Lung Cancer 116 (2018) 1–6

Contents lists available at ScienceDirect

Lung Cancer
journal homepage: www.elsevier.com/locate/lungcan

PD-L1 expression according to the EGFR status in primary lung T

adenocarcinoma
⁎
Kazuki Takadaa, , Gouji Toyokawaa, Tetsuzo Tagawaa, Kenichi Kohashib,
Mototsugu Shimokawac, Takaki Akaminea, Shinkichi Takamoria, Fumihiko Hiraia,
Fumihiro Shojia, Tatsuro Okamotoa, Yoshinao Odab, Yoshihiko Maeharaa
a
Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
b
Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
c
Clinical Research Institute, National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Objectives: It was reported that programmed cell death-ligand 1 (PD-L1) expression is associated with smoking
PD-L1 and wild-type epidermal growth factor receptor (EGFR) in lung adenocarcinoma. However, the association between
EGFR PD-L1 expression and EGFR mutation site in EGFR mutation-positive lung adenocarcinoma is unclear.
Lung adenocarcinoma Materials and methods: We retrospectively examined the relationship between PD-L1 expression and EGFR status
in 441 surgically resected primary lung adenocarcinomas. Membrane PD-L1 expression on tumor cells was
evaluated by immunohistochemical analysis using a PD-L1 antibody (clone SP142) and defined by tumor pro-
portion scores (TPSs) of 0%, 1–4%, 5–49%, and ≥50%, respectively.
Results: Two hundred and eighteen (49.4%) patients had wild-type EGFR, and 223 (50.6%) had mutant
EGFR—98 (44.0%) with exon 19 deletion, 116 (52.0%) with exon 21 L858R point mutation, and nine (4.0%)
with another EGFR mutation. Overall, Fisher’s exact test showed that PD-L1 positivity was associated with wild-
type EGFR, and there was only one case with PD-L1 TPS ≥50% among the cases with mutant EGFR. The analysis
of cases with mutant EGFR indicated no significant association between EGFR mutation site and PD-L1 ex-
pression. However, the prevalence of PD-L1 TPS 5–49% was higher among patients with EGFR exon 19 deletion
than with EGFR exon 21 L858R point mutation.
Conclusions: PD-L1 expression was significantly associated with wild-type EGFR, and PD-L1 TPS ≥50% seldom
overlaps with presence of driver oncogene EGFR. There was no significant difference in PD-L1 expression among
the EGFR mutation sites

1. Introduction the outcome of immunotherapy. For example, patients with smoking

Immune checkpoint inhibitors have been developed to target pro-
grammed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-
L1). PD-1 inhibitors such as nivolumab in the CheckMate study and
pembrolizumab in the KEYNOTE study, and PD-L1 inhibitors including
atezolizumab in the POPLAR and OAK studies, have provided survival
benefit in non-small cell lung cancer (NSCLC) compared with conven-
tional standard therapy [1–6]. Given these results, PD-1 inhibitors have
become the standard treatment for advanced-stage NSCLC patients.
Currently, there is no ideal biomarker for the response to immune
checkpoint inhibitors; however, the immunohistochemical analysis of
PD-L1 expression on tumor cells, which is associated with the response
to immune checkpoint inhibitors, is used as a predictive biomarker for
history and wild-type epidermal growth factor receptor (EGFR) associated
with high PD-L1 expression in lung adenocarcinoma, had a higher
sensitivity to immune checkpoint inhibitors in some clinical trials
[2,3,6–8]. However, some patients with EGFR mutation-positive lung
adenocarcinoma also responded to immunotherapy. However, why
there is a difference in benefit by immunotherapy among cases with
mutant EGFR is not clear.
Recently, a difference in the therapeutic effect of an EGFR-tyrosine
kinase inhibitor (TKI) according to the mutation site in cases with
mutant EGFR was reported [9]. Therefore, it is necessary to consider the
presence of mutant EGFR and the mutation site when managing the
therapeutic strategy for lung adenocarcinoma. If there is a difference in
benefit by immunotherapy among cases with mutant EGFR because of

⁎
Corresponding author at: Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan.
E-mail address: k_takada@surg2.med.kyushu-u.ac.jp (K. Takada).

https://doi.org/10.1016/j.lungcan.2017.12.003
Received 16 June 2017; Received in revised form 22 October 2017; Accepted 4 December 2017
0169-5002/ © 2017 Elsevier B.V. All rights reserved.

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K. Takada et al. Lung Cancer 116 (2018) 1–6

Fig. 1. Representative images of immunohistochemical staining for PD-L1 in surgically resected tumors from patients with primary lung adenocarcinoma. (A) PD-L1 TPS 0%. (B) PD-L1
TPS 1–4%. (C) PD-L1 TPS 5–49%. (D) PD-L1 TPS ≥50%. PD-L1: programmed cell death-ligand 1, TPS: tumor proportion score. Scale bar: 500 μm.

differential PD-L1 expression according to the mutation site, then in-
formation regarding the mutation site as well as the presence of mutant
EGFR should be considered when using immune checkpoint inhibitors.
However, the relationship between PD-L1 expression and EGFR muta-
tion site in lung adenocarcinoma harboring EGFR mutation is unclear.
In this translational study, we examined the expression of PD-L1
protein in 441 surgically resected primary lung adenocarcinoma pa-
tients, and investigated the association between PD-L1 expression and
EGFR status. Furthermore, we discuss the therapeutic strategy for the
management of advanced lung adenocarcinoma harboring EGFR mu-
tation.
2. Materials and methods
2.1. Patients and samples
We retrospectively examined patients who underwent surgical re-
section of their primary lung adenocarcinoma between January 2005
and December 2015 at the Department of Surgery and Science,
Graduate School of Medical Sciences, Kyushu University. Of these, only
those cases with available information regarding the EGFR mutation
site and presence of mutant EGFR were selected. Finally, 441 paraffin-
embedded specimens were available and retrieved from the registry of
the Department of Anatomic Pathology, Graduate School of Medical
Sciences, Kyushu University. EGFR status was determined in tumor
tissues using the peptide nucleic acid-locked nucleic acid (PNA-LNA)
polymerase chain reaction clamp method (Mitsubishi Chemical
Medience, Tokyo, Japan). All clinical information was obtained from
medical records. This study was approved by our institutional review
board (Kyushu University, IRB No. 28-100).
2.2. Immunohistochemical analysis
Immunohistochemistry was performed in 441 cases of surgically
resected primary lung adenocarcinoma using formalin-fixed tissue
sections according to our PD-L1 immunohistochemistry protocol as
described previously [7,8,10–13].
The primary antibody was an anti-human PD-L1 rabbit monoclonal
antibody (clone SP142, dilution 1:100; Spring Bioscience, Ventana,
Tucson, AZ). Carcinoma cells showing membranous staining for PD-L1
were evaluated as positive cells. The proportion of PD-L1-positive cells
was independently estimated as the percentage of total carcinoma cells
in whole sections by three investigators (K.T., K.K., and S.T.). If the
independent assessments did not agree, the slides were reviewed by all
three investigators together to achieve consensus. The consensus
judgments were adopted as the final results. In this study, membrane
PD-L1 expression on tumor cells was defined by tumor proportion
scores (TPSs) of 0%, 1–4%, 5–49%, and ≥50%, respectively, consistent
with the cut-offs used in atezolizumab clinical trials [1,4]. Fig. 1 shows
the representative images of (A) PD-L1 TPS 0%, (B) PD-L1 TPS 1–4%,
(C) PD-L1 TPS 5–49%, and (D) PD-L1 TPS ≥50%, respectively.
2.3. Statistical analysis
Associations between EGFR status, its mutation site, and smoking
status and PD-L1 expression were analyzed using Fisher’s exact test. We
examined the association between EGFR status and smoking status
using the Student’s t-test. All statistical analyses were performed by
JMP Statistical Discovery Software (v11.0; SAS Institute, Cary, NC,
USA). P values < 0.05 were statistically significant.

2
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K. Takada et al. Lung Cancer 116 (2018) 1–6

Table 1 (49.4%) were smokers; the median age of all patients was 69 years
Clinicopathological characteristics of all patients. (range, 29–88 years). Two hundred and eighteen (49.4%) patients had
wild-type EGFR, and 223 (50.6%) had mutant EGFR—98 (44.0%) with
Factors Value or number of patients
exon 19 deletion, 116 (52.0%) with exon 21 L858R point mutation, and
Age (years) Median 69 nine (4.0%) with another EGFR mutation (Table 1 and Supplementary
Range 29–88 Fig. 1).
Sex Male 214 Table 2 shows the association between EGFR status and PD-L1 ex-
Female 227 pression in all patients, and Supplementary Fig. 2A and 2B show a pie
Smoking status Never-smoker 223 chart representation of PD-L1 TPS in cases with or without EGFR mu-
Smoker 218 tation, respectively. In the cases with wild-type EGFR, there were 83
Grade G1 256 (38.1%) cases with PD-L1 expression, while there were 41 (18.4%)
G2 137 cases with PD-L1 expression in the cases with mutant EGFR. Fisher’s
G3 48 exact test showed that PD-L1 positivity was significantly associated
T T1 277 with wild-type EGFR (P < 0.0001). In cases with wild-type EGFR,
T2 138 there were 18 (8.3%) cases with PD-L1 TPS ≥50%, but there was only
T3 23
one (0.5%) case with PD-L1 TPS ≥50% in all cases with mutant EGFR.
T4 3

N N0 368 3.2. Association between EGFR mutation site and PD-L1 expression in
N1 34
N2 39
primary lung adenocarcinoma harboring EGFR mutation
N3 0
Next, we assessed the association between EGFR mutation site and
M M0 433
M1 8 PD-L1 expression in cases harboring EGFR mutation (Table 3). Sup-
plementary Fig. 2C and 2D show a pie chart representation of PD-L1
Stage IA 256
IB 83 TPS in cases with EGFR exon 19 deletion or cases with EGFR exon 21
IIA 31 L858R point mutation, respectively. There was only one (0.5%) case
IIB 23 with PD-L1 TPS ≥50% among cases with mutant EGFR, and that case
IIIA 38 had an exon 21 L858R point mutation. Regarding cases with a minor
IIIB 2
IV 8
mutation, there was only one (11.1%) case with a PD-L1 TPS of at least
1%. In the analysis (except cases with a minor mutation) there was no
pl* No 356
significant association between EGFR mutation site and PD-L1 expres-
Yes 84
sion (P = 0.5999). However, the prevalence of PD-L1 TPS 5–49% was
ly No 393
higher among patients with EGFR exon 19 deletion compared with
Yes 48
EGFR exon 21 L858R point mutation (12.2% vs 2.6%).
v No 337
Yes 104
3.3. Association between EGFR status and smoking status in primary lung
EGFR Wild-type 218 adenocarcinoma
Mutant
Exon 19 del 98
Exon 21 L858R 116 To determine the difference in prevalence of PD-L1 TPS 5–49%
Others** 9 between patients with EGFR exon 19 deletion and those with EGFR
exon 21 L858R point mutation, we examined the association between
pl: pleural invasion, ly: lymphatic invasion, v: vascular invasion, EGFR: epidermal growth EGFR status, its mutation site and smoking status. The pack year index
factor receptor.
was significantly higher in patients without EGFR mutation than in
* Cases for which data were available.
** Exon 21 L861Q and Exon 18 G719A/G719C/G719S. those with EGFR mutation (P < 0.0001; Fig. 2A). Moreover, there was
no significant correlation between EGFR mutation site and pack year
Table 2 index, although the pack year index was higher in patients with EGFR
Association between EGFR status and PD-L1 expression in primary lung adenocarcinoma. exon 19 deletion than in those with EGFR exon 21 L858R point muta-
tion (P = 0.0828; Fig. 2B). The average pack year index of the patients
PD-L1 TPS, N Wild-type EGFR Mutant EGFR P value with EGFR exon 19 deletion was 10.2 pack year index (range, 0–87.5
(%) (N = 218) (N = 223)
pack year index), while that of the patients with EGFR exon 21 L858R
0% 135 (61.9) 182 (81.6) < 0.0001* point mutation was 7.2 pack year index (range, 0–69 pack year index).
1–4% 24 (11.0) 25 (11.2) In cases with EGFR exon 19 deletion, there were 35 (35.7%) cases with
5–49% 41 (18.8) 15 (6.7) smoking history, while there were 34 (29.3%) cases with smoking
≥50% 18 (8.3) 1 (0.5)
history in cases with EGFR exon 21 L858R point mutation.
EGFR: epidermal growth factor receptor, PD-L1: programmed cell death-ligand 1, TPS:
tumor proportion score. 3.4. Association between smoking status and PD-L1 expression in primary
* PD-L1 TPS < 1% versus ≥ 1%. lung adenocarcinoma

3. Results Finally, we examined the association between smoking status and

3.1. Association between EGFR status and PD-L1 expression in primary
lung adenocarcinoma
Overall, 441 patients with primary lung adenocarcinoma who un-
derwent surgical resection were included in the present study (Table 1).
Two hundred and fourteen (48.5%) patients were male, and 218
PD-L1 expression in primary lung adenocarcinoma. Supplementary
Table 1 shows the association between smoking status and PD-L1 ex-
pression in primary lung adenocarcinoma, and Fisher’s exact test
showed that PD-L1 positivity was significantly associated with smoker
(P = 0.0021). However, in the subgroup analyses according to the
EGFR status, there were no significant associations between smoking
status and PD-L1 expression in primary lung adenocarcinoma with and
without EGFR mutation (Supplementary Tables 2 and 3).

3
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K. Takada et al. Lung Cancer 116 (2018) 1–6

Table 3 increase the number of mutations or specific immunogenic mutations,

EGFR mutation site and PD-L1 expression in primary lung adenocarcinoma harboring which in turn cause PD-L1 to be upregulated [21]. Therefore, PD-L1
EGFR mutation.
expression in NSCLC may be related to smoking and mutation burdens.
PD-L1 TPS, N Exon 19 del Exon 21 L858R P value Others* In this study, we did not assess mutation burdens, but the pack year
(%) (N = 98) (N = 116) (N = 9) index was higher in patients with EGFR exon 19 deletion than in those
with EGFR exon 21 L858R point mutation (P = 0.0828; Fig. 2B). These
0% 78 (79.6) 96 (82.8) 0.5999** 8 (88.9)
findings indicate there may be a difference in tumor microenvironment
1–4% 8 (8.2) 16 (13.8) 1 (11.1)
5–49% 12 (12.2) 3 (2.6) 0 (0.0) between cases with EGFR exon 19 deletion and those with EGFR exon
≥50% 0 (0.0) 1 (0.8) 0 (0.0) 21 L858R point mutation. Future, larger studies should examine im-
mune-related factors such as cluster of differentiation (CD) 3-positive,
EGFR: epidermal growth factor receptor, PD-L1: programmed cell death-ligand 1, TPS: CD4-positive, and CD8-positive cells.
tumor proportion score.
There may be another reason for the difference in PD-L1 expression
* Exon 21 L861Q and Exon 18 G719A/G719C/G719S.
** PD-L1 TPS < 1% versus ≥ 1%.
between cases with EGFR exon 19 deletion and those with EGFR exon
21 L858R point mutation. Banno et al. recently reported that EGFR exon
4. Discussion 19 deletion enhanced EGFR phosphorylation compared with EGFR exon
21 L858R point mutation [22]. The phosphorylation of EGFR leads to
In the current paper, there was no significant difference in PD-L1 activation of the AKT- mammalian target of rapamycin (mTOR)
expression between cases with EGFR exon 19 deletion and those with pathway, which increases PD-L1 protein expression in NSCLC [23].
EGFR exon 21 L858R point mutation, which are the most common Therefore, the difference in the degree of the phosphorylation of EGFR
EGFR mutations [15]. However, the prevalence of PD-L1 TPS 5–49% between EGFR exon 19 deletion and EGFR exon 21 L858R point mu-
was higher among patients with EGFR exon 19 deletion than with EGFR tation may contribute to the difference in PD-L1 expression.
exon 21 L858R point mutation (12.2% vs 2.6%). The reason for this This result may be important when determining the therapeutic
difference is not clear, but this finding might be important when de- strategy of advanced lung adenocarcinoma harboring EGFR mutation.
termining the therapeutic strategy for advanced lung adenocarcinoma Fig. 3A shows the standard therapeutic strategy of EGFR mutation-po-
harboring EGFR mutation. sitive advanced lung adenocarcinoma; after disease recurrence or
Recent genetic analyses showed that squamous cell carcinoma and during progression EGFR-TKI is used as a first line therapy. Patients
adenocarcinoma without EGFR mutation had larger somatic mutation undergo re-biopsy to identify T790M mutation, and then treatment
burdens than NSCLC in never-smokers, and tumors with a larger with EGFR-TKI or cytotoxic chemotherapy (+anti-PD-1/PD-L1) as a
number of somatic mutations were more sensitive to immunotherapy second line therapy is determined. Gainor et al. recently reported that
with PD-1/PD-L1 inhibitors [16–18]. Moreover, The Cancer Genome about 20% of EGFR mutation-positive NSCLC patients demonstrated
Atlas project revealed that melanoma and NSCLC, which showed sig- increased PD-L1 expression in the post-TKI specimens compared to the
nificant sensitivity to immune checkpoint inhibitors, had the greatest pre-TKI specimens [24]. Therefore, after EGFR-TKI is used as a first line
mutational burden per cell compared with other solid tumors [16,19]. therapy, we should examine both the presence of T790M mutation and
These data suggested that tumors with a greater number of somatic PD-L1 expression, especially in cases with EGFR exon 19 deletion, be-
mutations produce more immunogenic neoantigens, which might be cause there may be some cases with PD-L1 TPS ≥50%. Based on this
more frequently recognized and attacked by cytotoxic T-lymphocytes result, we should determine the treatment, EGFR-TKI, cytotoxic che-
[19]. One of the representative mechanisms of PD-L1 expression on motherapy (+anti-PD-1/PD-L1), or anti-PD-1/PD-L1, as a second line
tumor cells is termed adaptive immune resistance and describes cyto- therapy (Fig. 3B).
kines, particularly interferon-γ produced by cytotoxic T-lymphocytes, In the present study, there was only one (0.5%) case with PD-L1 TPS
which induces the expression of PD-L1 on tumor cells and inflammatory ≥50% in cases with mutant EGFR. Recently, Rangachari et al. reported
cells [20]. Moreover, Calles et al. recently reported that smoking might that a PD-L1 TPS of at least 50% seldom overlapped with the presence

Fig. 2. Smoking status (pack year index) according to (A) EGFR status and (B) EGFR mutation site. (A) Association between EGFR status and smoking status (pack year index). The pack
year index was significantly higher in patients without EGFR mutation than in those with EGFR mutation (P < 0.0001). (B) Association between EGFR mutation site and smoking status
(pack year index). There was no significant correlation between EGFR mutation site and the pack year index, but the pack year index was higher in patients with EGFR exon 19 deletion
than in those with EGFR exon 21 L858R point mutation (P = 0.0828). EGFR: epidermal growth factor receptor.

4
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K. Takada et al. Lung Cancer 116 (2018) 1–6

Fig. 3. Therapeutic strategy for patients with advanced lung adenocarcinoma harboring EGFR mutation. (A) Standard therapeutic strategy. After treatment with EGFR-TKI and disease
progression occurs, the patients undergo re-biopsy to identify T790 M mutation. (B) Proposed therapeutic strategy. After treatment with EGFR-TKI and disease progression occurs, the
patients undergo re-biopsy to identify T790M mutation and evaluate PD-L1 expression, especially in cases with EGFR exon 19 deletion. EGFR: epidermal growth factor receptor, TKI:
tyrosine kinase inhibitor, PD-L1: programmed cell death-ligand 1.

of driver oncogenes with approved targeted therapies, EGFR, anaplastic Acknowledgements

lymphoma receptor kinase gene (ALK), and ROS1 [25]. We used the
antibody clone SP142 to examine PD-L1 expression, which is different
from the antibody clone 22C3 used in the report by Rangachari et al.,
where most cases with mutant EGFR did not have PD-L1 TPS ≥50%.
The present study had several limitations. First, it was a single in-
stitutional retrospective study and not a trial-based correlative study;
thus, the possibility of bias could not be excluded. Second, PD-L1 im-
munohistochemistry was conducted using only one antibody. Recently,
McLaughlin et al. reported that discordance was observed in the ex-
pression of PD-L1 protein within a sample when using different anti-
bodies [26]. According to the report by the Blueprint Working Group,
the positive rate using clone SP142 was lower than that obtained using
other antibodies, such as 28-8, 22C3 and SP263 [27]. Several anti-
bodies should be evaluated in future studies.
In conclusion, PD-L1 expression was significantly associated with
wild-type EGFR. A PD-L1 TPS ≥50% seldom overlaps with the presence
of driver oncogene EGFR. There was no significant difference in PD-L1
expression among EGFR mutation sites. However, information about
the EGFR mutation site may lead us to use immunotherapy with PD-1/
PD-L1 inhibitors as a second line therapy for advanced lung adeno-
carcinoma harboring EGFR mutation.
Disclosure
The authors have declared no conflicts of interest.
Funding statement
This work was not supported by any funding sources.
None.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.lungcan.2017.12.003.
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