.smit@vumc.nl
Received 10 June 2016; revised 24 September 2016; accepted 15 October 2016; published online 22 November 2016
&2016 Cancer Research UK. All rights reserved 0007 – 0920/16
FULL PAPER
Keywords: NSCLC; classic EGFR mutation; non-classic EGFR mutation; EGFR-TKI; TKI; EGFR
British Journal of Cancer (2016) 115, 1504–1512 | doi: 10.1038/bjc.2016.372
1504 www.bjcancer.com | DOI:10.1038/bjc.2016.372
patients who harbour a classic EGFR mutation in their tumour is
well established (Lynch et al, 2004; Maemondo et al, 2010;
Mitsudomi et al, 2010; Fukuoka et al, 2011; Zhou et al, 2011; Han
et al, 2012; Rosell et al, 2012; Sequist et al, 2013; Wu et al, 2014).
However, resistance is inevitable and median progression-free
survival (PFS) on EGFR-TKI treatment for NSCLC patients with a
classic EGFR mutation is 8.0–13.1 months (Mok et al, 2009;
Maemondo et al, 2010; Mitsudomi et al, 2010; Fukuoka et al, 2011;
Zhou et al, 2011; Han et al, 2012; Rosell et al, 2012; Sequist et al,
2013; Wu et al, 2014, 2015).
The T790M mutation. The T790M mutation is a distinct EGFR
mutation that is located in exon 20. It interferes with binding of
EGFR-TKI to EGFR, thereby prohibiting the inhibitory effect of
these agents. Detection of the T790M mutation before EGFR-TKI
treatment is rare (0.5%; Yu et al, 2014), although the detection rate
of pretreatment T790M is higher with more sensitive detection
methods (Rosell et al, 2011). However, the T790M mutation is
detected in B60% of EGFR-mutated NSCLC patients on or post
treatment with an EGFR-TKI showing renewed tumour growth
(Yu et al, 2013).
Non-classic EGFR mutations. The EGFR mutations other than
the classic EGFR mutations and exon 20 T790M mutations are less
prevalent (hereafter referred to as ‘non-classic EGFR-mutations’)
(Supplementary Figure 1). The most prevalent non-classic EGFR
mutations are insertions or duplications in EGFR exon 20 (further
referred to as ‘EGFR exon 20 insertions’) that are detected in
B2.2–5.0% of NSCLC patients (Wu et al, 2008a; Arcila et al, 2013;
Oxnard et al, 2013; Beau-Faller et al, 2014). In the study of Arcila
et al (2013), EGFR exon 20 insertions were furthermore mutually
exclusive with mutations in other genes, such as KRAS and BRAF,
except for PIK3CA and there were no associations with age, sex,
race or stage. Patients with EGFR exon 20 insertions generally have
a lower response rate to EGFR-TKI treatment and a poorer
prognosis compared with NSCLC patients with classic EGFR
mutations (Wu et al, 2008a; Oxnard et al, 2013). Other non-classic
EGFR mutations include so-called uncommon mutations
(Supplementary Figure 1), for example, in EGFR exon 18
(e.g., G719X; X ¼A, S or C), EGFR exon 20 (e.g., S768I) and
EGFR exon 21 (e.g., L861Q). The proportion of uncommon EGFR
mutations among EGFR-mutated NSCLC patients might be as high
as 14%, but varies in different studies (Yokoyama et al, 2006;
Zhang et al, 2007; Wu et al, 2008b, 2011; Hata et al, 2010;
De Pas et al, 2011; Arcila et al, 2013; Kobayashi et al, 2013; Keam
et al, 2014).
Multiple uncommon EGFR mutations or an uncommon EGFR
mutation in combination with a classic EGFR mutation may
co-exist in the same tumour. These so-called ‘double’ (or complex,
or compound) mutations are reported to occur in 6.6% of EGFR-
mutated NSCLC patients (Hata et al, 2010).
Data on results of EGFR-TKI treatment in Caucasian patients
with non-classic EGFR mutations are scarce as they are commonly
reported in small series, whereas the larger series typically include
patients of Asian descent. We therefore evaluated a cohort of
Dutch (i.e., predominant Caucasian) EGFR-mutated NSCLC
patients retrospectively. The purpose of this study was to evaluate
the prevalence and genotype distribution of non-classic EGFR
mutations in this cohort, as well as clinical characteristics and
outcome on EGFR-TKI treatment.
MATERIALS AND METHODS
Patients. All NSCLC patients in whom an EGFR mutation was
detected in the VU University Medical Center (VUmc) between
May 2006 and November 2014 (N¼240) were retrospectively
evaluated. As the VUmc is a diagnostic referral centre, some
patients were diagnosed at our centre, but follow-up and treatment
were performed in other hospitals. Patients with missing data on
follow-up were excluded from analysis of clinical characteristics
and outcome on EGFR-TKI treatment. For all other patients, data
on demographics, clinical and tumour-related features, treatments
and clinical outcomes was extracted from the medical records.
Mutation analysis. All mutation analyses were part of the routine
diagnostic procedures in VU University Medical Center, Amster-
dam, The Netherlands. The molecular diagnostic modalities for
EGFR mutation analysis included Sanger sequencing, HRM
sequencing and cancer panel multiplexed targeted resequencing
(Janmaat et al, 2006; Heideman et al, 2009; Sie et al, 2014). All
assays are designed to identify deletions or insertions in EGFR
exons 19 and 20, and hot spot mutations in EGFR exons 18
through 21.
For analytical purposes, deletions in EGFR exon 19 and the
L858R point mutation in EGFR exon 21 are referred to as classic
EGFR mutations. Among non-classic EGFR mutations, a distinc-
tion between exon 20 insertions and ‘uncommon EGFR-mutations’
was made (Supplementary Figure 1). The post-treatment T790M
mutations are not included in our analyses, nor are common EGFR
polymorphisms. All alterations that were detected were checked in
Alamut Visual version 2.7 (Interactive Biosoftware, Rouen,
France), the mycancergenome database (www.mycancergen-
ome.org; accessed 1 April 2016) and the Cosmic database
(cancer.sanger.ac.uk/cosmic; accessed 23 April 2016).
Treatment and outcomes. Patients who were alive at closing date
(26 November 2015) or who were lost to follow-up were censored
at the last date of follow-up. The EGFR-TKI treatment included
treatment with erlotinib, gefitinib or afatinib in patients with
advanced-stage disease. Survival was calculated from date of
diagnosis of advanced-stage (stage IIIB or IV) disease until date of
death. Objective response rate (ORR) on EGFR-TKI treatment was
calculated as the proportion of patients with complete or partial
response according to Response Evaluation Criteria in Solid
Tumours (RECIST) 1.1 (Eisenhauer et al, 2009). Disease control
rate (DCR) on EGFR-TKI treatment was calculated as the
proportion of patients with an objective response or stable disease
(for at least 6 weeks) according to the RECIST 1.1 criteria
(Eisenhauer et al, 2009). Progression-free survival on EGFR-TKI
treatment was calculated as the time from first day of treatment
until progression of disease or date of death (from any cause).
Patients who had not progressed at data cutoff were censored at the
last day of follow-up. Overall survival (OS) on EGFR-TKI
treatment was either calculated as the time from the first day of
EGFR-TKI treatment until date of death (from any cause), or
patients were censored at last follow-up.
Statistical analyses. Comparison of categorical variables was
performed with Pearson’s w
2
test. Comparison of three or more
continuous variables was performed with one-way ANOVA. The
Kaplan–Meier method was used for survival analyses and the log
rank test was used to test for significance. Two-sided P-values of
p0.05 were considered significant and confidence intervals (CIs)
were calculated at a 95% CI. The SPSS for Windows (version 20;
SPSS Inc., Chicago, IL, USA) was used for statistical analyses. The
medical ethical committee of VU University Medical Center
(Amsterdam, The Netherlands) approved the protocol.
RESULTS
Classic EGFR mutations. In 186 out of 240 patients (77.5%), a
classic EGFR mutation was detected (Figure 1): 134 patients
(72.0%) with an exon 19 deletion and 52 patients (28.0%) with an
exon 21 L858R point mutation.
Unusual EGFR mutations and outcome in NSCLC BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2016.372 1505
Sixty-two patients with a classic EGFR mutation were not treated at
our centre and were excluded from further analysis. Clinical
characteristics of the remaining 124 EGFR-mutated NSCLC patients
are described in Table 1. Median follow-up was 31.6 months (95% CI,
26.1–27.3). The EGFR-TKI treatment was started in 111 patients
(89.5%) (Supplementary Table 1). Clinical outcome on EGFR-TKI
treatment of this group of patients is described in Table 2.
Supplementary Tables provide more detailed data on start and/or
progression on EGFR-TKI treatment (Supplementary Table 2),
survival after EGFR-TKI treatment (Supplementary Table 3) and
response setting (Supplementary Table 4).
Non-classic EGFR mutations. A total of 54 patients (22.5%)
harbouring a non-classic EGFR mutation were identified: 23
patients (9.6%) with an exon 20 insertion and 31 patients (12.9%)
with an uncommon EGFR mutation in exons 18, 19, 20 and/or 21.
In one patient, both an exon 20 insertion and an EGFR exon
20 V769L point mutation were detected. This patient was
categorised in the EGFR exon 20 insertion group. All EGFR exon
20 insertions concerned insertions located on regions V769-N771
or H773-V774.
Of the group with uncommon EGFR mutations, 15 patients
(6.3%) had a single uncommon EGFR mutation (Table 3) and 16
patients (6.7%) were identified with double uncommon EGFR
mutations (Table 4). In three patients (1.3%) with a single
uncommon EGFR mutation, a KRAS mutation was also detected
(Table 3). In four patients (1.7%) with double EGFR mutations,
one of these mutations concerned the classic EGFR mutation
L858R on exon 21 (Table 4). There were two patients with a single
G719X EGFR mutation and two patients with a single L861Q
EGFR mutation (Table 3). Nine patients were identified with a
double EGFR mutation that included G719X, L861Q and/or
S768I (further referred to as ‘double G719X/L861Q/S768I’ EGFR
mutations; Table 4).
Of the patients with non-classic EGFR-mutations, four were not
treated in our centre and excluded from further analysis (i.e., one
with an exon 20 insertion, one with L858R þV834L mutation, one
with an exon 19 insertion and one with L861Q mutation).
Clinical characteristics of the remaining 22 patients with an
EGFR exon 20 insertion and 28 patients with an uncommon EGFR
mutation are described in Table 1. Median follow-up of these
patients was 29.4 months (95% CI, 19.6–39.3). Baseline demo-
graphic characteristics were similar between the three groups,
except for smoking (Po0.01).
EGFR-TKI treatment in patients with an EGFR exon 20
insertion. Sixteen patients with advanced-stage disease and an
exon 20 insertion received EGFR-TKI treatment. Seven patients
(43.8%) received EGFR-TKI as first-line treatment, but most
patients received EGFR-TKI treatment as second-, third- or
fourth-line treatment (Supplementary Table 5). Median PFS on
EGFR-TKI treatment was 2.9 months (95% CI, 2.3–3.6). Median
OS on EGFR-TKI treatment was 9.7 months (95% CI, 0.00–21.1).
Both PFS and OS on EGFR-TKI treatment were significantly
shorter in patients with an EGFR exon 20 insertion compared with
patients with a classic EGFR mutation (Po0.01 and P¼0.01,
respectively; Figure 2A and B). The ORR was 0.0% and DCR
was 56.3%.
EGFR-TKI treatment in patients with an uncommon EGFR
exon 18, 19, 20 and 21 mutation. Twenty patients with
an uncommon EGFR mutation received EGFR-TKI treatment.
Sixteen patients (80%) received EGFR-TKI treatment as first-line
240 EGFR-mutated NSCLC patients
EGFR exon 20 insertion
N=23 (9.6%)
Uncommon EGFR mutation
N=31 (12.9%)
Excluded *
N=62
Excluded *
N=1
Excluded *
N=3
No (follow-up on) EGFR-TKI
treatment
N=26
No (follow-up on) EGFR-TKI
treatment
N=10
No (follow-up on) EGFR-TKI
treatment
N=6
Follow-up data on EGFR-TKI
treatment:
N=18
Follow-up data on EGFR-TKI
treatment:
N=16
Follow-up data on EGFR-TKI
treatment:
N=98
Classic EGFR mutation
N=186 (77.5%)
Non-classic EGFR mutation
N=54 (22.5%)
Patients with classic EGFR
mutation
N=124
Patients with exon 20 insertion
N=22
Patients with uncommon EGFR
mutation
N=28
Figure 1. Flowchart. *No treatment and follow-up in VUmc.
BRITISH JOURNAL OF CANCER Unusual EGFR mutations and outcome in NSCLC
1506 www.bjcancer.com | DOI:10.1038/bjc.2016.372
and four patients (20%) as second-line treatment. For two patients,
there was no registered date of progression. Median PFS on EGFR-
TKI treatment for the remaining 18 patients (all advanced-stage
disease) was 6.4 months (95% CI, 0.0–17.6). This was not
significantly different compared with median PFS in patients with
a classic EGFR mutation (P¼0.39). Median OS on EGFR-TKI
treatment in patients with an uncommon EGFR mutation was 20.2
months (95% CI, 0.0–41.7). This was significantly shorter
compared with the median OS on EGFR-TKI treatment in patients
with a classic EGFR mutation (P¼0.04).
For 15 patients with uncommon EGFR mutations, data on
response on EGFR-TKI treatment could be retrieved from the
medical records: ORR was 53.3% and DCR was 86.7%.
Ten patients with single or double G719X/L861Q/S768I EGFR
mutations were treated with an EGFR-TKI. Median PFS on
EGFR-TKI treatment for patients with a double G719X/L861Q/S768I
EGFR mutation (N¼7) was 6.4 months (95% CI, 0.0–17.6), and this
was significantly longer (P¼0.02) than for patients with single-mutant
status at these loci (N¼3; 1.6 months (95% CI, 1.5–1.7)). Median OS
on EGFR-TKI treatment for patients with a double G719X/L861Q/
S768I EGFR mutation was 28.6 months (95% CI, 11.3–45.8), and 3.9
months (95% CI, 0.5–7.4) for those with a single G719X/L861Q/S768I
EGFR mutation (P¼0.02).
DISCUSSION
Targeted agents are being developed rapidly and their clinical use is
increasing in NSCLC patients. Considering the toxicities and costs
Table 1. Patient characteristics
Patient characteristics Classic EGFR mutation
(N¼124)
EGFR exon 20 insertion
(N¼22)
Uncommon EGFR
mutation (N¼28) P-value
Median age
a
(years) 61.5 (range 29.5–83.0) 61.0 (range 41.4–81.5) 0.43
Frequency (percentage) Frequency (percentage) Frequency (percentage)
Gender
Male 29 23.4% 8 36.4% 9 32.1% 0.34
Female 95 76.6% 14 63.6% 19 67.9%
Ethnicity
Caucasian 110 89.4% 20 90.9% 24 85.7% 0.81
Other
b
13 10.6% 2 9.1% 4 14.3%
Unknown 1—0—0——
Smoking
c
Current or previous smoker 50 43.5% 6 33.3% 20 74.1% 0.01
Never smoker 65 56.5% 12 66.7% 7 25.9%
Unknown 9—4—1——
Performance Status (PS)
PS 0–1 95 92.2% 13 100.0% 18 92.6% 0.53
PS 418 7.8% 0 0.0% 2 7.4%
Unknown 21 — 9 — 8 — —
Histology
Adenocarcinoma 114 91.9% 21 95.5% 26 92.9% 0.79
Squamous cell carcinoma 1 0.8% 0 0.0% 1 3.6%
Adenosquamous carcinoma 2 1.6% 0 0.0% 0 0.0%
Large-cell neuroendocrine carcinoma 4 3.2% 0 0.0% 0 0.0%
Large-cell carcinoma 3 2.4% 1 4.5% 1 3.6%
Stage
a
I–IIIA 25 20.7% 9 40.9% 3 10.7% 0.03
IIIB–IV 96 79.3% 13 59.1% 25 89.3%
Unknown 3—0—0——
Abbreviation: EGFR ¼epidermal growth factor receptor.
a
At the time of first diagnosis of non-small-cell lung cancer (NSCLC).
b
Afro-American or Oriental.
c
Patients who smoked o100 cigarettes in a lifetime were considered as nonsmokers, patients who smoked within the last year before diagnosis were considered as current smokers and the
remainder was considered as previous smoker.
Table 2. Median PFS, OS, ORR and DCR on EGFR-TKI
treatment in advanced-stage NSCLC patients with classic
EGFR mutations
NMonths (95% CI) P-value
Median PFS
All patients 98
a
12.2 (10.8–13.5)
Exon 19 75 12.6 (11.2–14.1) 0.26
Exon 21 23 12.0 (7.5–16.6)
Median OS
All patients 107
b
26.4 (22.8–30.1)
Exon 19 79 28.2 (21.8–34.6) 0.04
Exon 21 28 21.0 (20.4–21.6)
N%P-value
ORR
All patients 94
c
84.0%
Exon 19 70 84.3% 0.91
Exon 21 24 83.3%
DCR
All patients 94
c
95.7%
Exon 19 70 97.1% 0.25
Exon 21 24 91.7%
Abbreviations: CI ¼confidence interval; DCR ¼disease control rate; EGFR ¼epidermal
growth factor receptor; NSCLC ¼non-small-cell lung cancer; ORR ¼objective response
rate; OS ¼overall survival; PFS ¼progression-free survival; TKI¼tyrosine kinase inhibitor.
a
For 13 patients, data on PFS on EGFR-TKI treatment were incomplete (Supplementary
Table 2).
b
For 4 patients, data on OS on EGFR-TKI treatment were incomplete (Supplementary
Table 3).
c
For 17, patients data on response on EGFR-TKI treatment were incomplete
(Supplementary Table 4).
Unusual EGFR mutations and outcome in NSCLC BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2016.372 1507
of these drugs, their usage should be restricted to patients who
truly benefit from them. In lung cancer, the efficiency of EGFR-
TKIs is well known for classic EGFR mutations, but less data are
available for patients with non-classic EGFR mutations. Moreover,
most studies were performed in Asian populations. This study,
among Dutch EGFR-mutated NSCLC-patients, adds to the current
knowledge on non-classic EGFR mutations and the outcome on
EGFR-TKI treatment in this subgroup of lung cancer patients.
In our cohort of 240 EGFR-mutated NSCLC patients, 54
patients (22.5%) were identified with a non-classic EGFR-
mutation: 23 patients (9.6%) with an EGFR exon 20 insertion
and 31 patients (12.9%) with an uncommon EGFR mutation.
Table 3. Patients with single uncommon EGFR mutations
EGFR mutation Clinical characteristics EGFR-TKI
treatment
Exon Codon Protein
KRAS
mutation Sex Histology Smoking Stage
a
PS
a
PFS Response
18 c.2124G4T p.K708N No F Adeno Current No stage IV Unknown No TKI
18 c.2155G4A p.G719S No M Adeno Previous Stage IV PS 1 1.6 PD
18 c.2156G4C p.G719A No F Adeno Previous Stage IV Unknown 1.5 NA
18 c.2161G4T p.G721C Yes F SqCC Previous Stage IV PS 1 No TKI
19 c.2232 C4G p.I744M No M Adeno Current Stage IV Unknown No TKI
20 c.2379 G4T p.M793I No F Adeno Previous Stage IV PS 1 16.2 SD
20 c.2327G4A p.R776H No M Adeno Previous Stage IV PS 1 9.8 PR
20 c.2327G4A p.R776H No F Adeno Unknown Stage IV Unknown No TKI
20 c.2327G4T p.R776L No F Adeno Current Stage IV PS 1 2.6 SD
20 c.2335_2336GG4TT p.G779F No M Adeno Current Stage IV Unknown No TKI
21 c.2582T4A p.L861Q No M Adeno Current Stage IV PS 2 1.8 PR
21 c.2513T4G p.L838P Yes F Adeno Previous Stage IV PS 0 2.2 SD
21 c.2495G4A p.R832H Yes M Adeno Previous Stage IV PS 1 No TKI
19 c.2231 2232ins18 p.I744 K745insKIPVAI No F Adeno No clinical data
21 c.2582T4A p.L861Q No F Adeno No clinical data
Abbreviations: Adeno¼adenocarcinoma; EGFR ¼epidermal growth factor receptor;F¼female; M ¼male; NA ¼not available; PD ¼progressive disease; PFS ¼progression-free survival;
PR ¼partial response; PS ¼performance status; SD ¼stable disease; SqCC¼squamous cell carcinoma; TKI¼tyrosine kinase inhibitor.
a
At the time of first diagnosis of non-small-cell lung cancer (NSCLC).
Table 4. Patients with double EGFR mutations (and at least one uncommon EGFR mutation)
EGFR mutation Clinical characteristics EGFR-TKI
treatment
Classic/uncommon Exon Codon Protein Gender Histology Smoking Stage
a
PS
a
PFS Response
Double uncommon 18 þ18 c.2155G4Aþc.2125G4A p.G719S þp.E709K F Adeno Nonsmoker Stage IV Unknown 15.0 PR
Double uncommon 18 þ20 c.2155G4Tþc.2303G4T p.G719C þp.S768I F Adeno Previous Stage IV Unknown 25.0 NA
Double uncommon 18 þ20 c.2155G4Aþc.2303G4T p.G719S þp.S768I F Adeno Previous Stage IV PS 0 No TKI
Double uncommon 18 þ20 c.2155G4Aþc.2327G4A p.G719S þp.R776H M Adeno Previous Stage IV PS 0 59.1 PR
Double uncommon 18 þ20 c.2156G4Cþc.2303G4T p.G719A þp.S768I F Adeno Previous Stage IV Unknown 1.9 PD
Double uncommon 19 þ21 c.2239-2253del15bp þ
c.2509G4T
p.del L747_T751 þ
D837T
F Adeno Previous Stage IV PS 0 15.0 CR
Double uncommon 18 þ21 c.2156G4Cþc.2582T4A p.G719A þp.L861Q F Adeno Current Stage IV PS 1 2.1 SD
Double uncommon 20 þ20 c.2303G4Tþc.2305G4C p.S768I þp.V769L F Adeno Previous Stage IV PS 1 1.2 NA
Double uncommon 20 þ20 c.2303G4Tþc.2320G4A p.S768Iþp.V774M F Adeno Nonsmoker Stage IV PS 1 No TKI
Double uncommon 21 þ21 c.2497T4Gþc.2504A4T p.L833V þp.H835L F Adeno Nonsmoker Stage IV PS 2 11.7 PR
Double uncommon 21 þ21 c.2497T4Gþc.2504A4T p.L833V þp.H835L F Adeno Nonsmoker Stage IV PS 1 NA NA
Double uncommon 21 þ21 c.2512C4Gþc.2582T4A p.L838V þp.L861Q F Large-cell Nonsmoker Stage IV PS 1 6.4 NA
Classic þuncommon 21 þ21 c.2573T4Gþc.2618G4A p.L858R þp.G873E M Adeno Previous Stage IV PS 1 NA PR
Classic þuncommon 21 þ21 c.2573T4Gþc.2612C4A p.L858R þp.A871E F Adeno Nonsmoker Stage IV PS 0 18.0 PR
Classic þuncommon 21 þ20 c.2573T4Gþc.2369C4T p.L858R þp.T790M
(pre-treatment T790M)
M Adeno Nonsmoker Stage IV PS 0 8.0 SD
Classic þuncommon 21 þ21 c.2573T4Gþc.2500G4T p.L858R þp.V834L F Large-cell No clinical characteristics
Abbreviations: Adeno ¼adenocarcinoma; CR ¼complete response; EGFR ¼epidermal growth factor receptor;F¼female; Large-cell ¼large cell carcinoma; M ¼male; NA¼not available;
PD ¼progressive disease; PFS ¼progression-free survival; PR ¼partial response; PS ¼performance status; SD ¼stable disease; TKI ¼tyrosine kinase inhibitor.
a
At the time of first diagnosis of non-small-cell lung cancer (NSCLC).
BRITISH JOURNAL OF CANCER Unusual EGFR mutations and outcome in NSCLC
1508 www.bjcancer.com | DOI:10.1038/bjc.2016.372
Previous studies on EGFR exon 20 insertions in predominantly
non-Asian EGFR-mutated NSCLC patients reported a rate of 9%,
4.0% and 9.2% (Arcila et al, 2013; Oxnard et al, 2013; Beau-Faller
et al, 2014), hence incidence of EGFR exon 20 insertions in our
cohort is approximately in line with these studies. The incidence of
uncommon EGFR mutations among non-Asian EGFR-mutated
NSCLC patients varies between 5.9% and 20.4% (Pallis et al, 2007;
De Pas et al, 2011; Beau-Faller et al, 2014; Stone et al, 2014; Arrieta
et al, 2015; Lohinai et al, 2015), and this is also in accordance with
results from our study. However, comparison to other studies is
difficult, as there is a large variance in ethnicity of patients
included, detection method of EGFR mutations and categorisation
of non-classic EGFR mutations.
Interestingly, we detected a numerical difference in PFS
and OS between patients with a classic EGFR mutation (exon
19 vs exon 21 mutation) that was significantly different for OS in
favour of patients with an EGFR exon 19 deletion. Although
originally it was thought that there was no difference between
these two subtypes of classical EGFR mutations (Igawa et al,
2014), a meta-analysis detected a difference in PFS in favour
of patients with an EGFR exon 19 deletion (Zhang et al, 2014).
We did not detect a significant difference in PFS, but we did
detect a difference in OS between these groups in accordance
with a recent study (Rossi et al, 2016). Further investigation is
warranted.
Several studies reported a higher prevalence of EGFR exon 20
insertions among women, nonsmokers and Asians (Huang et al,
2004; Kosaka et al, 2004; Shigematsu et al, 2005; Sasaki et al, 2007;
Wu et al, 2008a), although another study did not find a significant
difference in age, sex, ethnic origin or stage at diagnosis when
compared with both patients with a classic EGFR mutation as in
patients lacking a mutation (Arcila et al, 2013). Survival of
NSCLC patients with an EGFR exon 20 insertion has generally
been reported to be poor (Oxnard et al, 2013). Most exon 20
insertions are insensitive for treatment with both reversible and
irreversible EGFR-TKIs (except for the EGFR exon 20 insertion
A763_Y764insFQEA; Yasuda et al, 2013). This insensitivity is
probably the reason of the poorer survival of this category of
patients compared with NSCLC patients with classic EGFR
mutations (Wu et al, 2008a; Lund-Iversen et al, 2012; Woo et al,
2014). In our cohort, PFS of patients with an EGFR exon 20
insertion on EGFR-TKI treatment was 2.9 months, comparable to
the PFS of 1.5–2.0 months on erlotinib or gefitinib (Wu et al,
2008a; Jackman et al, 2009) and 2.7 months on afatinib (Yang et al,
2015b) that were reported previously. This suggests that
these patients should preferably be treated with cytotoxic
chemotherapy instead of first- and second-generation EGFR-TKIs.
Recently, favourable results of a clinical study with AUY922
in NSCLC patients with an EGFR exon 20 insertion were
reported and may hopefully provide a better treatment option
for EGFR-mutated NSCLC patients with exon 20 insertion
(Piotrowska et al, 2015).
Likewise, it has been reported that patients with uncommon
EGFR mutations have lower EGFR-TKI sensitivity (Arrieta et al,
2015). We did not detect a statistically significant difference
between patients with uncommon and classic EGFR mutations
with respect to PFS, but considering the large numerical difference
(6.4 vs 12.0 months, respectively), this is probably because of the
small sample size and wide variation in PFS among patients with
an uncommon EGFR mutation (Tables 3 and 4).
In our study, G719X/L816Q/S768I EGFR mutations are the
most frequently detected among uncommon EGFR mutations, in
AB
CD
PFS (proportion)PFS (proportion)
OS (proportion)OS (proportion)
Duration of PFS on EGFR-TKI
treatment (months)
Duration of OS on EGFR-TKI
treatment (months)
Duration of OS on EGFR-TKI
treatment (months)
Duration of PFS on EGFR-TKI
treatment (months)
P = 0.39
Classic EGFR
Uncommon
EGFR mutation
P < 0.01
Classic EGFR
Exon 20 insertion
P = 0.04
Classic EGFR
Uncommon
EGFR mutation
Uncommon
EGFR mutation
censored
Classic EGFR-
censored
P = 0.01
Classic EGFR
Classic EGFR-
censored
Exon 20 insertion
Exon 20 insertion-
censored
1.0
0.8
0.6
0.4
0.2
0.0
0 1020304050
1.0
0.8
0.6
0.4
0.2
0.0
0 20 40 60 80 100
1.0
0.8
0.6
0.4
0.2
0.0
0 102030405060 0 20406080100
1.0
0.8
0.6
0.4
0.2
0.0
Figure 2. The PFS and OS on EGFR-TKI treatment in patients with a classic EGFR mutations vs EGFR exon 20 insertions or uncommon EGFR
mutations. Difference between classic EGFR mutations vs EGFR exon 20 insertions in PFS (A) and OS (B) and between classic EGFR mutations and
uncommon EGFR mutations in PFS (C) and OS (D). A full colour version of this figure is available at British Journal Of Cancer online.
Unusual EGFR mutations and outcome in NSCLC BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2016.372 1509
line with previous reports (Mitsudomi and Yatabe, 2007; Shi et al,
2014). The G719X and the L861Q EGFR mutations were reported
to have a shorter OS on gefitinib compared with classic EGFR
mutations (Watanabe et al, 2014), although a recent study reported
a PFS and OS of 13.8 and 26.9 months, respectively, for patients
with a G719X EGFR mutation on first-line afatinib (Yang et al,
2015a). Chiu et al (2015) detected a statistically significant difference
in median PFS on EGFR-TKI between patients (N¼161) with single
and double G719X/L816Q/S768I EGFR mutations. In our cohort,
patients with double G719X/L816Q/S768I EGFR mutations not only
had a statistically significant longer PFS on EGFR-TKI treatment
compared with patients with single-mutant status at these loci, but
also a longer OS. However, groups were small in our cohort, and
hence interpretation should be with caution. Further investigation on
the difference between single and double G719X, L816Q and/or
S768I EGFR mutations is warranted.
It has been suggested that platinum-doublet treatment might be
the best first-line treatment option for patients with (both single
and double) G719X/L816Q/S768I EGFR mutations (Watanabe
et al, 2014). However, taking into account the durable responses on
EGFR-TKI treatment of several patients with a double EGFR
mutation that included G719X, L861Q and/or S768I, in our
opinion EGFR-TKI treatment could be considered as first-line
treatment for these patients. In addition, high response rates of
patients with G719X/L816Q/S768I EGFR mutations to first-line
afatinib were recently reported (Yang et al, 2015a). Prospective
trials are needed to elucidate this question.
Several limitations should be taken into account for this study.
Because of the retrospective design, bias cannot be excluded. In
addition, in a considerable part of the patients (especially in
patients with an EGFR exon 20 insertion) data on performance
score and smoking could not be retrieved from the medical records
and the line of EGFR-TKI treatment (i.e., first-, second-line and so
on) varied. Perhaps therefore, we detected a significant difference
between the groups in smoking. In addition, in the early days of
EGFR testing, clinical characteristics were taken into account.
Therefore, there might have been a screening bias for women and
nonsmokers. However, from 2012, all stage IV adenocarcinoma
patients were tested for EGFR mutations, irrespective of gender,
smoking status and race. A large molecular heterogeneity existed
among patients with non-classic EGFR mutations. The results of
the subgroup analyses should therefore be interpreted with caution.
Furthermore, in routine pathology, solely tumour tissue is
evaluated, and for most cases of our study no normal DNA was
available to confirm the somatic origin of the mutations identified.
The R776H mutation (detected in three patients in our cohort), for
example, has both been reported as somatic and germline
(Nagalakshmi et al, 2013; van Noesel et al, 2013). For one of our
patients, analysis of normal DNA confirmed somatic nature (data
not shown), but for the others because of absence of normal DNA
a germline nature cannot be excluded.
To summarise, in this cohort of Dutch EGFR-mutated NSCLC
patients, the prevalence and genotype distribution of non-classic
EGFR mutations was in accordance with previously published
studies among non-Asian, EGFR-mutated NSCLC patients. Out-
come on EGFR-TKI treatment was poor for patients with EGFR
exon 20 insertions and varied widely in patients with uncommon
EGFR mutations. Further (prospective) studies on patients with
non-classic EGFR mutations are warranted to hopefully improve
prognosis of these patients.
CONFLICT OF INTEREST
D Heideman has occasionally been member of the scientific
advisory boards of Amgen and Pfizer.
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Supplementary Information accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc)
BRITISH JOURNAL OF CANCER Unusual EGFR mutations and outcome in NSCLC
1512 www.bjcancer.com | DOI:10.1038/bjc.2016.372
