PD-L1 expression in human cancers and its association with clinical outcomes

TLDR: Better understanding of tumor microenvironment and use of other biomarkers such as gene marker and combined index are necessary to better identify patients who will benefit from PD-1/PD-L1 checkpoint blockade therapy.

Abstract: PD-L1 is an immunoinhibitory molecule that suppresses the activation of T cells, leading to the progression of tumors. Overexpression of PD-L1 in cancers such as gastric cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, pancreatic cancer, ovarian cancer, and bladder cancer is associated with poor clinical outcomes. In contrast, PD-L1 expression correlates with better clinical outcomes in breast cancer and merkel cell carcinoma. The prognostic value of PD-L1 expression in lung cancer, colorectal cancer, and melanoma is controversial. Blocking antibodies that target PD-1 and PD-L1 have achieved remarkable response rates in cancer patients who have PD-L1-overexpressing tumors. However, using PD-L1 as an exclusive predictive biomarker for cancer immunotherapy is questionable due to the low accuracy of PD-L1 immunohistochemistry staining. Factors that affect the accuracy of PD-L1 immunohistochemistry staining are as follows. First, antibodies used in different studies have different sensitivity. Second, in different studies, the cut-off value of PD-L1 staining positivity is different. Third, PD-L1 expression in tumors is not uniform, and sampling time and location may affect the results of PD-L1 staining. Therefore, better understanding of tumor microenvironment and use of other biomarkers such as gene marker and combined index are necessary to better identify patients who will benefit from PD-1/PD-L1 checkpoint blockade therapy.

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http://dx.doi.org/10.2147/OTT.S105862
PD-L1 expression in human cancers and its
association with clinical outcomes
Xin Wang
1,2,
*
Feifei Teng
2,3,
*
Li Kong
2
Jinming Yu
2
1
School of Medicine and Life
Sciences, University of Jinan –
Shandong Academy of Medical
Sciences,
2
Department of Radiation
Oncology, Shandong Cancer
Hospital and Institute,
3
School of
Medicine, Shandong University,
Jinan, People’s Republic of China
*These authors contributed equally
to this work
Abstract: PD-L1 is an immunoinhibitory molecule that suppresses the activation of T cells,
leading to the progression of tumors. Overexpression of PD-L1 in cancers such as gastric
cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, pancreatic cancer,
ovarian cancer, and bladder cancer is associated with poor clinical outcomes. In contrast, PD-L1
expression correlates with better clinical outcomes in breast cancer and merkel cell carcinoma.
The prognostic value of PD-L1 expression in lung cancer, colorectal cancer, and melanoma
is controversial. Blocking antibodies that target PD-1 and PD-L1 have achieved remarkable
response rates in cancer patients who have PD-L1-overexpressing tumors. However, using
PD-L1 as an exclusive predictive biomarker for cancer immunotherapy is questionable due to
the low accuracy of PD-L1 immunohistochemistry staining. Factors that affect the accuracy
of PD-L1 immunohistochemistry staining are as follows. First, antibodies used in different
studies have different sensitivity. Second, in different studies, the cut-off value of PD-L1 stain-
ing positivity is different. Third, PD-L1 expression in tumors is not uniform, and sampling
time and location may affect the results of PD-L1 staining. Therefore, better understanding
of tumor microenvironment and use of other biomarkers such as gene marker and combined
index are necessary to better identify patients who will benefit from PD-1/PD-L1 checkpoint
blockade therapy.
Keywords: PD-L1, prognostic value, checkpoint blockade, immunotherapy, clinical outcome
Introduction
The classic T cell activation is regulated by two signal transduction pathways: one
is antigen dependent, and the other is antigen independent. The antigen-independent
signaling includes positive and negative second signals. PD-1 and CTLA-4 are two
immune-inhibitory checkpoint molecules that suppress T cell-mediated immune
responses, leading to the development of tumors.
1
Cancer immunoediting is a pro-
cess that consists of immunosurveillance and tumor progression.
2
It has three phases:
elimination, equilibrium, and escape. In elimination phase, tumor cells are recognized
by upregulated tumor antigen expression and killed by different types of immune
effector cells. In equilibrium phase, tumor cells change into variants and induce immu-
nosuppression to avoid constant immune pressure, resulting in a state of functional
dormancy of the tumor. In escape phase, various immunosuppressive molecules and
cytokines are activated by the tumor cells and contribute to tumor outgrowth, causing
clinically apparent disease. PD-L1 is a PD-1 ligand that plays an important role in the
inhibition of T cell-mediated immune response. Binding of PD-L1 to PD-1 causes
the exhaustion of effector T cells and immune escape of tumor cells, leading to poor
prognosis. In rare cases, positive PD-L1 expression has been reported to be associ-
ated with better clinical outcome. Clinical trials have demonstrated that monoclonal
Correspondence: Jinming Yu
Department of Radiation Oncology,
Shandong Cancer Hospital and Institute,
440 Jiyan Road, Jinan 250117, Shandong,
People’s Republic of China
Tel +86 531 8798 4729
Fax +86 531 8798 4079
Email sdyujinming@163.com
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antibodies (mAbs) that target PD-L1 or its receptor PD-1
prevent the inhibitory effects of PD-1/PD-L1 pathway and
enhance T cell functions, leading to impressive outcomes
in patients with melanoma, renal cell carcinoma (RCC),
non-small-cell lung cancer (NSCLC), and bladder cancer.
3–5
However, the predictive effects of PD-L1 in response to
PD-1/PD-L1 antibodies in some tumors are not conclusive,
and the indication of PD-L1 expression in tumors remains
controversial and needs to be understood profoundly. This
review focuses on PD-L1 expression and its association with
clinical outcomes in different cancers and factors affecting
the accuracy of prediction of PD-L1. We also discuss the
value of PD-L1 in predicting the clinical efficacy of PD-1/
PD-L1 checkpoint blockades in cancer patients.
Expression and biological function
of PD-L1
PD-L1 is mainly expressed on the surface of tumor cells and
antigen-presenting cells in various solid malignancies such as
squamous cell carcinoma of the head and neck, melanoma,
and carcinomas of the brain, thyroid, thymus, esophagus,
lung, breast, gastrointestinal tract, colorectum, liver, pan-
creas, kidney, adrenal cortex, bladder, urothelium, ovary,
and skin.
6–12
In tumor microenvironment, PD-L1 expression
on tumor cells and other tumor-promoting cells is caused
by two mechanisms, constitutive mechanism and induced
mechanism, both of which depend on two binding sites of
IRF-1.
13
For example, in BRAFV600-mutated melanoma,
PD-L1 expression is a result of cancer cells’ adaptive
response to immune attack evoked by cytokines, or a consti-
tutive expression which is a result of oncogenic processes.
14
PD-L1 is rarely expressed on normal tissues but inducibly
expressed on tumor site, which makes PD-L1 pathway
uniquely different from other coinhibitory pathways,
15
indi-
cating that the selective expression of PD-L1 may have some
association with clinical outcomes of the cancer patients and
can be a selective target for antitumor therapy.
PD-1 (CD279), a PD-L1 receptor, is expressed on
CD4
-
CD8
-
thymocytes and CD4
+
CD8
+
T cells during thymic
development and is selectively expressed on CD4
+
and
CD8
+
T cells, monocytes, natural killer T cells, B cells,
and dendritic cells upon induction by TCR and cytokine
arousal.
16,17
In chronically infected mice model, high expres-
sion of PD-1 on T cells leads to T cell exhaustion and makes
the exhausted CD8
+
T cells lose effector function of secret-
ing cytokines such as IL-2, IFN-γ, and TNF-α.
18
Binding
of PD-L1 to PD-1 leads to the formation of PD-1/TCR
inhibitory microcluster that recruits SHP1/2 molecule and
dephosphorylates multiple members of TCR signaling
pathway, leading to the shut-off of T cell activation through
induction of apoptosis, reduction of proliferation, and inhibi-
tion of cytokine secretion (Figure 1). However, whether all
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Figure 1 PD-1/PD-L1 signaling: decreased CD8
+
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Abbreviations: DC, dendritic cell; Treg, regulatory T cell; ICOS, inducible costimulator; ICOS-L, inducible costimulator-ligand; CD28, cluster of differentiation 28; CTLA-4,
cytotoxic T lymphocyte-associated antigen-4; PD-L1, programmed death-ligand 1; PD-1, programmed death-1; MHC, major histocompatibility complex; TCR, T cell receptor;
IFN-γ, interferon-γ; IFN-γR, interferon-γ Receptor.
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Association of PD-L1 expression with cancers and clinical outcomes
kinds of cancers utilize the same action mechanism of PD-L1
signaling, that is, whether different prognosis of different
cancers is caused by different PD-L1 mechanisms, remains
inconclusive and needs to be further explored.
PD-1/PD-L1 pathway plays a prominent role in immune
regulation by delivering inhibitory signals to maintain the
balance in T cell activation, tolerance, and immune-mediated
tissue damage. It exerts significant inhibitory functions in per-
sistent antigenic stimulation environment such as exposure
to self-antigen, chronic viral infection, and tumor.
19–21
PD-L1
can also serve as a receptor transmitting antiapoptotic signal
to tumor cells to protect them from apoptosis. Moreover,
Shi et al
22
have demonstrated that PD-L1 may have onco-
genic function during colon cancer carcinogenesis. PD-L1
not only inhibits T cell proliferation and cytokine production
but also enhances T cell activation.
23
The explanation of this
contradictory phenomenon is unknown.
In normal tissues, PD-1 signaling in T cells regulates
immune responses to decrease damage to adjacent tissue, and
counteracts the development of autoimmunity by promoting
tolerance to self-antigens. PD-L1 receptor is also expressed
on the surface of CD4
+
Foxp3
+
regulatory T cells (Tregs), a
subset of CD4
+
T cells that play a critical role in maintain-
ing immune tolerance and weakening immune responses, to
promote the development, maintenance, and immunosuppres-
sive function of Tregs through inhibiting mTOR and AKT
phosphorylation.
24
Prognostic value of PD-L1 in
malignancies
Table 1 summarizes the studies on the prognostic value
of PD-L1 in different malignancies. Some malignancies
such as hepatocellular carcinoma, pancreatic cancer
(PC), gastric cancer, RCC, esophageal cancer (EC), and
ovarian cancer can generate an immunosuppressive tumor
microenvironment by expressing high-aggregate PD-L1
to avoid cytolysis by activated T cells. It may explain why
overexpression of high-aggregate PD-L1 in tumors leads
to poor prognosis in cancer patients. Interestingly, several
long-term follow-up investigations have found an inverse
correlation between PD-L1 expression on tumor cells and
poor prognosis of patients. Additionally, in lung cancer,
colorectal cancer (CRC), and melanoma, PD-L1 expression
has both positive and negative prediction values. In thy-
moma and thymic carcinoma, squamous cell carcinoma
of the lung, and cervical cancer, PD-L1 expression alone
is not of prognostic value but is of significant prediction
value when combined with other indicators such as CD8
+
/
Foxp3
+
T cell ratio.
Lung cancer
Two studies demonstrated that PD-L1 expression on tumor
cells is correlated with poor prognosis in NSCLC patients.
Both the studies detected PD-L1 expression both on mem-
brane and in cytoplasm of tumor cells, but the cut-off value
for PD-L1 immunohistochemistry (IHC) positivity was not
mentioned.
25,26
Azuma et al
26
revealed that expression of high-
aggregate PD-L1 on tumor cells is associated with EGFR
gene mutations. Mu et al
25
evaluated the intensity of PD-L1
expression in 109 NSCLC specimens. By IHC analysis,
strong association was found between PD-L1 expression and
shorter survival time in adenocarcinoma patients.
Two other studies showed a positive correlation between
better clinical outcomes and PD-L1 expression. Velcheti et al
27
assessed the predictive value of PD-L1 expression in two
cohorts with 204 and 340 specimens, respectively. Tumor
PD-L1 expression was found in 36% (Greek) and 25%
(Yale) of the cases. Patients with PD-L1 expression above the
detection threshold showed significantly better outcome. In
2014, Yang et al
28
reported that patients with positive PD-L1
expression on tumor cell membrane had better relapse-free
survival. Importantly, Boland et al
29
showed that PD-L1 is not
of independent prognostic value in squamous cell carcinoma
of the lung. Based on the above studies, we conclude that
PD-L1 has controversial predictive function in lung cancer.
Gastric cancer
Three articles reported the negative prediction value of
PD-L1 in gastric cancer patients. Hou et al
30
found that 70
of 111 patients had positive PD-L1 expression either on
membrane or in the cytoplasm of tumor cells, and there was
a positive correlation between the expression of PD-L1 and
poor prognosis. The cut-off value was 10% in their study.
The study by Hou et al also demonstrated that the combina-
tion of increased number of Foxp3
+
Tregs and increased
expression of PD-L1 is an even stronger predictor of lower
overall survival (OS) rate and worse prognosis as compared
to each individual factor alone. The other two studies did
not mention the cut-off values of PD-L1 IHC staining; both
of them showed that PD-L1 is of independent prognostic
value.
31,32
Wu et al
31
examined 43 of 102 specimens and
found that PD-L1 expression is mainly in the cytoplasm of
tumor cells. Qing et al
32
showed that 54 of 107 cases had
positive PD-L1 expression. Both studies demonstrated that
PD-L1 expression is significantly associated with invasion
and lymph node metastasis, which are poor prognostic factors
of tumors. In conclusion, gastric cancer patients with positive
PD-L1 expression have a significantly poorer prognosis than
PD-L1-negative patients.
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Table 1 Prognostic value of PD-L1 in different malignancies
Disease N Detection method; PD-L1
detection antibody
Location of PD-L1 expression PD-L1 tumor
surface expression
cut-off for
positivity
Thymoma and
thymic carcinoma
101 and 38 Parafn IHC; anti-PD-L1 (clone E1L3N) 70% of thymic carcinoma (type C) and 23%
of thymoma (types A, AB, and B) samples
were positive for PD-L1
1%
features, including WHO classication type
PD-L1 positivity was not a signicant
Adrenocortical
carcinoma
28 Parafn IHC; monoclonal anti-PD-L1
antibody (405.9A11)
PD-L1 expression was detected both on
tumor cell membrane and in TIMCs
5%
membrane or in TIMCs was not signicantly
Head and neck
squamous cancer
24 Frozen IHC; anti-PD-L1 (5H1) Eleven of 24 specimens had intracytoplasmic
staining, eleven of 24 tumors had membrane
reactivity, and ten of 24 had both
NA
Malignant brain
tumors
83 FACS; anti-PD-L1 (BD Pharmingen Inc.,
San Diego, CA, USA)
61% of brain tumors (but no WHO grade 1
tumors) expressed PD-L1
NA
Glioma 54 Frozen IHC; anti-PD-L1 (clone MIH1;
eBioscience, San Diego, CA, USA)
NA NA
10 Frozen IHC; anti-PD-L1 (5H1) Strong PD-L1 expression was detected in all
ten glioma samples examined
NA
Lung cancer 109 Parafn IHC;
anti-PD-L1 (clone not specied)
PD-L1 expression on membrane and in
cytoplasm of tumors with cluster and
scattered patterns
NA
α
++
+
+
164 Parafn IHC; anti-PD-L1 (Lifespan
Biosciences, Seattle, WA, USA);
ow cytometry
PD-L1 was detected on membrane or in
the cytoplasm (or both) of tumor cells and
stromal lymphocytes in the surgically resected
tumor specimens
NA PD-L1 expression is signicantly higher
with a signicantly shorter OS for NSCLC
204 and 340 Parafn IHC; anti-PD-L1 (clone 5H1) NA NA
lymphocytic inltrate
inammation, while in Yale cohort,
with histology and inammation
signicant better outcome
163 Parafn IHC; anti-PD-L1 (Proteintech
Group Inc., Chicago, IL, USA)
PD-L1 expressed on membrane of tumor cells 5%
inltration was slightly higher in
signicant risk factors for poor prognosis
214 Parafn IHC; anti-PD-L1 (clone 5H1,
laboratory developed)
PD-L1 predominately expressed on membrane
and minimally expressed in cytoplasm
5% PD-L1 expression had no signicant
correlation with inltrating
Boland et al
120 and 10 Parafn IHC; anti-PD-L1 NA NA
+
52 Frozen IHC; anti-B7-H1 (MIH1) PD-L1 expression in cytoplasm or on
membrane or both, in focal or scattered
patterns in all 52 specimens of NSCLC
NA In a subset of ve patients, the
amount of T lymphocytes inltration
was signicantly reduced in PD-L1-
Gastric cancer 111
Parafn IHC; anti-Foxp3
+
(polyclonal
antibody, Sigma-Aldrich Co., St Louis, MO,
USA) and anti-PD-L1 (polyclonal antibody;
Abcam, Cambridge, UK)
70 of 111 cases demonstrated PD-L1
expression on the membrane or in the
cytoplasm
10%
Signicant correlation was found
between the inltration of Foxp3
+
++
102 Parafn IHC; anti-B7-H1 (2H11) PD-L1 was expressed mainly in the cytoplasm;
some nuclear membrane localization was also
present
NA
+
107 Parafn IHC; anti-PD-L1
(polyclonal antibody)
54 of 107 cases had positive PD-L1 expression NA Positive PD-L1 expressions were signicantly
signicantly associated with a poor
( )
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Association of PD-L1 expression with cancers and clinical outcomes
TIL or other immunocytes
associated with PD-L1
expression
Other clinicopathological factors
associated with PD-L1 expression
Clinical outcomes Study
Parafn IHC; anti-PD-L1 (clone E1L3N) 70% of thymic carcinoma (type C) and 23%
of thymoma (types A, AB, and B) samples
NA Higher PD-L1 expression was more likely
to exhibit male, more advanced pathological
features, including WHO classication type
and Masaoka–Koga stage
PD-L1 positivity was not a signicant
negative factor of OS
Katsuya et al
6
Parafn IHC; monoclonal anti-PD-L1
antibody (405.9A11)
NA PD-L1 positivity on either tumor cell
membrane or in TIMCs was not signicantly
associated with higher stage at diagnosis,
higher tumor grade, and excessive hormone
secretion
PD-L1 expressed on both tumor
cell membrane and in TIMCs with no
relationship with 5-year OS
Fay et al
9
Frozen IHC; anti-PD-L1 (5H1) NA 16 of 24 specimens had PD-L1 staining NA Strome et al
10
FACS; anti-PD-L1 (BD Pharmingen Inc.,
San Diego, CA, USA)
61% of brain tumors (but no WHO grade 1
tumors) expressed PD-L1
NA NA NA Jacobs et al
11
Frozen IHC; anti-PD-L1 (clone MIH1;
eBioscience, San Diego, CA, USA)
NA PD-L1 expression was closely correlated
with the grade of the tumor
NA Wilmotte et al
12
Frozen IHC; anti-PD-L1 (5H1) NA NA NA Wintterle et al
82
Parafn IHC;
anti-PD-L1 (clone not specied)
CD1α
+
TIDC was increased in PD-L1
+
portions of tumor and had higher
expression of PD-L1 than CD83
+
DCs
PD-L1
+
cells in adenocarcinoma were
more
than in squamous cell carcinoma
PD-L1 positivity correlated with survival
shorter than 3 years after lobectomy
Mu et al
25
Parafn IHC; anti-PD-L1 (Lifespan
Biosciences, Seattle, WA, USA);
ow cytometry
the cytoplasm (or both) of tumor cells and
NA PD-L1 expression is signicantly higher
in females, never smokers, those with
adenocarcinoma histology, and those
with EGFR mutations
PD-L1 overexpression was associated
with a signicantly shorter OS for NSCLC
patients and had independent negative
prognostic value
Azuma et al
26
Parafn IHC; anti-PD-L1 (clone 5H1) PD-L1 protein and mRNA
expression was consistently
associated with increased local
lymphocytic inltrate
In Greek cohort, PD-L1 expression
had association with tumor stage and
inammation, while in Yale cohort,
with histology and inammation
Patients with PD-L1 expression above the
detection threshold showed statistically
signicant better outcome
Velcheti et al
27
Parafn IHC; anti-PD-L1 (Proteintech
Group Inc., Chicago, IL, USA)
The degree of T lymphocytes
inltration was slightly higher in
PD-L1-positive tumors than in
PD-L1-negative ones
Higher PD-L1 expression was correlated
with higher grade differentiation and
vascular invasion
Positive PD-L1 expression had better RFS.
Advanced-stage and positive VPSI were
signicant risk factors for poor prognosis
of OS
Yang et al
28
Parafn IHC; anti-PD-L1 (clone 5H1,
laboratory developed)
PD-L1 expression had no signicant
correlation with inltrating
lymphocytes
NA PD-L1 was not of independent prognostic
value in squamous cell carcinoma of the lung
Boland et al
29
Parafn IHC; anti-PD-L1 NA PD-L1 overexpression was related to poor
tumor cell differentiation and advanced
TNM stage
PD-L1
–
NSCLC patients had longer overall
5-year survival than PD-L1
+
patients. PD-L1
status had independent prognostic value of
NSCLC
Chen et al
62
Frozen IHC; anti-B7-H1 (MIH1) In a subset of ve patients, the
amount of T lymphocytes inltration
was signicantly reduced in PD-L1-
expressing tumor regions
No correlation was observed between
PD-L1 expression and clinicopathological
characteristics
No correlation between PD-L1 expression
and patient survival
Konishi et al
81
Parafn IHC; anti-Foxp3
+
(polyclonal
USA) and anti-PD-L1 (polyclonal antibody;
Abcam, Cambridge, UK)
Signicant correlation was found
between the inltration of Foxp3
+
Tregs and the expression of PD-L1
on the tumor cells and TILs
High-level Foxp3
+
Tregs and PD-L1
expression led to lymph node metastasis
and an advanced clinicopathological stage
Enhanced expression of Foxp3
+
Tregs and
PD-L1 exhibited a lower OS
rate and a
worse prognosis
Hou et al
30
Parafn IHC; anti-B7-H1 (2H11) NA 42.2% of gastric carcinoma tissues were
PD-L1
+
. PD-L1 correlated with tumor size,
invasion, and lymph node metastasis
PD-L1 expression was an independent
prognostic factor and correlated with
poorer survival
Wu et al
31
Parafn IHC; anti-PD-L1
(polyclonal antibody)
NA Positive PD-L1 expressions were signicantly
associated with depth of invasion, lymph
node metastasis, pathological type, higher
T stage, and higher differentiation
PD-L1-positive gastric cancers were
signicantly associated with a poor
prognosis
Qing et al
32
(Continued)
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