The immune checkpoint regulator PD-L1 is highly expressed in aggressive primary ... › content › ... · 1 The immune checkpoint regulator PD-L1 is highly expressed in aggressive

1

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]

Research. on July 20, 2020. © 2015 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on November 16, 2015; DOI: 10.1158/1078-0432.CCR-15-2042

http://clincancerres.aacrjournals.org/

2

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.




3

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




4

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




5

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.




6

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




7

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




8

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




9

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).




10

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




11

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.




12

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




13

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.




14

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.




15

References

1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359-86. 2. Siegel R, DeSantis C, Virgo K, Stein K, Mariotto A, Smith T, et al. Cancer treatment and survivorship statistics, 2012. CA Cancer J Clin. 2012;62:220-41. 3. Heidenreich A, Bastian PJ, Bellmunt J, Bolla M, Joniau S, van der Kwast T, et al. EAU guidelines on prostate cancer. Part II: Treatment of advanced, relapsing, and castration-resistant prostate cancer. Eur Urol. 2014;65:467-79. 4. Horwich A, Parker C, de Reijke T, Kataja V. Prostate cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2013;24 Suppl 6:vi106-14. 5. Laufer M, Denmeade SR, Sinibaldi VJ, Carducci MA, Eisenberger MA. Complete androgen blockade for prostate cancer: what went wrong? J Urol. 2000;164:3-9. 6. Beer TM, Armstrong AJ, Rathkopf DE, Loriot Y, Sternberg CN, Higano CS, et al. Enzalutamide in metastatic prostate cancer before chemotherapy. N Engl J Med. 2014;371:424-33. 7. Scher HI, Fizazi K, Saad F, Taplin ME, Sternberg CN, Miller K, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med. 2012;367:1187-97. 8. de Bono JS, Logothetis CJ, Molina A, Fizazi K, North S, Chu L, et al. Abiraterone and increased survival in metastatic prostate cancer. N Engl J Med. 2011;364:1995-2005. 9. Fizazi K, Scher HI, Molina A, Logothetis CJ, Chi KN, Jones RJ, et al. Abiraterone acetate for treatment of metastatic castration-resistant prostate cancer: final overall survival analysis of the COU-AA-301 randomised, double-blind, placebo-controlled phase 3 study. Lancet Oncol. 2012;13:983-92. 10. Sweeney CJ, Chen YH, Carducci M, Liu G, Jarrard DF, Eisenberger M, et al. Chemohormonal Therapy in Metastatic Hormone-Sensitive Prostate Cancer. N Engl J Med. 2015;373:737-46. 11. Kwek SS, Cha E, Fong L. Unmasking the immune recognition of prostate cancer with CTLA4 blockade. Nat Rev Cancer. 2012;12:289-97. 12. Schweizer MT, Drake CG. Immunotherapy for prostate cancer: recent developments and future challenges. Cancer Metastasis Rev. 2014;33:641-55. 13. Zou W, Chen L. Inhibitory B7-family molecules in the tumour microenvironment. Nat Rev Immunol. 2008;8:467-77. 14. Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000;192:1027-34. 15. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol. 2008;26:677-704. 16. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366:2455-65. 17. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012;366:2443-54. 18. Cho DC, Sosman JA, Sznol M, Gordon MS. Clinical activity, safety, and biomarkers of MPDL3280A, an engineered PD-L1 antibody in patients with metastatic renal cell carcinoma (mRCC). J Clin Oncol 2013;31, (suppl; abstr 4505). 19. Choueiri TK, Fishman MN, Escudier BJ, Kim JJ. Immunomodulatory activity of nivolumab in previously treated and untreated metastatic renal cell carcinoma (mRCC): Biomarker-based results from a randomized clinical trial. J Clin Oncol 2014;32:5s:(suppl; abstr 5012). 20. Powles T, Eder JP, Fine GD, Braiteh FS, Loriot Y, Cruz C, et al. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature. 2014;515:558-62. 21. Seiwert T, Seiwert T, Seiwert T. A phase Ib study of MK-3475 in patients with human papillomavirus (HPV)-associated and non-HPV-associated head and neck (H/N) cancer. J Clin Oncol. 2014;32:5s, 2014 (suppl; abstr 6011)




16

22. Ribas A, Hodi FS, Kefford R. Efficacy and safety of the anti-PD-1 monoclonal antibody MK-3475 in 411 patients (pts) with melanoma (MEL). J Clin Oncol. 2014;2014;32(Suppl):5S. 23. Rizvi N, Rizvi N, Rizvi N. Clinical trials of MPDL3280A (anti-PDL1) in patients (pts) with non-small cell lung cancer (NSCLC). J Clin Oncol. 2014;32:5s, 2014 (suppl; abstr TPS8123). 24. Chen BJ, Chapuy B, Ouyang J, Sun HH, Roemer MG, Xu ML, et al. PD-L1 expression is characteristic of a subset of aggressive B-cell lymphomas and virus-associated malignancies. Clin Cancer Res. 2013;19:3462-73. 25. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002;8:793-800. 26. Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med. 1999;5:1365-9. 27. Liang SC, Latchman YE, Buhlmann JE, Tomczak MF, Horwitz BH, Freeman GJ, et al. Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses. Eur J Immunol. 2003;33:2706-16. 28. Lyford-Pike S, Peng S, Young GD, Taube JM, Westra WH, Akpeng B, et al. Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPV-associated head and neck squamous cell carcinoma. Cancer Res. 2013;73:1733-41. 29. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012;12:252-64. 30. Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, et al. Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med. 2012;4:127ra37. 31. Thompson RH, Kuntz SM, Leibovich BC, Dong H, Lohse CM, Webster WS, et al. Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up. Cancer Res. 2006;66:3381-5. 32. Kristiansen G, Fritzsche FR, Wassermann K, Jager C, Tolls A, Lein M, et al. GOLPH2 protein expression as a novel tissue biomarker for prostate cancer: implications for tissue-based diagnostics. Br J Cancer. 2008;99:939-48. 33. Meller S, Bicker A, Montani M, Ikenberg K, Rostamzadeh B, Sailer V, et al. Myoglobin expression in prostate cancer is correlated to androgen receptor expression and markers of tumor hypoxia. Virchows Arch. 2014;465:419-27. 34. Stein J, Majores M, Rohde M, Lim S, Schneider S, Krappe E, et al. KDM5C Is Overexpressed in Prostate Cancer and Is a Prognostic Marker for Prostate-Specific Antigen-Relapse Following Radical Prostatectomy. Am J Pathol. 2014;184:2430-7. 35. Epstein JI, Zelefsky MJ, Sjoberg DD, Nelson JB, Egevad L, Magi-Galluzzi C, et al. A Contemporary Prostate Cancer Grading System: A Validated Alternative to the Gleason Score. Eur Urol. 2015. 36. Soliman H, Khalil F, Antonia S. PD-L1 expression is increased in a subset of basal type breast cancer cells. PLoS One. 2014;9:e88557. 37. Hamanishi J, Mandai M, Iwasaki M, Okazaki T, Tanaka Y, Yamaguchi K, et al. Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer. Proc Natl Acad Sci U S A. 2007;104:3360-5. 38. Karim R, Jordanova ES, Piersma SJ, Kenter GG, Chen L, Boer JM, et al. Tumor-expressed B7-H1 and B7-DC in relation to PD-1+ T-cell infiltration and survival of patients with cervical carcinoma. Clin Cancer Res. 2009;15:6341-7. 39. Konishi J, Yamazaki K, Azuma M, Kinoshita I, Dosaka-Akita H, Nishimura M. B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression. Clin Cancer Res. 2004;10:5094-100. 40. Ohigashi Y, Sho M, Yamada Y, Tsurui Y, Hamada K, Ikeda N, et al. Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand-2 expression in human esophageal cancer. Clin Cancer Res. 2005;11:2947-53.




17

41. Strome SE, Dong H, Tamura H, Voss SG, Flies DB, Tamada K, et al. B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma. Cancer Res. 2003;63:6501-5. 42. Thompson RH, Gillett MD, Cheville JC, Lohse CM, Dong H, Webster WS, et al. Costimulatory B7-H1 in renal cell carcinoma patients: Indicator of tumor aggressiveness and potential therapeutic target. Proc Natl Acad Sci U S A. 2004;101:17174-9. 43. Bigelow E, Bever KM, Xu HY, Yager A, Wu A, Taube J, et al. Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue. Jove-J Vis Exp. 2013. 44. Boland JM, Kwon ED, Harrington SM, Wampfler JA, Tang H, Yang P, et al. Tumor B7-H1 and B7-H3 Expression in Squamous Cell Carcinoma of the Lung. Clin Lung Cancer. 2013;14:157-63. 45. Nakanishi J, Wada Y, Matsumoto K, Azuma M, Kikuchi K, Ueda S. Overexpression of B7-H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers. Cancer Immunol Immunother. 2007;56:1173-82. 46. Zhang Y, Huang S, Gong D, Qin Y, Shen Q. Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small cell lung cancer. Cell Mol Immunol. 2010;7:389-95. 47. Botticelli AR, Casali AM, Botticelli L, Zaffe D. Immunohistochemical detection of cell-cycle associated markers on paraffin embedded and formalin fixed needle biopsies of prostate cancer: correlation of p120 protein expression with AgNOR, PCNA/cyclin, Ki-67/MIB1 proliferation-scores and Gleason gradings. Eur J Histochem. 1998;42:41-8. 48. Kaibuchi T, Furuya Y, Akakura K, Masai M, Ito H. Changes in cell proliferation and apoptosis during local progression of prostate cancer. Anticancer Res. 2000;20:1135-9. 49. Scalzo DA, Kallakury BV, Gaddipati RV, Sheehan CE, Keys HM, Savage D, et al. Cell proliferation rate by MIB-1 immunohistochemistry predicts postradiation recurrence in prostatic adenocarcinomas. Am J Clin Pathol. 1998;109:163-8. 50. Visakorpi T. Proliferative activity determined by DNA flow cytometry and proliferating cell nuclear antigen (PCNA) immunohistochemistry as a prognostic factor in prostatic carcinoma. J Pathol. 1992;168:7-13. 51. Bishop JL, Sio A, Angeles A, Roberts ME, Azad AA, Chi KN, et al. PD-L1 is highly expressed in Enzalutamide resistant prostate cancer. Oncotarget. 2015;6:234-42.




18

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.




19

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]

Research.

on July 20, 2020. © 2015 A

merican A

ssociation for Cancer

clincancerres.aacrjournals.org D

ownloaded from

Author m

anuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author M

anuscript Published O

nlineFirst on N

ovember 16, 2015; D

OI: 10.1158/1078-0432.C

CR

-15-2042


20

† 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]

Research.

on July 20, 2020. © 2015 A

merican A

ssociation for Cancer

clincancerres.aacrjournals.org D

ownloaded from

Author m

anuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Author M

anuscript Published O

nlineFirst on N

ovember 16, 2015; D

OI: 10.1158/1078-0432.C

CR

-15-2042


21

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


p-value


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













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

Updated version

10.1158/1078-0432.CCR-15-2042doi:

Access the most recent version of this article at:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/early/2015/11/14/1078-0432.CCR-15-2042To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-15-2042

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/early/2015/11/14/1078-0432.CCR-15-2042


The immune checkpoint regulator PD-L1 is highly expressed in aggressive primary ... › content › ... · 1 The immune checkpoint regulator PD-L1 is highly expressed in aggressive

Documents