So Yamaki

PD-L1 Expression in Pancreatic Ductal Adenocarcinoma is a Poor Prognostic Factor in Patientswith High CD8+ Tumor-Infiltrating Lymphocytes: Highly Sensitive Detection using PhosphorIntegrated Dot Staining

So Yamaki1, Hiroaki Yanagimoto1*, Koji Tsuta2, Hironori Ryota1,2, Masanori Kon1

1

Department of Surgery, Kansai Medical University, Hirakata, Osaka, Japan2

Department of Pathology and Laboratory Medicine, Kansai Medical University, Hirakata, Osaka,

Japan*Correspondence author: Hiroaki Yanagimoto
Department of Surgery, Kansai Medical University Hirakata Hospital
2-3-1 Shinmachi, Hirakata-shi, Osaka 573-1191, Japan
TEL: +81-72-804-0101; FAX: +81-72-804-0131
E-mail: yanagimh@hirakata.kmu.ac.jp

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So Yamaki

Abstract
Background. Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. For thedevelopment of more effective immunotherapies, it is first necessary to elucidate the immunologicalescape mechanisms. In this study, we applied our recently developed highly sensitive
immunostaining method employing fluorescent phosphor-integrated dot (PID) nanoparticles toevaluate the prevalence of programmed death ligand 1 (PD-L1) in patients with PDAC.
Methods. This study included 42 patients with PDAC who underwent pancreatectomy. We evaluatedPD-L1 expression in the patients with PDAC using PID staining, and correlated the PD-L1
expression level with the patients' clinico-pathological features.
Results. PD-L1 expression was detected in 61.9% (26/42) of the patients with PDAC by PID
staining. There was a significant difference in overall survival between PD-L1-positive and PD-L1negative cases (hazard ratio [HR] = 2.07; 95% confidence interval [CI] = 1.00–4.54; P = 0.049).
Among CD8+ tumor-infiltrating lymphocyte-positive cases, the overall survival of PD-L1-positive
patients was significantly poorer than that of PD-L1-negative patients (HR = 3.84; 95% CI = 1.59–
10.35; P = 0.003). Univariate and multivariate analyses indicated that PD-L1 expression was anindependent predictive poor prognostic factor in patients with PDAC.
Conclusions. PD-L1 expression appears to be an important prognostic factor in patients with PDAC
who underwent surgical resection.

Key word: pancreatic adenocarcinoma, PD-L1, immunohistochemistry, phosphor-integrated dots

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Introduction
Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a poor prognosis; it is thefifth leading cause of cancer-related deaths in Japan and the fourth most common malignancy in the
United States [1,2]. At the time of PDAC diagnosis, less than 20% of the tumors are resectable, andthe actual 5-year survival rate is reported to range from 15% to 25% [3]. Current clinical treatments
for PDAC have limited efficacy; thus, improved treatment strategies are required to prolong patient
survival.
Expression of programmed death-1 (PD-1) is significantly upregulated on activated cancerspecific T cells. PD-1 receptor attaches to its ligand PD-L1, which is expressed by tumor cells andinfiltrating immune cells. The interaction of PD-1 and PD-L1 inhibits T-cell activation and promotes
tumor immune escape.[4–7] The escape mechanism acquired by tumor cells to avoid immune
recognition and destruction is a major contributor to the limitations of the therapeutic efficacy.
However, recently developed therapeutic antibodies against PD1/PD-L1 show promising clinicalresults for several tumors such as melanoma, renal cancer, and non-small cell lung cancer [8].
Although the efficacy of PD-1/PD-L1 antibody therapy should be correlated with PD-L1
protein expression in tumor cells, approximately 10–40% of PD-L1 immuno-negative cases also
respond to anti-PD-1/PD-L1 therapy [9–11]. This contradiction may be caused by the performanceof the PD-L1 immunostaining assay based on the color intensity visualized using the chromogen
dye diaminobenzidine (DAB). We recently developed an immunohistochemistry method using
fluorescence-emitting phosphor-integrated dot (PID) nanoparticles as a fluorescent dye. PID showsa higher luminance and dynamic range than those of conventional fluorescent dyes and DAB [12].
Specifically, the fluorescence intensity of the PID particles was found to be approximately 100-foldhigher than that of a conventional fluorescent dye. Furthermore, the ratio of particle to antibody
binding is 1:1. Thus, this technique is highly sensitive as well as quantitative as compared to theconventional DAB-based method.
The aim of the present study was to evaluate the expression of PD-L1 in patients with PDAC
by immunostaining using PID technology, and to compare the results with those obtained afterconventional DAB staining.

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Materials and methods
Patients and samples
This study included 42 patients with PDAC, 31 of whom underwent pancreaticoduodenectomy
and 11 underwent distal pancreatectomy at the Department of Surgery at Kansai Medical University
Hospital (Osaka, Japan) between May 2001 and December 2007. All patients were histologicallyconfirmed as positive for PDAC. The tumors were classified according to the TNM classiﬁcation[13]. A prospectively maintained institutional PDAC database was used to collect all the clinicalparameters of the patients.
Surgically resected specimens were fixed with formalin and embedded in paraffin, and serial
sections were stained with hematoxylin and eosin for histological evaluation. The mostrepresentative tumor areas were sampled for the tissue microarray using 2-mm-diameter samples(Azumaya, Tokyo, Japan).
This study was conducted in accordance with the Declaration of Helsinki, and the study
protocol was approved by the institutional review board of our hospital (Protocol No. H151043 and27-14).

ImmunohistochemistryFour-micrometer-thick sections were routinely deparaffinized. To block endogenous
peroxidase activity, deparaffinized sections were subjected to 3% hydrogen peroxide treatment for
15 min and were then washed in deionized water for 2 to 3 min. Then, the sections were boiled in
10 mM sodium citrate buffer for 10 min at 121°C. The sections were allowed to cool at room
temperature for 40 min, followed by rinsing with deionized water and washing with phosphatebuffered saline for 5 min.

Measurement of the fluorescence properties of PID
The sections were incubated with the primary antibody toward PD-L1 (E1L3N, 1:1000, Cell
Signaling Technology, Danvers, MA, USA). Sections were incubated with 2 μg/mL biotinylated
anti-rabbit antibody (LO-RG-1) for 30 min, and then with PID-conjugated streptavidin (0.06 nM)

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for 2 h, both at room temperature. The sections were then irradiated at 580 nm, and the fluorescenceintensities were measured using a BX53 fluorescence microscope (Olympus, Tokyo, Japan) andimages were acquired with a DP73 CCD camera (Olympus) (Fig. 1). The number of PID particles
per cell was measured with an automated PID Analyzer (Konica Minolta, Tokyo, Japan). The PDL1 particle number was evaluated only on the tumor cells. Five fields at 400× magnification wereselected randomly, and the number of PD-L1 particles on each tumor cell was counted and theaverage number of particles per cell was calculated for each field. The highest value among the five
fields was determined to be the PID staining value. The negative control was prepared with PID
staining but without the primary antibody.

Measurement of DAB intensity
The sections were incubated with the primary antibody toward PD-L1 (E1L3N, 1:200, Cell
Signaling Technology) diluted in antibody diluent (Signal Stain Antibody Diluent #8112, Cell
Signaling Technology). The sections were treated with peroxidase-labeled secondary antibody(EnVision/HRP system, DAKO, Carpinteria, CA, USA) after linker reagent treatment for 15 min.
The sections were then rinsed in the buffer and immersed in DAB to observe color development.
For DAB-naked eye evaluation, three observers (S.Y., H.R., and K.T.) assessed theimmunostaining results in a blinded manner without knowledge of the clinical or histopathologicaldiagnoses. Intensity was graded on a 3-tier scale (1+, negative to weak; 2+, moderate; and 3+,strong). The percentage of staining was recorded, and a semi-quantitative (H-score) approach [14]was used for analysis. PD-L1 expression scores were calculated (from 0 to 300) by multiplying thepercentage of the stained tumor area by the staining intensity score.

Double staining of PD-L1 using the PID method and of CD8+ lymphocytes using the DAB method
Double staining, i.e., DAB staining for CD8+ lymphocytes and PD-L1 staining with the PID
method, was performed to count the tumor infiltrating lymphocytes (TILs) (primary antibody for
PD-L1: E1L3N, 1:100; primary antibody for CD8: C8/114B, 1:250; DAKO; secondary antibody:
EnVision/HRP system) (Fig. 2).
The correlations between PD-L1 expression, overall survival, and clinico-pathological data

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were evaluated.

Statistical analysis
Statistical analysis was performed using the JMP® 10 software (SAS institute Inc., Cary, NC,
USA). The Student’s t-test was used to analyze continuous variables and the chi-square test wasused to analyze categorical variables. Cumulative survival rates were calculated by the Kaplan–
Meier method. Significant differences in survival status were evaluated using the log-rank test. The
Cox proportional hazards model was used in the multivariate analysis and expressed by the hazard
ratio (HR) with a 95% confidence interval (CI). P < 0.05 was considered statistically significant.

Results
Patient characteristics
The patient background and clinico-pathological parameters are shown in Table 1. Among all thepatients, 16 were women and 26 were men. The patients’ median age at the time of diagnosis was
65 years (range 50−83 years). This study comprised 3, 6, 33, and 0 patients diagnosed at the T1, T2,
T3, and T4 stages, respectively. Lymph node metastases were detected in 26 of 42 patients (61.9%).
Seven patients were identified as M1 because of No. 16 lymph node metastasis without other organ
metastasis. Considering the pathological stage (pStage) as defined by the Union for International
Cancer Control classification, 3, 4, 9, 19, and 7 cases were at pStage Ia, Ib, IIa, IIb, and IV,
respectively. The median survival time (MST) of the 42 patients was 26 months (Fig. 3).
Pathological curative resection (R0) was performed in 27 of the 42 patients (64.3%). Neoadjuvant chemo-radiation therapy was performed in 8 of the 42 patients (19.0%), and adjuvant
chemotherapy was performed in 23 of the 42 patients (54.8%).

PD-L1 expression in the 42 PDAC patients by DAB and PID staining
To judge the rate of positive PID staining, the threshold of the PID staining value wasestablished. As the average of the highest value of the negative control of PID staining was 3.01,
the threshold value for judging positive PID staining was set to 3.0. Using this threshold, PD-L1

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expression was detected in 26 of the 42 patients (61.9%).
By contrast, PD-L1 expression measured by DAB-naked eye evaluation was detected in only 6 of
the 42 patients (14.3%). This expression rate was measured to be between the PID positive staining
value derived from the threshold set at 4.0 (28.6%, 12/42) and 5.0 (11.9%, 5/42).

Relationship between PD-L1 expression and clinico-pathological features
The correlations between pathological features and PD-L1 expression are shown in Table 2.
The ratio of males was significantly higher in the PD-L1-positive group (male/female: 20/6) than in
the PD-L1-negative group (male/female: 6/10; P = 0.021). There was no significant correlation
between PD-L1 expression and tumor size, lymph node metastasis (including distant lymph nodemetastasis [M1]), pre-operative CA19-9 level, and R0/1 status. CD8+ TILs counts were notsignificantly correlated to PD-L1 expressions.

Survival analysis
Survival curves obtained using the Kaplan-Meier method are shown in Fig 4a,b. Among the 42
patients, there was a significant difference in the overall survival rate between the PD-L1-positive
cases and PD-L1-negative cases using PID staining (HR = 2.07; 95% CI = 1.00–4.54; P = 0.049).
The MST was 23.5 months in the PD-L1-positive group and 51.6 months in the PDL-1-negative
group. Among 29 patients who had positive CD8+ TIL (more than 3 cells per 400× field), the PDL1-positive group showed a significantly poorer overall survival rate than the PD-L1-negative groupbased on detection with the PID method (HR = 3.84; 95% CI = 1.59–10.35; P = 0.003).
The results of univariate and multivariate analyses in the 42 patients are shown in Table 3.
Univariate and multivariate analysis revealed that PD-L1 expression determined by the PID method(PID staining value > 3.0) was an independent prognostic factor (HR = 2.34; 95% CI = 1.02–5.74;
P = 0.045). In the 29 TIL-positive patients, PD-L1 expression determined by the PID method wasan independent predictive poor prognostic factor (Table 4). Moreover, there was a stronger
prognostic effect of PD-L1 expression among these 29 TIL-positive cases compared to the analysis
including all 42 patients (HR = 4.39; 95% CI = 1.64–13.34; P = 0.003).

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Discussion
In this study, we investigated PD-L1 expression in patients with PDAC. The PD-L1-positive
detection rate in PID staining was higher than that in DAB staining, which was possibly due to thefact that digital immunostaining can detect proteins at lower concentrations. The higher
fluorescence intensity of the nanoparticles contributes to the higher sensitivity of PID compared to
DAB. In addition, quantitative analysis was possible even in cells with strong positive expression,
without saturation.
In this study, the PD-L1 positive expression rate detected using the PID method in PDAC cases
was higher than that reported in previous studies (4–49%) [15-17] using the DAB method. Amongthe current cases, no case was found to be negative for PID and positive for DAB. The PD-L1
positive expression rate was 14.3% using the DAB method and was 61.9% using the PID method
when the PID staining threshold value was set to 3.0. Thus, this result supported the current PD-L1
protein expression data using PID, which was found to be more sensitive than DAB. However, theDAB staining positive rate was similar to the PID staining positive rate when using the threshold of
4.0 or 5.0. In other words, cells with slightly positive expression showing 3–5 PID particles could
not be detected by conventional methods.
Immunotherapeutic approaches, most notably, immune checkpoint inhibitors epitomized by
antibodies directed against T-lymphocyte regulators, cytotoxic T-lymphocyte-associated protein 4(CTLA-4), and PD-1 have demonstrated efficacy in a variety of solid tumors, including metastaticmelanoma and lung cancer, and have already received US Food and Drug Administration approval.
PD-1/PD-Ll pathway blockade has resulted in significant and durable clinical responses in patientswith several malignancies such as melanoma, renal cell carcinoma, lung cancer, mismatch repairdeficient colorectal cancer, and bladder cancer [18]. However, PDAC has generally been considered
as a non-immunogenic malignancy, given that tumor-infiltrating effector T lymphocytes do not
represent a histopathological hallmark of this disease [19,20]. Investigators have been actively
exploring the mechanisms underlying the evasion of immune surveillance by pancreatic cancer cells,
and several potential strategies have been proposed to overcome resistance to immune checkpoint
inhibitors. In this study, TILs reached tumor cells in 29 of 42 surgical PDAC specimens.

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In a clinical setting, patients with positive PD-L1 expression tend to show significantly
unfavorable outcomes. Previous studies using DAB methods have also demonstrated that patientswith PD-L1-positive PDAC showed unfavorable outcomes. Wang et al showed the correlation
between B7-H1 (PD-L1) expressions and pathological grade and TNM stage [16]. Nomi et alreported that PD-L1 positive PDAC patients had significant poor prognosis than PD-L1 negative
patients [17]. In their data, as same as our result, there was no significant correlation between tumor
PD-L1 status and clinical indicators including tumor status, nodal status, metastatic status andpathologic stage. In the current study, among the TIL-positive patients, there was PD-L1 expression
showed a stronger prognostic correlation than observed in the analysis including all patients.
Univariate and multivariate analyses showed that positive PD-L1 expression (PID staining value >3.0) was an independent poor prognostic factor in PDAC cases with positive CD8+ TILs. This result
suggests that an immunosuppressive tumor microenvironment with high PD-L1 expression caninterfere with the attack by TILs on the tumor cells. In these cases, there is a possibility that blockageof the PD-1/PD-L1 signaling pathway can make the attack of TILs dramatically more effective.
Although there have been no objective responses observed in patients with PDAC who received
anti-PD-L1 antibody therapy [21], our results suggest that PD-L1-positive patients with TILs might
be good candidates for anti-PD-L1 antibody therapy.

Conclusions
PID staining provided superior results as compared to those obtained by the canonical DABstaining method. We have shown for the first time that PD-L1, detected using PID staining, is a
novel prognostic marker for human PDAC. Digital immunostaining is a promising tool forcompanion diagnostics to evaluate the therapeutic effects of molecular-targeted drugs andimmunotherapy.

Acknowledgements

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The authors would like to thank Aoi Nozawa (KONICA MINOLTA, INC.) for helpful suggestions. We
would also like to thank Editage (www.editage.jp) for English language editing.

Conflict of Interest Statement
The authors declare that have no conflict of interest.

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Table 1. Patients’ characteristics and clinicopathological data.
n=42
Age
65 (50-83)
Male : Female
26 : 16
Tumor location (Ph : Pbt)
31 : 11
R0 : R1
27 : 15
Neoadjuvant therapy (+:-)
8 : 34
Adjuvant chemotherapy (+:-)
23 : 19
Pre-operative CA19-9 (U/ml)
134.2 (1.6–8116)
T stage (1 : 2 : 3)
3 : 6 : 33
N stage (0 : 1)
16 : 26
M stage (0 : 1)
35 : 7
Tumor diameter (mm)30 (16-75)
The data are expressed as median and range.
Ph: pancreatic head, Pbt: pancreatic body or tailT and N stage were based on the TNM classification of malignant tumors, sixth edition.
All M1 cases had No.16 lymph node metastasis without other organ metastasis.

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Table 2. Relationships between PD-L1 expression and pathological features
Parameter
PD-L1 expression (+) (n=26)
PD-L1 expression (-) (n=16)Age64(51-82)
66(50-78)
Male : Female
20 : 6
6 : 10Tumor location (Ph : Pbt)
19 : 7
12 : 4R0 : R1
15 : 11
12 : 4
Neoadjuvant therapy (+:-)
5 : 21
3 : 13
Adjuvant chemotherapy (+:-)
15 : 118:8Pre-operative CA19-9 (U/ml)
143.0(1.6-8116)
113.0(20.3-1712)
T stage (1,2 : 3)
5 : 21
4 : 12
N stage (0 : 1)
10: 16
6 : 10
M stage (0 : 1)
21 : 5
14: 2
Tumor diameter (mm)29.5(18-75)
32.5(16-45)
The data are expressed as median and range.
Ph: pancreatic head, Pbt: pancreatic body or tailT and N stage were based on the TNM classification of malignant tumors, sixth edition.
All M1 cases had No.16 lymph node metastasis without other organ metastasis.

P
0.385
0.021
0.891
0.256
0.969
0.627
0.421
0.711
0.950
0.570
0.583

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Table 3. The results of univariate and multivariate analysis for overall survival in all cases. (n=42)
Univariate

Multivariate

HR(95% CI)

P

HR(95% CI)

P

R0/1

1.81(0.87-3.64)

0.108

1.16(0.51-2.59)

0.710

Neoadjuvant therapy (+ / -)1.75(0.74-5.18)

0.220

Adjuvant chemotherapy (+ / -)1.28(0.64-2.61)

0.480

CA19-9 > 138 U/ml

1.57(0.76-3.22)

0.216

T stage (1-2 / 3)

1.28(0.58-3.21)

0.555

N stage (0 / 1)2.06(1.01-4.44)

0.046

2.21(0.91-5.62)

0.081

M stage (0 / 1)2.25(0.88-5.08)

0.087

1.09(0.38-2.86)

0.863

ly (0-1 / 2-3)

1.23(0.60-2.65)

0.573

v (0-1 / 2-3)

1.15(0.57-2.50)

0.697

PD-L1 expression (+) (PID)

2.07(1.00-4.54)

0.049

2.34(1.02-5.74)

0.045

PD-L1 expression (+) (DAB)

1.28(0.50-4.33)

0.633

CD8+ TILs >3.0

1.31(0.62-3.15)

0.490

HR: Hazard Ratio, CI: confidence interval, PID: phosphor-integrated dots , DAB: diaminobenzidine
T and N stage were based on the TNM classification of malignant tumors, sixth edition.
All M1 cases had No.16 lymph node metastasis without other organ metastasis.

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Table 4. The results of univariate and multivariate analysis for overall survival in patients with positive CD8 +TILs. (n=29)
Univariate

Multivariate

HR(95% CI)

P

HR(95% CI)

P

R0/1

1.90(0.82-4.24)

0.131

1.55(0.63-3.77)

0.334

Neoadjuvant therapy (+ / -)1.42(0.53-4.88)

0.512

Adjuvant chemotherapy (+ / -)1.00(0.45-2.21)

0.999

CA19-9 > 138 U/ml

1.14(050-2.60)

0.751

T stage (1-2 / 3)

1.08(0.39-2.60)

0.864

N stage(0 / 1)1.64(0.73-3.94)

0.233

1.80(0.66-5.12)

0.250

M stage(0 / 1)

3.09(0.97-8.58)

0.056

1.63(0.47-5.05)

0.420

ly (0-1 / 2-3)

1.18(0.48-2.69)

0.710

v(0-1 / 2-3)

1.05(0.43-2.43)

0.904

PD-L1 expression(+) (PID)

3.84(1.59-10.35)

0.003

4.39(1.64-13.34)

0.003

PD-L1 expression(+) (DAB)

1.89(0.44-5.84)

0.349

HR: Hazard Ratio, CI: confidence interval, PID: phosphor-integrated dots , DAB: diaminobenzidine
T and N stage were based on the TNM classification of malignant tumors, sixth edition.
All M1 cases had No.16 lymph node metastasis without other organ metastasis.

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Figure Captions
Fig 1. Immunohistochemistry of pancreatic ductal adenocarcinoma tissue using phosphor-integrated
dot (PID) staining. Red spots on tumor cells indicate PID particles.
Fig 2. Immunohistochemical double staining for PD-L1 on tumor cells and CD8+ lymphocytes.
Phosphor-integrated dot (PID) staining was used for PD-L1 detection and diaminobenzidine (DAB)
staining was used for CD8+ lymphocytes. The number of PID particles measured by an automated
PID analyzer is indicated for each tumor cell (white numbers) and lymphocytes (yellow numbers).
Fig 3. Overall survival of 42 patients with pancreatic ductal adenocarcinoma.
Fig 4. (a) Overall survival rates of 42 patients with pancreatic ductal adenocarcinoma correlated to
PD-L1 expression. Overall survival in PD-L1-positive cases was significantly poorer than that in
PD-L1-negative cases (P = 0.049). (b) Overall survival rate of 29 patients showing positive CD8+
tumor-infiltrating lymphocytes (TILs) correlated to PD-L1 expression; there was a significant
difference in prognosis between PD-L1-positive and -negative cases (P = 0.003).

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