PD-L1 expression in papillary renal cell carcinoma
Motoshima et al. BMC Urology
PD-L1 expression in papillary renal cell carcinoma
Takanobu Motoshima 0 2
Yoshihiro Komohara 0
Chaoya Ma 0
Arni Kusuma Dewi 0 1
Hirotsugu Noguchi 6
Sohsuke Yamada 6
Toshiyuki Nakayama 6
Shohei Kitada 5
Yoshiaki Kawano 2
Wataru Takahashi 2
Masaaki Sugimoto 3 4
Motohiro Takeya 2
Naohiro Fujimoto 5
Yoshinao Oda 4
Masatoshi Eto 2 3
0 Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University , Kumamoto 860-8556 , Japan
1 Department of Anatomy Histology, Faculty of Medicine Airlangga University , Surabaya , Indonesia
2 Department of Urology, Graduate School of Medical Sciences, Kumamoto University , Kumamoto , Japan
3 Department of Urology, Graduate School of Medical Sciences, Kyushu University , Fukuoka , Japan
4 Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University , Fukuoka , Japan
5 Department of Urology, School of Medicine, University of Occupational and Environmental Health , Kitakyushu , Japan
6 Department of Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health , Kitakyushu , Japan
Background: The immune escape or tolerance of cancer cells is considered to be closely involved in cancer progression. Programmed death-1 (PD-1) is an inhibitory receptor expressed on activating T cells, and several types of cancer cells were found to express PD-1 ligand 1 (PD-L1) and ligand 2 (PD-L2). Methods: In the present study, we investigated PD-L1/2 expression in papillary renal cell carcinoma (pRCC). Result: We found PD-L1 expression in 29 of 102 cases, but no PD-L2 expression was seen. PD-L1 expression was not significantly correlated with any clinicopathological factor, including progression-free survival and overall survival. The frequency of PD-L1-positive cases was higher in type 2 (36%) than in type 1 (22%) pRCC; however, there was no significant difference in the percentages of score 0 cases (p value = 0.084 in Chi-square test). The frequency of high PD-L1 expression cases was higher in type 2 (23%) than in type 1 (11%), and the frequency of high PD-L1 expression cases was higher in grade 3/4 (21%) than in grade 1/2 (13%). However, no significant association was found between PD-L1 expression and all clinicopathological factors in pRCC. Conclusion: High expression of PD-L1 in cancer cells was potentially associated to highly histological grade of malignancy in pRCC. The evaluation of the PD-L1 protein might still be useful for predicting the efficacy of anti-cancer immunotherapy using immuno-checkpoint inhibitors, however, not be useful for predicting the clinical prognosis.
Renal cell carcinoma (RCC) is a common cancer of the
kidney, and the three most frequent histological subtypes
are clear cell RCC (ccRCC, 70 to 80%), papillary RCC
(pRCCc, 10 to 20%), and chromophobe RCC (5%) .
Although patients with sporadic RCC of any histological
subtype usually show a good clinical outcome, patients
with metastatic pRCC show a significantly worse clinical
course than patients with ccRCC or chromophobe RCC
[2–4]. Recent findings have indicated that MET gene
activation, which is known to promote proliferative
activity and cell survival, is frequently observed in pRCC,
and MET inhibitors have become a new type of
therapeutic agent for patients with advanced pRCC [5, 6].
Anti-cancer immune responses were considered to
play important roles in preventing cancer progression in
RCC ; however, immunotherapy against advanced
RCC such as interferon therapy and vaccine therapy
has shown limited anti-cancer effects over the past
fewdecades . In recent years, immuno-checkpoint
inhibitors have attracted much attention.
Anti-CTLA4 antibodies, which are used to treat advanced
melanoma patients, have been reported to have an excellent
therapeutic effect . Subsequently, anti-PD-1
antibody was discovered and used to treat kidney
cancer, non-small cell lung cancer and malignant
melanoma patients, and superior therapeutic effects
have been reported . Some clinical trials
demonstrated that combination therapy using anti-CTLA-4
antibody and anti-PD-1 antibody produced significant
anti-cancer effects . However, although the
antibodies produced excellent therapeutic effects in most
patients, no therapeutic effect was observed in some
patients. PD-1 ligand 1 (PD-L1) expression in cancer
tissues is considered to be a biomarker for predicting the
therapeutic effect of immuno-checkpoint inhibitors .
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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Although some studies have demonstrated that a high
expression of PD-L1 was associated with poor clinical
outcomes [13–17], few studies have investigated PD-L1
expression in pRCC. Therefore, we analyzed the
correlation between PD-L1 expression and clinicopathological
factors in pRCC.
Patients and Samples
We reviewed 102 cases of pRCC that were excised at
Kumamoto University, the University of Occupational
and Environmental Health, and Kyushu University
between 2001 and 2014. All samples were obtained with
informed consent from patients in accordance with the
study protocols that were approved by the review board
of each university (Kumamoto University Hospital
Review Board, Kyushu University Review Board, Review
Board of University of Occupational and Environmental
Health). Tissue samples of primary site were fixed in
10% neutral buffered formalin and were embedded in
paraffin as per a routine method. Nuclear grade and T
classification were assessed according to the World Health
Organization classification. Patient characteristics, such as
age, gender, Fuhrman grade, pathological TNM stage and
follow-up data were retrospectively collected.
Rabbit monoclonal antibodies against PD-L1 (clone
E1L3N) and PD-L2 (clone D7U8C) were purchased from
Cell Signaling Technology (Danvers, MA, USA). Briefly,
after samples were reacted with primary antibodies, they
were incubated with horseradish peroxidase
(HRP)-labeled goat anti-rabbit secondary antibodies (Nichirei,
Tokyo, Japan). Can Get Signal Solution (TOYOBO,
Tokyo, Japan) was used to dilute the antibodies for
enhancing the immunoreaction. Reactions were visualized
using the diaminobenzidine substrate system (Nichirei).
Two pathologists (TM and YK), who were blinded to
information about the samples, evaluated the
immunostaining of PD-L1/2.
Two lymphoma cell lines (PD-L1/2-positive cell line;
ATL-T, PD-L1/2-negative cell line; DAUDI) were
obtained from RIKEN Cell Bank (Tsukuba, Japan), and
cell block specimens were used for confirmation of
specificities of anti-PD-L1/2 antibodies. Cells were fixed in
10% neutral buffered formalin and cell block samples
were embedded in paraffin.
Statistical analysis was carried out with StatMate III
(Atoms, Tokyo, Japan). The simultaneous relationships
between multiple prognostic factors for survival were
assessed using the Cox proportional hazards model with
a stepwise backwards reduction. A value of p < 0.05 was
considered to be statistically significant.
PD-L1/B7-H1 expression in pRCC and correlations with
At first, we confirmed the specificities of anti-PD-L1 and
PD-L2 monoclonal antibodies used for immunostaining
using cell block samples of two cell lines (Fig. 1a). In
total, 102 resected specimens of pRCC were used for
immunostaining of PD-L1/B7-H1; the characteristics of
the patients are shown in Table 1. PD-L1-positive cancer
cells were detected in 29 of the 102 cases, and the
positive signals were detected on the cell surface membrane
and cytoplasm of the cancer cells (Fig. 1b). Macrophages
stained positive for PD-L1 in 4 cases (Fig. 1c), but no
infiltrated lymphocytes were positive for PD-L1. Notably,
neurons were strongly positive for PD-L1 in all cases
(Fig. 1d). In contrast, no expression of PD-L2 was
observed on any of the cancer cells or infiltrating
macrophages and lymphocytes (Fig. 1e).
Next, we classified the PD-L1 staining patterns into three
scores according to the percentage of PD-L1-positive cancer
cells: score 0, negative or less than 2%; score 1, 2 to 30%;
and score 2, more than 30% (Fig. 2a, Additional file 1 table
S1). Overall, the most frequent pattern was score 0 (71%),
while score 1 and score 2 comprised 11 and 18%,
respectively, of the cases. Since there are two histological
subtypes of pRCC (types 1 and 2), the frequencies of scores
0 1 and 2 were compared between the subtypes. The
frequency of score 1/2 (PD-L1-positive) was higher in
type 2 (36%) than in type 1 (22%) pRCC; however,
there was no significant difference in the percentages
of score 0 cases (p value = 0.084 in Chi-square test,
Fig. 2). Next, the frequencies of scores 0, 1 and 2
were compared between the nuclear grades. The
frequency of score 1/2 (PD-L1-positive) was slightly
higher in grade 3/4 (32%) than in grade 1/2 (25%);
however, there was no significant difference between
grade 1/2 and grade 3/4 (p value = 0.47 in Chi-square
test, Fig. 2). The percentage of score 2 seemed to be
higher in Type 2 (23%) or grade 3/4 (21%) than that
in Type 1 (11%) or grade 1/2 (13%); however,
however, there was no significant difference (p value =
0.42 or 0.36 in Chi-square test respectively, Fig. 2).
Although score 0 cases seemed to have shorter
progression-free survival and longer cancer specific
overall survival, there was no significant correlation
between PD-L1 scores and clinical course (Fig. 3).
Expression of PD-L1 is associated with a poor clinical
course in colorectal cancer, lung cancer, ovarian cancer,
Fig. 1 PD-L1/2 expression in pRCC a Pictures of immunostaining of
PDL1/2 in two cell lines (PD-L1/2-positive cell line; ATL-T, PD-L1/2-negative
cell line; DAUDI) b Pictures of H.E. staining and immunostaining of PD-L1
in 2 cases of pRCC. Positive signals are detected most strongly in cancer
cells c Positive signals for PD-L1 are also detected in macrophages
d Positive signals for PD-L1 are also detected in neuronal fibers e PD-L2 is
detected in dendritic cells in the lymph node, whereas no PD-L2 is
observed in the cancer cells of any of the pRCC cases
and ccRCC [17–22]. In this study, we demonstrated that
PD-L1 expression on cancer cells is not useful as a
biomarker in pRCC. PD-L1 expression is positive in
approximately 50% of ccRCC cases [13, 17, 18], which is
much higher than in the pRCC cases of the present
study. PD-L1 expression is known to be induced by
infiltrating T cell-derived interferon γ. It is well known that
ccRCC is an immunogenic cancer and many T-cell
infiltrations are detected in ccRCC . However, T-cell
Table 1 Characteristic of pRCC patients
Cancer specific death 14
infiltration was lower in pRCC than in ccRCC in our
preliminary observations (data not shown). PD-L1
expression and the density of infiltrating CD8-positive T
cells have been shown to be well correlated in ccRCC
. The differences in the frequencies of
PD-L1positive cells between ccRCC and pRCC might be due to
the immunogenicity of the cancer cells.
PD-L1 expression is also known to be detected in
infiltrating leukocytes, including macrophages ; however,
PD-L1 expression was observed only in macrophages in
4 of the 102 cases. PD-L1 expression in macrophages is
induced by cancer-derived factors, and
macrophagederived interleukin 10 is involved in the induction of
PD-L1 expression , suggesting that cell-cell
interaction with cancer cells is necessary for PD-L1
expression in macrophages. The density of macrophages is
closely associated with poor clinical prognosis in ccRCC
, but no report has yet described the relationship
between macrophages and clinical course in pRCC. The
discrepancy in PD-L1 expression in macrophages
between ccRCC and pRCC might be due to the induction
of cell-cell interactions between cancer cells and
Although PD-L2 expression in cancer cells has been
reported in ovarian cancer , there are much fewer
studies on PD-L2 than on PD-L1 in cancer tissues. This
might be due to the fact that no monoclonal antibody
suitable for use on paraffin sections had been
commercially available. However, a new monoclonal antibody
against PD-L2 has recently been made commercially
Fig. 2 The frequencies of scores 0, 1, and 2 in pRCC. Cases were divided into two groups by histological subtype (a) or nuclear grade (b), and the
frequencies of scores 0, 1, and 2 are shown
available , and as a result, some research articles
related to PD-L2 expression have just been published. One
such article stated that PD-L2 expression was seen in
49% of pRCC cases; however, no pictures were published
. The staining density of PD-L2 expression seemed
to be lower than that of PD-L1 expression (Fig. 1a),
therefore, appropriate positive or negative controls are
required and should be presented in research articles to
indicate whether the immunostaining procedure was
In the present study, PD-L1 expression was observed in
28% of the pRCC cases. The frequency of score 2 was
high in type 2 or higher nuclear grade cases. Although
PD-L1 expression appeared to be related to worse
Fig. 3 Kaplan-Meier analysis of cancer-specific overall survival and progression-free survival
overall survival, there was no significant correlation
between PD-L1 expression and clinical prognosis.
Further studies are necessary to evaluate if PD-L1
expression might be useful for predicting the efficacy of
anticancer immunotherapy using immuno-checkpoint
Availability of data and materials
Due to ethical restrictions, the raw data underlying this paper is available
upon request from the corresponding author.
TM, YK, CM, and AKD carried out the immunostaining and evaluate the
results. HN, SY, TN, SK, YK, MS, YO and WT participated in the collection of
tissue sections and clinicopathological data. TM, TK, MT, NF, and ME
participated in the design of the study, performed the statistical analysis, and
drafted the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All samples were obtained with informed consent from patients in
accordance with the study protocols that were approved by the review
board of each university (Kumamoto University Hospital Review Board,
Kyushu University Review Board, Review Board of University of Occupational
and Environmental Health) and written informed consent was obtained from
the patients for their data to be used for research purposes.
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