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The immune checkpoint regulator PD-L1 is highly expressed in aggressive primary prostate cancer
Heidrun Gevensleben1,#, Dimo Dietrich1,#, Carsten Golletz1, Susanne Steiner1, Maria Jung1,
Thore Thiesler1, Michael Majores2, Johannes Stein1, Barbara Uhl1, Stefan Müller3, Jörg
Ellinger3, Carsten Stephan4, Klaus Jung4, Peter Brossart5, and Glen Kristiansen1
Author Affiliations: 1 Institute of Pathology, University Hospital Bonn, Bonn, Germany 2 Institute of Dermatopathology, Bonn, Germany 3 Department of Urology, University Hospital Bonn, Bonn, Germany 4 Department of Urology, Charité University Hospital Berlin, Berlin, Germany 5 Department of Hematology and Oncology, University Hospital Bonn, Bonn
# Contributed equally to this work.
Running title: PD-L1 is highly expressed in primary prostate cancer
Keywords: PD-L1, prostate cancer, immune therapy, biomarker, immunohistochemistry
Word count: 2.924
Figures and Tables: 5
Corresponding author: Prof. Dr. Glen Kristiansen
Institute of Pathology, University Hospital Bonn,
Sigmund-Freud-Str. 25, 53127 Bonn, Germany
Phone: +49-228-287-15375
Fax: +49-228-287-1530
Email: [email protected]
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Abstract
Purpose: Therapies targeting the programmed death 1 (PD-1)/programmed death ligand 1
(PD-L1) pathway promote anti-tumor immunity and have shown promising results in various
tumors. Preliminary data further indicate that immunohistochemically detected PD-L1 may be
predictive for anti-PD-1 therapy. So far, no data are available on PD-L1 expression in primary
prostate cancer.
Experimental design: Following validation of a monoclonal antibody, immunohistochemical
analysis of PD-L1 expression was performed in two independent, well-characterized cohorts
of primary prostate cancer patients following radical prostatectomy (RP), and resulting data
were correlated to clinicopathological parameters and outcome.
Results: In the training cohort (n=209), 52.2% of cases expressed moderate to high PD-L1
levels, which positively correlated with proliferation (Ki67, p<0.001), Gleason score
(p=0.004), and androgen receptor (AR) expression (p<0.001). Furthermore, PD-L1-positivity
was prognostic for biochemical recurrence (BCR; p=0.004; HR=2.37 [95%CI=1.32–4.25]. In
the test cohort (n=611) moderate to high PD-L1 expression was detected in 61.7% and
remained prognostic for BCR in univariate Cox analysis (p=0.011; HR=1.49 [95%CI=1.10–
2.02]. The correlation of Ki-67 and AR with PD-L1 expression was confirmed in the test
cohort (p<0.001). In multivariate Cox analysis of all patients, PD-L1 was corroborated as
independently prognostic for BCR (p=0.007; HR=1.46 [95%CI=1.11–1.92].
Conclusion: We provide first evidence that expression of the therapy target PD-L1 is not
only highly prevalent in primary prostate cancer cells but is also an independent indicator of
BCR, suggesting a biological relevance in primary tumors. Further studies need to ascertain,
if PD-1/PD-L1 targeted therapy might be a treatment option for hormone-naive prostate
cancers.
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Statement of translational relevance
Cancer immunotherapy represents a breakthrough in oncology. Among immunotherapeutical
approaches, blocking the PD-1/PD-L1 pathway, and thereby promoting activity of tumor-
specific effector T cells, has shown outstanding results. However, high response rates are
only seen in a few cancer entities, e.g. melanomas. In patients with hormone refractory
metastatic prostate cancer, previous studies have not been able to provide evidence of
response to anti-PD-L1 therapy. Our study revealed that PD-L1 is differentially expressed
among primary prostate cancer patients. Furthermore, high PD-L1 expression was
associated with a poor prognosis. Accordingly, PD-1/PD-L1 targeted therapy might be a
promising novel treatment option for hormone-naive prostate cancers.
Introduction
Prostate cancer is the second most frequently diagnosed cancer in men accounting for ~15%
of all newly diagnosed male cancers worldwide (1). Overall, it is the fifth most common cause
of death from cancer in men with 307.000 estimated deaths (6.6% of all estimated deaths) in
2012 (1). First-line therapies for early stage localized prostate cancer include surgery and
radiotherapy, and the 5-year relative survival rate approaches 100% (2). For patients
affected by metastatic prostate cancer, androgen deprivation therapy (ADT) still is the
mainstay of treatment (3). Although surgical or chemical castration can initially be effective
delaying disease progression, the majority of patients eventually develops castration
resistant prostate cancer (CRPC) and has an adverse prognosis (4-5). Recent advances and
the arrival of several new agents in the field of CRPC, however, have improved overall
survival in this patient population. Enzalutamid, a novel androgen receptor (AR) inhibitor that
reduces nuclear translocation of the AR complex and subsequent DNA binding, was shown
to significantly prolong overall survival in two phase III randomized, placebo-controlled trials
(6-7). Furthermore, Abiraterone, an inhibitor of cytochrome P450, family 17, subfamily A,
polypeptide 1 (CYP17A1), suppresses androgen biosynthesis in combination with
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prednisone, increasing survival time and time to prostate-specific antigen (PSA) progression
(8-9). Above all, the early addition of docetaxel to ADT has recently been shown to extend
survival for men with newly diagnosed hormone-sensitive prostate cancer by more than 13
months (10).
Among many concepts under investigation, augmenting immune responses to cancer has
been proposed as a valid therapeutic option providing an alternative approach to improve
survival. In particular, checkpoint inhibitors have emerged as a complementary avenue of
clinical research in prostate cancer (11-12). These treatments target checkpoint molecules in
the regulation of the immune system harnessing pre-existing anti-cancer immune responses.
The programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway plays a
pivotal role in the regulation of T cell activity at the time of inflammatory response. The PD-1
receptor thereby acts as a negative checkpoint regulator to prevent off-target immune
activation of T-lymphocytes (13). Binding of PD-1 to its ligand PD-L1, the predominant
mediator of immunosuppression, inhibits proliferation of activated T cells leading to “T-cell
exhaustion” (14). PD-L1 itself is an immunomodulatory cell-surface glycoprotein primarily
expressed by antigen-presenting cells on myeloid dendritic cells, activated T cells, and some
non-hematopoietic tissues (15).
Recent evidence strongly suggests that the activation of the PD-1/PD-L1 pathway represents
a mechanism allowing tumors to elude the host’s immune system. Therapies targeting this
signaling pathway promote marked anti-tumor immunity and have shown promising results in
a subset of solid tumors (16-17). As reported by Topalian et al., blockade of PD-L1 using an
anti-PD-1 antibody induced objective response rates of 6 to 17% and prolonged stabilization
of disease in patients with several advanced cancers, i.e. malignant melanoma, non-small-
cell lung cancer, and renal-cell cancer (17). Further, immunohistochemical assessment of
PD-L1 in pretreatment cancer specimens from 42 patients revealed that response to
treatment was seen exclusively in PD-L1 positive tumors (9/25, 36%), indicating that PD-L1
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expression might be a predictive biomarker for anti-PD-1 therapy (17). However, several
studies have demonstrated response to treatment in patients with low or absent PD-L1
expression, although these patients seem to be in the minority (18-23). So far, no data are
available to support that PD-1/PD-L1 targeted therapy may be effective in prostate cancer.
This prompted us to analyze PD-L1 expression in primary prostate cancer.
Currently, only few PD-L1 antibodies have been validated for FFPE material, only one of
which being commercially available ((E1L3N®) XP®) (14, 24-31). The utilization of this
antibody for predicting response to anti-PD1 or anti-PD-L1 therapy, however, remains
unknown. The majority of clinical trials targeting the PD-1/PD-L1 pathway are using
proprietary antibodies, implying that validation data for these particular antibodies is
undefined. So far, no immunohistochemical detection method for quantifying PD-L1
expression is uniformly accepted as standard. We here comprehensively validated a novel
monoclonal rabbit antibody against PD-L1 (clone EPR1161(2)) amenable to FFPE tissue and
analyzed two large, well-characterized cohorts of primary prostate cancer after radical
prostatectomy (RP) for PD-L1 expression.
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Methods
Patients
Two previously described RP cohorts were analyzed in the present study (32-34).
Clinicopathological details including Gleason grade grouping according to the latest
International Society of Urological Pathology (ISUP) consensus (35) are given in Table 1.
The training cohort included 262 patients having undergone radical prostatectomy at the
University Hospital of Bonn between 1998 and 2008. Immunohistochemical PD-L1 staining
was available for 209 cases. The test cohort comprised 640 patients diagnosed at the
Charité University Hospital, Berlin between 1999 and 2005, and a total of 611 cases were
eligible for PD-L1 immunhistochemistry. All research conducted was approved by the Charité
Ethics Committee (EA1/06/2004) and the University Hospital of Bonn.
Tissue Microarray Construction
Tissue microarrays (TMAs) were constructed as previously described (32). Briefly, formalin-
fixed paraffin embedded tissue specimens were selected according to tissue availability and
retrieved from the archive of the Charité University Hospital Berlin and the University Hospital
Bonn. Each case was represented by one to three tissue cores with a core diameter of 1.2
mm or 1.8 mm.
Cell Line Controls and Fluorescence-Activated Cell Sorter (FACS) Analysis
The prostate cancer cell line DU145 was obtained from American Type Culture Collection
(ATCC, Rockville, MD, USA) and maintained in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal calf serum (FBS), 1% l-glutamine, and 1% antibiotics
(Life Technologies, Carlsbad, CA, USA). Cells were grown at 37°C in a humidified 5% CO2
atmosphere. PD-L1 expression was analyzed using flow cytometric analysis. Briefly, 5 x 105
cells were washed with 5 ml phospate-buffered saline (PBS) and resuspended in 300 ul of
PBS. FACS analysis was performed on Navios Flow Cytometer (Beckman Coulter, Miami,
CA, USA) following incubation with FITC-tagged anti-human PD-L1 or mouse IgG isotype
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control (BD Pharmingen, Erembodegem, Belgium) for 20 min at room temperature, a second
wash, and resuspension in PBS.
Western Blot Analysis
Cells were lysed in RIPA buffer (Sigma-Aldrich, Munich, Germany) in the presence of
protease inhibitors. Cleared lysates were separated on NuPAGE 4–12% Bis-Tris gels (1.0
mm, 15 well; Life Technologies, Carlsbad, CA, USA) and transferred onto an Invitrolon PVDF
membrane (Life Technologies, Carlsbad, CA, USA). Membranes were stained using anti-PD-
L1 rabbit monoclonal antibody (mAb) clone EPR1161(2) (Abcam, Cambridge, UK; 1:500) in
5% nonfat dry milk, and protein concentrations were determined by a colorimetric assay (Bio-
Rad Laboratories Inc., Richmond, CA, USA). Blots were stripped and reprobed with anti-b-
actin mAb (Sigma- Aldrich, Munich, Germany; 1:500). Western blot analysis with the
validated anti-PD-L1 rabbit mAb (E1L3N®) XP® (Cell Signaling Technology, Frankfurt,
Germany; 1:500) was performed for comparison.
siRNA Transfection for Transient Knockdown of PD-L1
DU145 cells were seeded with 2 × 105 cells in 6 well plates and incubated for 72h in DMEM
in the presence of 10 to 20 nM siRNA directed against PD-L1 (FlexiTube Gene Solution
GS29126, Qiagen, Hilden, Germany) or non-targeting control siRNA (Qiagen, Hilden,
Germany) complexed with HiPerFect Transfection Reagent (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions. Cells treated with HiPerFect Transfection
Reagent only served as controls. siRNA-induced knockdown was evaluated using western
blot analysis as described above.
Peptide Neutralization
Prior to standard proceedings, the anti-PD-L1 antibody clone EPR1161(2) was incubated
with a 10fold concentration excess of blocking peptide (Abcam, Cambridge, UK) that
corresponds to the epitope recognized by the primary antibody. Western blot analysis was
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carried out with the neutralized antibody side-by-side with the antibody alone. In analogy to
this experiment, immunohistochemical staining was performed on placental and tonsillar
tissue, following primary antibody neutralization with a 10fold concentration excess of
blocking peptide, and compared to standard staining results.
Immunohistochemistry and Evaluation
Tissue sections were freshly cut (3μm), mounted on Super Frost Plus slides (Thermo Fisher,
Waltham, MA, USA), and rehydrated in descending gradient alcohols. Immunohistochemical
staining was carried out on the Ventana BenchMark Ultra automated staining system
(Ventana, Tucson, AZ, USA) and visualized with the Ventana amplifier detection kit using the
following antibodies: PD-L1, clone EPR1161(2) (Abcam, Cambridge, UK; 1:75), Ki-67, clone
MIB-1 (Dako, Glostrup, Denmark; 1:500), and AR, clone AR441 (Dako, Glostrup, Denmark;
1:400). Omission of the primary antibody was used as a negative control.
Immunohistochemical stainings were evaluated independently by two pathologists (GK, HG)
who were blinded to patients’ clinical outcome. Specific membranous and cytoplasmic
staining of epithelial tumor cells was considered positive. Since the staining was uniformly
homogenous, the intensity of PD-L1 positive cells was scored semiquantitatively as negative
(0), weak (1), moderate (2), or strong (3). Discrepant cases were reviewed at a multi-headed
microscope and the consensus was reported.
Statistical Analysis
Following dichotomization by median, associations between PD-L1 protein expression and
clinicopathologic variables, including AR and Ki-67 expression, were analyzed using the Χ2
test. The correlation of Ki-67 and AR with PD-L1 expression as continuous variables was
further tested using Kendall’s τ rank correlation. BCR-free survival was calculated for PD-L1
expression dichotomized by median using the Kaplan-Meier method, and survival time
differences were compared using the log-rank test. PD-L1 expression as continuous variable
was also examined within univariate and multivariate Cox proportional hazards regression
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models. P-values refer to the Wald test. An interrater reliability analysis using the Kappa (κ)
statistic was conducted to determine consistency among raters. All statistical tests were two-
sided. P-values <0.05 were considered to be statistically significant. All analyses were
carried out using the SPSS 21 software package (IBM, Armonk, NY).
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Results
Validation of PD-L1 Antibody Specificity
For immunohistochemical analysis of PD-L1 expression in formalin-fixed paraffin-embedded
(FFPE) tissue, we validated a novel monoclonal antibody (mAb) against PD-L1 (clone
EPR1161(2)) and compared its performance to the previously validated PD-L1 mAb
(E1L3N®) XP® (14, 24-31). The prostate cancer cell line DU145 served as positive control.
Flow cytometric analysis (FACS) of untreated DU145 cells compared to concentration
matched mouse IgG isotype control demonstrated positive PD-L1 membrane expression in
40% of cells (Figure 1A). For western blot analysis cell lysates from untreated DU145 and
MCF7 cells were probed with PD-L1 mAbs. Both antibodies detected a band at the expected
size of glycosylated PD-L1 (~55 kDa) for the FACS-positive cell line DU145 (Figure 1B). As
reported previously (36), PD-L1 was not detected in MCF7 cells. Antibody specificity was
confirmed by small interfering RNA (siRNA) knockdown of PD-L1 and western blot analysis
(Figure 1C). Cross-reactivity was excluded using immunizing peptide blocking experiments.
Primary antibody neutralization with a specific blocking peptide prior to western blot and
immunohistochemical proceedings abolished immunoreactivity and thus verified specificity of
this PD-L1 mAb clone EPR1161(2) (Figure 1D).
PD-L1 Expression in Prostate Cancer
Detailed clinicopathological characteristics of the patient cohorts included in the present
study are shown in Table 1. Tumoral immunoreactivity of PD-L1 was uniformly homogenous,
and scoring results were highly concordant (κ=0.75, p<0.001). PD-L1 expression was
detected at the membrane or in the cytoplasm of the tumor cells (Figure 2). Additionally
stromal and tumor infiltrating lymphocytes displayed a strong PD-L1 immunoreactivity.
In the training cohort, 52.2% of cases (109/209) expressed moderate to high levels of PD-L1
which positively correlated with proliferation (Ki67, p<0.001, τ=0.26) AR expression (p<0.001,
τ=0.21). These results were confirmed by performing the X2-test (Table 1). Furthermore, an
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association of PD-L1 with Gleason score was found (p=0.004; Table 1). The introduction of a
cut-off for patients' stratification is required for a Kaplan-Meier analysis. However, data
dichtotomization based on a cut-off leads to a loss of information on the one hand and a
multiple testing problem due to the multitude of possible cut-offs on the other hand.
Therefore, PD-L1 expression was initially analyzed as a continuous variable. In univariate
proportional hazards model analysis, semiquantitatve PD-L1 expression was strongly
prognostic for biochemical recurrence (BCR; p=0.004; HR=2.37 [95%CI=1.32–4.25]; Table
2). Kaplan-Meier survival analysis of PD-L1 expression dichotomized by median confirmed
that high PD-L1 expression was associated with significantly reduced BCR-free survival
(p=0.022, Figure 3A).
In the independent test cohort, moderate to high PD-L1 expression was detected in 61.7% of
cases (377/611). The strong association of PD-L1 expression with AR (p<0.001, τ=0.16), and
Ki-67 (p<0.001, τ=0.22) was substantiated in this cohort, whereas no association with
Gleason score was verified. Along with known prognostic factors (pT status, Gleason score,
surgical margins, and preoperative PSA levels), PD-L1 expression was shown to be
significantly prognostic for BCR in univariate Cox proportional hazards model analysis
(p=0.011; HR=1.49 [95%CI=1.10–2.02]; Table 2). After dichtomization, PD-L1 expression
conferred a significantly shorter PSA relapse-free survival in Kaplan-Meier survival analysis
(p=0.009, Figure 3B). An association of age and pre-surgical PSA with PD-L1 could only be
shown in either the training or test cohort, respectively (Table 1).
In multivariate Cox proportional hazards model analysis of all patients, PD-L1 furthermore
remained an independent prognostic factor of BCR (p=0.007; HR=1.46 [95%CI=1.11–1.92];
Table 2) when tested together with pT status, Gleason score, preoperative PSA, and surgical
margins.
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Discussion
This study is the first to demonstrate that PD-L1 expression is not only highly prevalent in
primary prostate cancer but is also an independent prognostic factor of disease progression
in cohorts of primary tumors following RP. Consistent with previous studies, our results
suggest an association of PD-L1 with aggressive tumor behavior in primary prostate cancer,
indicating that PD-1/PD-L1 pathway activation assists the evasion of anti-tumor immune
response, driving tumor proliferation and progression. Further studies in watchful waiting
cohorts need to ascertain, whether PD-L1 might also contribute to the identification of lethal
prostate cancer.
We here comprehensively validated a novel monoclonal rabbit antibody against PD-L1 (clone
EPR1161(2)) amenable to FFPE tissue and analyzed two large, well-characterized cohorts of
primary prostate cancer after RP for PD-L1 expression. Our study shows that the
immunoreactivity of clone EPR1161(2) for the detection of PD-L1 on FFPE tissue is specific
and robust. In preliminary experiments (not shown), which we routinely conduct to establish a
new antibody in our laboratory, we employed different immunohistochemical platforms and
found this antibody's performance to be independent of the detection system. It necessitates
conventional antigen retrieval, but yields a crisp and clean signal with no background issues
at all.
Increased expression of PD-L1 on tumor cells has previously been described for several
malignancies, including glioblastoma, pancreatic, ovarian, breast, renal, head and neck,
esophageal, and non-small cell lung cancer (37-42). Moreover, PD-L1 expression has been
associated with poor prognosis and adverse clinicopathological characteristics (28, 31, 43-
46). In line with these findings, we have found moderate to high PD-L1 expression in 52.2-
61.7% of primary prostate cancers after RP. Multivariate analysis further revealed that high
PD-L1 expression was significantly associated with reduced BCR-free survival independently
of other clinicopathological factors, suggesting that expression of PD-L1 on tumor cells
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promotes tumor recurrence by interrupting antitumor immunity (29). In addition, we found a
strong correlation of PD-L1 with tumor cell proliferation as estimated by Ki-67, which has
been associated with a highly adverse prognosis and primary therapy failure in prostate
cancer (47-50).
Preliminary data from a phase I study on an anti-PD1 monoclonal antibody treatment
(nivolumab) also indicated that immunohistochemically detected PD-L1 is a potential
predictive biomarker for therapeutic blockade of the PD1/PD-L1 pathway (17). In the
landmark study by Topalian et al. immunohistochemical assessment of PD-L1 in
pretreatment cancer specimens from 42 patients revealed that objective response to
treatment was seen exclusively in PD-L1 positive tumors (9/25, 36%; p=0.006). More recent
data suggest that PD-L1 positive tumors have higher response rates to agents targeting the
PD1/PD-L1 pathway, although response to treatment was also recorded for PD-L1 negative
cases (18-23). Notably, none of the 17 patients with metastatic CRPC included in the original
study by Topalian and colleagues responded to anti-PD-1 treatment. For two cancers which
were eligible for immunohistochemical analysis no PD-L1 expression was detected (17); all
of which suggested that PD-1/PD-L1 targeted treatment was not particularly promising for
prostate cancer patients. Although the sample size with only two prostate cancer specimens
eligible for immunohistochemical analysis is clearly limited, the absence of PD-L1 expression
might as well be due to the fact that only heavily pretreated CRPC were enrolled in this
study. So far, it is speculative if the strong correlation of AR and PD-L1 observed in our study
provides a viable explanation for low expression rates of PD-L1 and lack of response to anti-
PD-1 treatment in CRPC (17). However, the high rate of PD-L1-positivity found in primary,
and hence hormone-naive prostate cancers, indicates that PD-1/PD-L1 pathway targeted
therapy might potentially be a novel treatment option, and immunohistochemical assessment
of PD-L1 might accordingly represent a biomarker for the identification of patients eligible for
this therapy.
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Strikingly, Bishop et al. recently reported that CRPC patients resistant to enzalutamide
showed elevated levels of PD-L1 expressing dendritic cells in blood (51). Additional cell line
and xenograft experiments suggested that enzalutamide resistant tumors might suppress
immune response not only trough intrinsic PD-L1 expression, but also via induction of PD-L1
expression on circulating dendritic cells. The authors concluded that PD-L1 expression on
tumor cells might be a mechanism of non-AR driven resistance to enzalutamide. In addition
to patients with hormone-sensitive prostate cancer, these patients might therefore also
potentially respond to anti-PD-L1 checkpoint blockade immunotherapy.
In order to elucidate the potential of PD-1/PD-L1 targeted therapy in prostate cancer, further
studies are needed to clarify: a) if PD-L1 expression can contribute to identifying insignificant
prostate cancer cases that may be spared immediate definitive therapy, b) if PD-L1
expression in primary tumors is predictive for response to anti-PD-L1 therapy, c) if castration-
sensitive prostate cancers have low PD-L1 expression levels due to androgen suppression
and subsequently d) how much ADT itself downregulates PD-L1, and finally, e) if anti-PD-L1
therapy is effective at all in prostate cancer.
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Figure legends
Figure 1. Validation of PD-L1 antibody specificity. A. Flow cytometric analysis of
untreated DU145 cells compared to concentration matched mouse IgG isotype control
showed PD-L1 membrane expression in 40% of cells. B. Western blot analysis of untreated
DU145 and MCF7 cells probed with the PD-L1 mAb clone EPR1161(2) (left) and the
validated PD-L1 mAb (E1L3N®) XP® (right). Equal loading was shown by probing for β-Actin.
C. Western blot analysis (left) and quantification of protein levels (right) of siRNA-mediated
transient knockdown of PD-L1 in DU145 cells. Cells were treated with siRNA directed against
PD-L1 (siPD-L1), non-targeting control siRNA (siCtrl), or transfectant reagent only (HPF) for
72h. Lysates were probed with the PD-L1 mAb clone EPR1161(2) (top) and PD-L1 mAb
(E1L3N®) XP® (bottom). Protein levels were normalized against β-Actin and quantified using
a colorimetric assay. D. Western blot analysis (top) of DU145 cells using PD-L1 mAb clone
EPR1161(2) with and without blocking peptide preincubation. Equal loading was shown by
probing for β-Actin. Immunohistochemical staining (bottom) of tonsillar and placental tissue
using PD-L1 mAb clone EPR1161(2) with distinct membranous staining of tonsillar
epithelium and placental trophoblasts (left) and primary antibody neutralization using
blocking peptide (right).
Figure 2. Immunohistochemical analysis of PD-L1 in prostate cancer. Representative
immunohistochemical staining showing strong (A), moderate (B), and weak (C) membranous
PD-L1 expression in epithelial tumor cells. Negative staining (D) was observed in a minority
of cases.
Figure 3. Survival analysis in primary prostate cancer after radical prostatectomy.
Kaplan-Meier analysis of BCR-free survival in 209 (training cohort, A) and 611 (test cohort,
B) prostate cancer patients stratified by PD-L1 expression.
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Tables Table 1: Baseline characteristics. Clinicopathologic variables and PD-L1 expression dichtotomized by median (high=above median, low=below median) of the training (n=209) and test cohort (n=611). All patients had a localized prostate cancer (M0) with a postoperative decrease of PSA serum levels below 0.1 ng/ml.
Training Cohort Test Cohort
All
Patients [%] PD-L1 high [%] PD-L1
low [%] P-Value All Patients [%] PD-L1
high [%] PD-L1 low [%] P-Value
Patient Number 209 [100.0] 109 [52.2] 100 [47.8] 611 [100.0] 377 [61.7] 234 [38.3] Mean Follow-up [Months]
62.8 49.5
Median Follow-up [Months]
61.0 49.6
Range Follow-up [Months]
0-140 0-129
Age Range 45-83 43-74 Mean 64 62 Median 65 62 ≤ Median 108 [51.7] 47 [22.5] 61 [29.2] 307 [50.2] 190 [31.1] 117 [19.1] > Median 100 [47.8] 61 [29.2] 39 [18.7] p = 0.010† 304 [49.8] 187 [30.6] 117 [19.1] p = 0.92† Unknown 1 [0.5] 0 [0.0] <50 5 [2.4] 0 [0.0] 5 [2.4] 19 [3.1] 11 [1.8] 8 [1.3] 50-54 5 [2.4] 3 [1.4] 2 [1.0] 55 [9.0] 31 [5.1] 24 [3.9] 55-59 28 [13.4] 14 [6.7] 14 [6.7] 105 [17.2] 62 [10.1] 43 [7.0] 60-64 58 [27.8] 24 [11.5] 34 [16.3] 222 [36.3] 149 [24.4] 73 [11.9] 65-69 74 [35.4] 49 [23.4] 25 [12.0] 175 [28.6] 100 [16.4] 75 [12.3] ≥70 38 [18.2] 18 [8.6] 20 [9.6] p = 0.077‡ 35 [5.7] 24 [3.9] 11 [1.8] p = 0.58‡ Pathological Stage pT2 124 [59.3] 63 [30.1] 61 [29.2] 418 [68.4] 251 [41.1] 167 [27.3] pT3/pT4 85 [40.7] 46 [22.0] 39 [18.7] p = 0.64† 193 [31.6] 126 [20.6] 67 [11.0] p = 0.22† Preoperative PSA [ng/ml]
Range 0.7 - 163
0.8 - 39.0
Mean 11.6
8.5
Median 7.5
7.1
< Median 98 [46.9] 54 [25.8] 44 [21.1] 301 [49.3] 170 [27.8] 131 [21.4]
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† Χ2-test (Pearson) ‡Χ2-test (linear-by-linear)
≥ Median 101 [48.3] 51 [24.4] 50 [23.9] p = 0.571† 302 [49.4] 201 [32.9] 101 [16.5] p = 0.011† Unknown 10 [4.8] 8 [1.3] ≤4.0 16 [7.7] 9 [4.3] 7 [3.3] 46 [7.5] 29 [4.7] 17 [2.8] 4.1 – 10 119 [56.9] 66 [31.6] 53 [25.4] 395 [64.6] 234 [38.3] 161 [26.4] 10.1 – 20 43 [20.6] 18 [8.6] 25 [12.0] 142 [23.2] 101 [16.5] 41 [6.7] > 20 21 [10.0] 12 [5.7] 9 [4.3] p = 0.53‡ 20 [3.3] 7 [1.1] 13 [2.1] p = 0.76‡ Surgical Margin Unknown 2 [1.0] 3 [0.5] R1 83 [39.7] 50 [23.9] 33 [15.8] 169 [27.7] 107 [17.5] 62 [10.1] R0 124 [59.3] 57 [27.3] 67 [32.1] p = 0.044† 439 [71.8] 268 [43.9] 171 [28.0] p = 0.61† Gleason Grade Unknown 5 [2.4] G1 (<7) 99 [47.4] 42 [20.1] 57 [27.3] 216 [35.4] 126 [20.6] 90 [14.7] G2 (7) 61 [29.2] 37 [17.7] 24 [11.5] 286 [46.8] 180 [29.5] 106 [17.3] G3 (>7) 44 [21.1] 29 [13.9] 15 [7.2] p = 0.004‡ 109 [17.8] 71 [11.6] 38 [6.2] p = 0.20‡ Gleason grading group
Unknown 5 [2.4] I (<7) 99 [47.4] 42 [20.1] 57 [27.3] 216 [35.4] 126 [20.6] 90 [14.7] 2 (3+4) 41 [19.6] 24 [11.5] 17 [8.1] 228 [37.3] 137 [22.4] 91 [14.9] 3 (4+3) 20 [9.6] 13 [6.2] 7 [3.3] 58 [9.5] 43 [7.0] 15 [2.5] 4 (8) 30 [14.3] 20 [9.6] 10 [4.8] 69 [11.3] 45 [7.4] 24 [3.9] 5 (>8) 14 [6.7] 9 [4.3] 5 [2.4] p = 0.007‡ 40 [6.5] 26 [4.3] 14 [2.3] p = 0.11‡ Nodal Status (pN) Unknown 1 [0.5] 303 [49.6] N0 192 [91.9] 102 [48.8] 90 [43.1] 299 [48.9] 201 [32.9] 98 [16.0] N1 16 [7.7] 7 [3.3] 9 [4.3] p = 0.47† 9 [1.5] 5 [0.8] 4 [0.7] p = 0.46† Androgen Receptor Unknown 5 [2.4] 67 [11.0] None 5 [2.4] 1 [0.5] 4 [1.9] 40 [6.5] 18 [2.9] 22 [3.6] Weak 61 [29.2] 22 [10.5] 39 [18.7] 144 [23.6] 79 [12.9] 65 [10.6] Moderate 80 [38.3] 45 [21.5] 35 [16.7] 266 [43.5] 179 [29.3] 87 [14.2] Strong 58 [27.5] 39 [18.7] 19 [9.1] p < 0.001‡ 94 [15.4] 69 [11.3] 25 [4.1] p < 0.001‡ Ki-67 Unknown 5 [2.4] 17 [2.8] Negative (≤ Median) 118 [56.5] 49 [23.4] 69 [33.0] p < 0.001† 301 [49.3] 153 [25.0] 148 [24.2] p < 0.001† Positive (> Median) 86 [41.1] 58 [27.8] 28 [13.4] 293 [48.0] 214 [35.0] 79 [12.9]
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Table 2: Univariate and multivariate Cox analyses on BCR-free survival in the training (n=209) and validation cohort (n=611) of prostate cancer cases treated by radical prostatectomy.
Univariate Cox Analysis Multivariate Cox Analysis
Training Cohort Test Cohort All Patients
Hazard ratio [95% CI]
p-value
Hazard ratio [95% CI]
p-value
Hazard ratio [95% CI]
p-value
pT Category (pT2 reference)
pT3 and pT4 1.93 [1.08 – 3.45] 0.027 4.62 [3.03 – 7.03] <0.001 2.20 [1.49 – 3.24] <0.001
Gleason Score (7 reference)
<7 0.37 [0.16 – 0.86] 0.37 [0.20 – 0.67] 0.43 [0.26 – 0.71]
>7 4.08 [2.02 – 8.21] <0.001 2.39 [1.55 – 3.69] <0.001 1.95 [1.32 – 2.86] <0.001 Surgical Margin (R0 reference)
R1 1.94 [1.07 – 3.52] 0.030 2.84 [1.90 – 4.25] <0.001 1.44 [1.00 – 2.09] 0.053
PD-L1 (continuous variable) 2.37 [1.32 – 4.25] 0.004 1.49 [1.10 – 2.02] 0.011 1.46 [1.11 – 1.92] 0.007
Preoperative PSA level 1.01 [0.99 – 1.02] 0.41 1.05 [1.02 – 1.09] 0.002 1.00 [0.99 – 1.01] 0.75
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Published OnlineFirst November 16, 2015.Clin Cancer Res Heidrun Gevensleben, Dimo Dietrich, Carsten Golletz, et al. aggressive primary prostate cancerThe immune checkpoint regulator PD-L1 is highly expressed in
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