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Safety and Antitumor Activity of Anti-PD-1 Antibody, Nivolumab, in

Patients With Platinum-Resistant Ovarian Cancer

Article  in  Journal of Clinical Oncology · September 2015

DOI: 10.1200/JCO.2015.62.3397

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 Published Ahead of Print on September 8, 2015 as 10.1200/JCO.2015.62.3397
The latest version is at http://jco.ascopubs.org/cgi/doi/10.1200/JCO.2015.62.3397
JOURNAL OF CLINICAL ONCOLOGY
Junzo Hamanishi, Masashi Kanai,
Yukiko Mori, Shigemi Matsumoto,
Shunsuke Chikuma, Noriomi
Matsumura, Kaoru Abiko, Tsukasa
Baba, Ken Yamaguchi, Akihiko Ueda,
Yuko Hosoe, Tasuku Honjo, and Ikuo
Konishi, Kyoto University Graduate
School of Medicine, Kyoto, Japan;
Masaki Mandai, Kinki University Faculty
of Medicine, Osaka, Japan; and Taka-
fumi Ikeda, Manabu Minami, Atsushi
Kawaguchi, Toshinori Murayama,
Satoshi Morita, Masayuki Yokode, and
Akira Shimizu, Kyoto University Hospi-
tal, Kyoto, Japan.
Published online ahead of print at
www.jco.org on September 8, 2015.
Supported by research funds from
Health and Labour Sciences Research
Grant No. 11104959 (to J.H. and I.K.);
by Translational Research Network
Program of the Ministry of Education,
Culture, Sports, Science and Technol-
ogy of Japan Grant No. 2013037 (to
J.H. and I.K.); and by Kyoto University
grants.
Both J.H. and M.M. contributed nearly
equally to this work.
Terms in blue are defined in the glos-
sary, found at the end of this article
and online at www.jco.org.
Authors’ disclosures of potential
conflicts of interest are found in the
article online at www.jco.org. Author
contributions are found at the end of
this article.
Corresponding author: Junzo Hamanishi,
MD, PhD, Department of Gynecology and
Obstetrics, Kyoto University Graduate
School of Medicine, 54 Kawahara-cho,
Shogoin, Sakyo-ku, Kyoto, Kyoto 606-8507,
Japan; e-mail: jnkhmns@kuhp.kyoto-u.ac.jp.
© 2015 by American Society of Clinical
Oncology
0732-183X/15/3399-1/$20.00
DOI: 10.1200/JCO.2015.62.3397
Copyright 2015 by American Society of Clinical Oncology
O R I G I N A L R E P O R T
Safety and Antitumor Activity of Anti–PD-1 Antibody,
Nivolumab, in Patients With Platinum-Resistant
Ovarian Cancer
Junzo Hamanishi, Masaki Mandai, Takafumi Ikeda, Manabu Minami, Atsushi Kawaguchi,
Toshinori Murayama, Masashi Kanai, Yukiko Mori, Shigemi Matsumoto, Shunsuke Chikuma,
Noriomi Matsumura, Kaoru Abiko, Tsukasa Baba, Ken Yamaguchi, Akihiko Ueda, Yuko Hosoe,
Satoshi Morita, Masayuki Yokode, Akira Shimizu, Tasuku Honjo, and Ikuo Konishi
A B S T R A C T
Purpose
Programmed death-1 (PD-1), a coinhibitory immune signal receptor expressed in T cells, binds to
PD-1 ligand and regulates antitumor immunity. Nivolumab is an anti–PD-1 antibody that blocks
PD-1 signaling. We assessed the safety and antitumor activity of nivolumab in patients with
platinum-resistant ovarian cancer.
Patients and Methods
Twenty patients with platinum-resistant ovarian cancer were treated with an intravenous infusion
of nivolumab every 2 weeks at a dose of 1 or 3 mg/kg (constituting two 10-patient cohorts) from
October 21, 2011. This phase II trial defined the primary end point as the best overall response.
Patients received up to six cycles (four doses per cycle) of nivolumab treatment or received doses
until disease progression occurred. Twenty nivolumab-treated patients were evaluated at the end
of the trial on December 7, 2014.
Results
Grade 3 or 4 treatment-related adverse events occurred in eight (40%) of 20 patients. Two
patients had severe adverse events. In the 20 patients in whom responses could be
evaluated, the best overall response was 15%, which included two patients who had a durable
complete response (in the 3-mg/kg cohort). The disease control rate in all 20 patients was
45%. The median progression-free survival time was 3.5 months (95% CI, 1.7 to 3.9 months),
and the median overall survival time was 20.0 months (95% CI, 7.0 months to not reached) at
study termination.
Conclusion
This study, to our knowledge, is the first to explore the effects of nivolumab against ovarian
cancer. The encouraging safety and clinical efficacy of nivolumab in patients with platinum-
resistant ovarian cancer indicate the merit of additional large-scale investigations (UMIN Clinical
Trials Registry UMIN000005714).
J Clin Oncol 33. © 2015 by American Society of Clinical Oncology
Studies have demonstrated that ovarian can-
INTRODUCTION
cers express cancer-specific antigens and elicit
Ovarian cancer is the leading cause of death among spontaneous antitumor immune responses from
patients with gynecologic malignancies. Greater immunotherapy.4-7 As we and others have shown,
than 50% of patients with ovarian cancer are di- the number of tumor-infiltrating lymphocytes is a
agnosed at an advanced stage.1,2 Despite cytore- significant prognostic factor,5-10 which suggests
ductive surgery and platinum- and taxane-based that host immunity plays a pivotal role in the
chemotherapies, greater than 70% of patients clinical course of ovarian cancer. However, it has
with advanced ovarian cancer who achieve remis- recently been shown that ovarian cancer cells ac-
sion ultimately experience relapse. Because there quire the potential to escape from host immunity
are few effective treatments for these patients,3 the in the tumor microenvironment, and new treat-
development of new treatment strategies is ur- ment strategies to overcome this phenomenon
gently required.3,4 are needed.3,11-13
© 2015 by American Society of Clinical Oncology 1
 Hamanishi et al
Programmed cell death-1 (PD-1), an immune checkpoint recep-
tor expressed by T cells, binds to two PD-1 ligands (PD-Ls), PD-L1
(B7-H1) and PD-L2 (B7-DC), and suppresses antigen-specific cancer
immune reactions.14-16 Many types of human tumors, such as mela-
noma, renal cancer, and ovarian cancer, express PD-L1 and have the
ability to escape from the host immune system via PD-1/PD-L1 sig-
naling.7, 17-19 We previously reported that PD-L1 expression was asso-
ciated with poor prognosis in ovarian cancer5 and that PD-L1
promoted progression of ovarian cancer by inducing host immuno-
suppression of peripheral cytotoxic CD8⫹ T cell lymphocytes.20 These
findings suggest that PD-1/PD-L1 signaling might be a new treatment
target for ovarian cancer.
Therefore, we conducted a phase II clinical trial in 20 patients
with platinum-resistant, recurrent, or advanced ovarian cancer to
evaluate the safety and antitumor efficacy of nivolumab, a fully
human immunoglobulin G4 anti–PD-1 receptor– blocking mono-
clonal antibody.
PATIENTS AND METHODS
Study Design and Patients
This single-center, open-label, phase II trial was conducted in two co-
horts of patients with advanced or relapsed, platinum-resistant ovarian cancer.
Eligible patients were characterized by the following: documented platinum-
resistant (defined by a platinum-free interval of ⬍ 6 months from the most
recent platinum-based treatment regimen), recurrent or advanced ovarian
cancer, peritoneal cancer, or tubal cancer; a treatment history of at least
two chemotherapy regimens that included platinum and taxane agents; age
from 20 to 79 years; life expectancy of 3 months or greater; an Eastern
Cooperative Oncology Group performance status of 0 or 1; and lesions
evaluable by RECIST. Patients were excluded if they had any of the follow-
ing: complications of autoimmune disease; previous systemic corticoste-
roid therapy or immune suppression; or a history of anti–PD-1 antibody
therapy. The study was approved by the institutional review board of
Kyoto University Hospital. Written informed consent for participation in
the study was obtained from all participants.
Procedure
Eligible patients were allocated sequentially to the low-dose cohort (niv-
olumab 1 mg/kg; n ⫽ 10) followed by the high-dose cohort (nivolumab 3
mg/kg; n ⫽ 10) and received nivolumab intravenously every 2 weeks for up to
1 year or until progressive disease (PD) or disease progression occurred. One
cycle comprised four doses administered 2 weeks apart (over a total of 8
weeks). Patients received a maximum of six cycles (24 doses over 48 weeks).
Dose modification was not permitted. The lesions of all patients were evalu-
ated by computed tomography every 2 months for up to 1 year or until PD or
disease progression occurred. Response rate (RR) was assessed by RECIST
version 1.1, and adverse events were evaluated by the National Cancer Institute
Common Terminology Criteria for Adverse Events version 4.0. The trial
scheme and flow diagram are shown in Appendix Figures A1 and A2, respec-
tively (online only).
Outcomes
The primary end point was the best overall response for each patient as
assessed by RECIST version 1.1. Antitumor response was independently eval-
uated by three central reviewers, including a radiologist involved in this study.
The secondary end points were the following: drug safety, progression-free
survival, overall survival, disease control rate, and adverse events.
Statistical Analysis
The sample size was based on the precision of the estimate of the primary
end point (RR), because the main purpose of this trial was to provide prelim-
2 © 2015 by American Society of Clinical Oncology
inary evidence of drug efficacy before embarking on a larger trial. For study
sample sizes of 20, the corresponding upper limit of the one-sided 97.5% CI
(95% CI) that was calculated on the basis of the binomial distribution if one
patient had a response of 24.9% (21.6%). The next phase (a larger-scale,
randomized, phase II or III trial) would be considered worth undertaking if
greater than one patient had a response. Because this is the first time niv-
olumab has been studied for ovarian cancer, two dose cohorts were imple-
mented. Dose-specific evaluation and the dose-response relationship were
only secondary end points, for which statistical accuracy has not been assured
in this study. Neither the precisions of the estimates for each cohort nor a
comparison between cohorts was primarily evaluated in this trial, but the
precision for 10 patients would be considered guaranteed on the basis of the
upper limit values presented in the Appendix (online only). Thus, for this trial
with two sequential cohorts, 10 patients were included per cohort, for a total
sample size of 20.
Binary end points were used to estimate the best overall response, with a
two-sided 95% CI that was based on the exact binomial distribution. Time-to-
event end points were used to estimate median survival times with the Kaplan-
Meier method. The two-sided 95% CIs were calculated with the Brookmeyer
and Crowley method for the median survival time. These analyses were imple-
mented for the pooled cohort and for each cohort individually.
Drug and Drug Supply
Nivolumab is a fully human immunoglobulin G4 anti–PD-1 monoclo-
nal antibody that binds PD-1, inhibits the engagement with PD-Ls (PD-L1 or
PD-L2), and can enhance antitumor activity of T cells.21 Nivolumab was
provided by Ono Pharmaceutical (Osaka, Japan). All trial drugs were packaged
identically in this study.
Immunohistochemical Analysis of PD-L1
Immunohistochemical analysis of PD-L1 was performed using
formalin-fixed, paraffin-embedded tumor specimens with murine anti–
human PD-L1 monoclonal antibody (clone 27A2; MBL, Nagoya, Japan)5 by
LSI Medience (Tokyo, Japan). The staining for PD-L1 was evaluated and scored in
a third-party review (LSI Medience) by two independent pathologists who were
unaware of the outcomes. The methods are detailed in the Appendix.
RESULTS
Baseline Patient Characteristics
A total of 20 patients with advanced or relapsed, platinum-
resistant ovarian cancer were eligible for the protocol and were treated
with anti–PD-1 antibody (nivolumab) from October 21, 2011, to
December 7, 2014 (trial completion date). The median patient age was
60 years (range, 47 to 79 years; standard deviation [SD], 9.0). Histo-
logic tumor subtypes were as follows: serous (n ⫽ 15; 75%), endo-
metrioid (n ⫽ 3; 15%), and clear cell (n ⫽ 2; 10%; Table 1;
International Federation of Gynecology and Obstetrics (FIGO) stag-
ing included as well). Eleven (55%) of the 20 patients were previously
treated with at least four regimens (Table 1). The median follow-up
period and the duration of treatment with nivolumab were 11.0
months (range, 3 to 32 months; SD, 8.7) and 3.5 months (range, 1 to
12 months; SD, 3.6), respectively (Appendix Table A1, online only).
Safety
The most common treatment-related adverse events (ie, in ⬎
four patients [ⱖ 20%]) were increased serum AST, hypothyroidism,
lymphocytopenia, decreased serum albumin, fever, increased serum
ALT, maculopapular rash, arthralgia, arrhythmia, fatigue, and anemia
(Table 2). Grade 3 or 4 treatment-related adverse events occurred in
eight of 20 patients (40%; 95% CI, 19.9% to 64.0%; Table 2).
JOURNAL OF CLINICAL ONCOLOGY
 Anti–PD-1 Antibody for Ovarian Cancer

Table 1. Patient Characteristics

No. (%) of Patients

Enrolled Patient Data Total (N ⫽ 20) 1 mg/kg (n ⫽ 10) 3 mg/kg (n ⫽ 10)

Median (range) age, years 60.0 (47-79) 62.5 (49-79) 60.0 (47-76)
Cancer type
Ovarian cancer 18 (90) 8 (80) 10 (100)
Peritoneal cancer 2 (10) 2 (20) 0 (00)
Tubal cancer 0 (0) 0 (0) 0 (0)
FIGO stage [subtype, No. of patients]
I 2 (10) [Ic, 2] 0 (0) 2 (20) [Ic, 2]
II 0 (0) 0 (0) 0 (0)
III 14 (70) [IIIb, 1; IIIc, 13] 9 (90) [IIIb, 1; IIIc, 8] 5 (50) [IIIc, 5]
IV 4 (20) 1 (10) 3 (30)
Histology
Serous 15 (75) 8 (80) 7 (70)
Endometrioid 3 (15) 2 (20) 1 (10)
Clear cell 2 (10) 0 (0) 2 (20)
Prior chemotherapy regimens
ⱖ4 11 (55) 5 (50) 6 (60)
⬍4 9 (45) 5 (50) 4 (40)
ECOG performance status
0 18 (90) 10 (100) 8 (80)
1 2 (10) 0 (0) 2 (20)

NOTE. Summary of exposure to nivolumab, all treated patients: A total of 20 patients with advanced or relapsed, platinum-resistant ovarian cancer were treated
with anti–programmed death-1 (nivolumab). International Federation of Gynecology and Obstetrics (FIGO) staging of disease in all patients was performed at initial
diagnosis postoperatively.
Abbreviation: ECOG, Eastern Cooperative Oncology Group.

Treatment-related adverse events in this study included several events
that were expected, including hypothyroidism and lymphocytopenia
without neutropenia. The frequency and severity of treatment-related
adverse events were not different between the 1- and 3-mg/kg cohorts
(Table 2). Treatment-related serious adverse events were observed in
two patients (10%; 95% CI, 1.2% to 31.7%; Appendix Table A2,
online only). One patient in the 1-mg/kg cohort had two grade 3
events, disorientation and gait disorder, after developing a fever
that lasted longer than 1 month. Another patient in the 3-mg/kg
cohort had grade 3 fever and deep vein thrombosis. After treatment
for these conditions, objective antitumor regression was observed
in this patient. Nivolumab treatment was discontinued in two
(11%) of 18 patients as a result of treatment-related thyroiditis
(Appendix Table A1).
The most frequently observed adverse events were those related
to thyroid function: hypothyroidism (n ⫽ 8; Table 2), thyroiditis (n ⫽
2), hyperthyroidism (n ⫽ 1), high thyroid-stimulating hormone (n ⫽
1), and low thyroid-stimulating hormone (n ⫽ 1). Almost all events
were grade 1 except for one occurrence of grade 2 thyroiditis. Niv-
olumab 1 mg/kg treatment for this patient with nivolumab was ceased
after one dose in the first course because of thyroiditis-induced fever
and tachycardia, and subsequent tumor progression occurred. The

Table 2. Treatment-Related Adverse Events of Special Interest That Occurred in at Least 20% of All Treated Patients

No. (%) of Patients With Adverse Event by Treatment Cohort and Grade

Total (N ⫽ 20) 1 mg/kg (n ⫽ 10) 3 mg/kg (n ⫽ 10)

Event All Grades Grade 3 or 4 All Grades Grade 3 or 4 All Grades Grade 3 or 4
Any adverse event 19 (95) 8 (40) 9 (90) 4 (40) 10 (100) 4 (40)
AST increased 8 (40) 0 (0) 6 (60) 0 (0) 2 (20) 0 (0)
Hypothyroidism 8 (40) 0 (0) 4 (40) 0 (0) 4 (40) 0 (0)
Lymphocytopenia 7 (35) 3 (15) 4 (40) 3 (30) 3 (30) 0 (0)
Albumin decreased 6 (30) 2 (10) 2 (20) 1 (10) 4 (40) 1 (10)
Fever 6 (30) 1 (5) 2 (20) 1 (10) 4 (40) 0 (0)
ALT increased 5 (25) 1 (5) 4 (40) 1 (10) 1 (10) 0 (0)
Maculopapular rash 5 (25) 1 (5) 3 (30) 0 (0) 2 (20) 1 (10)
Arthralgia 5 (25) 0 (0) 5 (50) 0 (0) 0 (0) 0 (0)
Arrhythmia 6 (30) 0 (0) 4 (40) 0 (0) 2 (20) 0 (0)
Fatigue 4 (20) 0 (0) 2 (20) 0 (0) 2 (20) 0 (0)
Anemia 4 (20) 3 (15) 3 (30) 2 (20) 1 (10) 1 (10)

www.jco.org © 2015 by American Society of Clinical Oncology 3

 Hamanishi et al

Table 3. Clinical Effect of Anti–PD-1 Antibody Treatment

Response No. (%) of Patients [95% CI]ⴱ Survival, Months (95% CI)
Dose of No. of
Nivolumab Patients CR PR SD PD NE RR DCR PFS OS
1 mg/kg 10 0 1 4 4 1 1 (10) [0.3 to 44.5] 5 (50) [18.7 to 81.3] 3.5 (0.7 to 3.9) 16.1 (2.6 to NR)
3 mg/kg 10 2 0 2 6 0 2 (20) [2.5 to 55.6] 4 (40) [12.2 to 73.8] 3.0 (1.5 to 3.9) NR (2.9 to NR)
Total 20 2 1 6 10 1 3/20 (15%) [3.2 to 37.9] 9/20 (45%) [23.1 to 68.5] 3.5 (1.7 to 3.9) 20.0 (7.0 to NR)

Abbreviations: CR, complete response; DCR, disease control rate (ie, CR, PR, and SD); NE, not evaluated; NR, not reached; OS, point estimate of overall survival;
PD-1, programmed death-1; PFS, point estimation of progression-free survival; PR, partial response; RR, response rate (ie, CR and PR); SD, stable disease.
ⴱ
No. of patients of the total listed in the same row.

patient data, therefore, were classified as not evaluated. In another
patient with thyroiditis, treatment with nivolumab 1 mg/kg also ended
during the third course, although the patient experienced a partial
response (PR) for 5 months. The most common adverse events are
described in Appendix Table A3 (online only).
Clinical Activity
The best overall RR across the two cohorts, confirmed by inde-
pendent central review, was 15% (three of 20 patients; 95% CI, 3.2% to
37.9%), and the disease control rate was 45% (nine of 20 patients; 95%
CI, 23.1% to 68.5%; Table 3).
In the 1-mg/kg cohort, one patient experienced a PR and four
had stable disease. The objective RR was 10% (95% CI, 0.3% to
44.5%), and the disease control rate was 50% (95% CI, 18.7% to
81.3%). The partial responder experienced an antitumor response up
to 5 months; however, nivolumab treatment was stopped after three
courses because of adverse events, and disease recurred 3 months after
withdrawal from the trial.

A
150

CA-125 (U/mL) 111

100

50
28
9 7 8 7 6 7

0 250 500
Baseline 4 Months
Time (days)

B 316
CA-125 (U/mL)

300

200

100

16 10 10 8 8 8

0 200 400

Baseline 4 Months Time (days)

Fig 1. Activity of anti–programmed death 1 (PD-1) antibody in two patients with a complete response to recurrent ovarian cancer. (A) Complete response of
platinum-resistant and recurrent serous ovarian cancer in a 59-year-old patient who received anti–PD-1 antibody (nivolumab) at a dose of 3.0 mg/kg. This patient had
previously undergone primary surgery, and progressive disease had developed after treatment with systemic chemotherapies (ie, cyclophosphamide, adriamycin, and
cisplatin; or docetaxel and carboplatin). Although multiple pelvic lymph node metastases were observed on a baseline computed tomographic image (left), these lesions
were no longer apparent at 4 months after the start of nivolumab treatment (middle). The white circles show regression of recurrent lymph node metastases. Tumor
marker cancer antigen 125 (CA-125) decreased to normal range after one course of nivolumab (right). (B) Complete response of platinum-resistant and recurrent ovarian
clear cell carcinoma in a 60-year-old patient who received nivolumab at a dose of 3.0 mg/kg. This patient had previously undergone right salpingo-oophorectomy, and
progressive disease had developed after treatment with systemic chemotherapies (ie, mitomycin and irinotecan; irinotecan and cisplatin; or paclitaxel and carboplatin)
and supracervical hysterectomy plus left salpingo-oophorectomy. The circles show complete regression of recurrent peritoneal dissemination (left, baseline; middle,
4 months after the start of nivolumab treatment). Tumor marker CA-125 decreased to normal range after one course of nivolumab (right). The horizontal dashed line
represents the cutoff level for CA-125 (35 U/mL).

4 © 2015 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY


In the 3-mg/kg cohort, two patients had a complete response
(CR), and two had stable disease. The objective RR was 20% (95% CI,
2.5% to 55.6%), and the disease control rate was 40% (95% CI, 12.2%
to 73.8%). One of the two patients who experienced a CR was a
59-year-old person with ovarian serous adenocarcinoma (Fig 1A).
After two courses of nivolumab treatment, multiple pelvic lymph
node metastatic lesions completely disappeared, and levels of tumor
marker cancer antigen 125 (CA-125) decreased to the normal range.
This patient survived without experiencing tumor relapse for 2
months after completion of the 1-year trial of nivolumab. The other
complete responder was a 60-year-old patient who had clear cell
carcinoma with recurrent disease and peritoneal dissemination (Fig
1B). After two courses of nivolumab treatment, these peritoneal le-
sions disappeared, and CA-125 decreased to the normal range. This
patient recently completed a 1-year trial of nivolumab and continues
to experience a CR.
As shown in Figure 2A, in the four patients who had antitumor
responses, the responses were durable and evident. Three of four
patients had an objective response according to RECIST version 1.1
(CR, n ⫽ 2; PR, n ⫽ 1), whereas one patient experienced progressive
disease with a new recurrent lesion even though the target lesion had
completely regressed (Figs 2A and 2B).
A 200
Change in Target Lesions
150
From Baseline (%)
100
50
0
50 100 150 200 250
-50
-100
Time (days)
B showed the same finding.21 According to the National Comprehen-
150
Change in Target Lesions
PD
From Baseline (%)
100
50 PD PD PD PD
PD PD SD SD PD* PD* SD SD SD NE PD*
0 antitumor effect.22-24 In an early phase II trial in 44 patients with
-50
-100
Fig 2. Best overall responses in all patients in two cohorts with anti–
programmed death 1 (PD-1) antibody. The blue lines represent the 1-mg/kg
cohort, and the gold lines represent the 3-mg/kg cohort. (A) Duration of best
overall responses and changes in target lesions from baseline in 20 patients who
received anti–PD-1 antibody at a dose of 1.0 or 3.0 mg/kg every 2 weeks. Two
patients with a complete response (CR; [*] one patient with lymph node
metastasis evaluated as CR after anti–PD-1 antibody treatment [Fig 1A]; [†]
another patient with peritoneal dissemination evaluated [Fig 1B]) were still
receiving treatment with anti–PD-1 antibody. (B) Changes in target lesions from
baseline in 20 patients. Although four of 20 patients had data below the horizontal
dashed lines, three of these four patients were considered to have an objective
response (ie, two CRs and oen partial response [PR]). In one patient with
progressive disease (PD) who had a new recurrent lesion, best overall response
was measured as stable disease (SD), although the target lesion had completely
regressed after SD. In three patients who had new recurrent lesions (indicated by
PD*), PD was determined, although the target lesions were measured as SD.
The two horizontal dashed lines mark the thresholds for objective response
(lower line, ⫺30%) and PD (higher line, 20%), according to RECIST (version 1.1).
NE, not evaluable.
www.jco.org
Anti–PD-1 Antibody for Ovarian Cancer
The median progression-free survival times were 3.5 months
(95% CI, 1.7 to 3.9 months) for the pooled cohort, 3.5 months (95%
CI, 0.7 to 3.9 months) for the 1-mg/kg cohort, and 3.0 months (95%
CI, 1.5 to 3.9 months) for the 3-mg/kg cohort (Table 3; Fig 3A). The
median overall survival times were 20.0 months (95% CI, 7.0 to
months) for the pooled cohort and 16.1 months (95% CI, 2.6 to
months) for the 1-mg/kg cohort; overall survival was not evaluated for
the 3-mg/kg cohort, because data for greater than one half of the
patients were censored (Table 3; Fig 3B). The upper limits could not be
evaluated for all 20 patients, because too few events occurred after the
median overall survival times (Table 3; Fig 3).
PD-L1 Expression on Tumor Cells
Ovarian cancer specimens from all 20 patients were analyzed for
PD-L1 expression in tumor cells by immunohistochemistry (Figs 4A
to 4D). Tumor specimens from 16 (80%) of 20 patients showed high
expression of PD-L1 (n ⫽ 15 patients with scores of ⫹2; n ⫽ 1 patient
with a score of ⫹3), whereas four (20%) of 20 patients showed low
expression of PD-L1 (n ⫽ 4 patients with scores of ⫹1). An objective
response (ie, CR and PR) occurred in two of the 16 patients with
tumors that showed high expression of PD-L1, whereas nonresponse
occurred in two of the four patients with tumors that expressed low
levels of PD-L1. However, the expression of PD-L1 was not signifi-
cantly correlated with objective response (P ⫽ .509; Fig 4E).
1 mg/kg (n = 10)
DISCUSSION
3 mg/kg (n = 10)
This study, to our knowledge, is the first investigator-initiated, phase II
clinical trial to test the safety and efficacy of nivolumab against
300 350 400 platinum-resistant ovarian cancer. The 3-mg/kg dose of nivolumab
PR SD* may be more favorable than the 1-mg/kg dose for ovarian cancer,
CR*
because this dose showed better efficacy without a significant increase
CR†
in toxicity. A previous clinical trial of nivolumab in other solid tumors
1 mg/kg (n = 10)
sive Cancer Network guidelines, the current standard treatment for
3 mg/kg (n = 10) platinum- and taxane-resistant ovarian cancer is single-agent chemo-
therapy with pegylated liposomal doxorubicin, topotecan, or gemcit-
abine; however, these agents did not demonstrate a satisfactory clinical
platinum-resistant ovarian cancer, monotherapy with bevacizumab, a
PR
SD* CR
vascular endothelial growth factor inhibitor, no patients experienced a
*
CR† CR, and the RR was 15.9%. In this trial, the median progression-free
survival time was 4.4 months, and the median overall survival time
was 10.7 months at study termination.25
In the context of these previous findings, the durable antitumor
response of the 3-mg/kg dose of nivolumab in our exploratory study
was clinically significant enough to warrant large-scale studies of niv-
olumab in patients with platinum-resistant ovarian cancer. Notably,
in one of the two patients who experienced a CR, the tumor was
histologically identified as clear cell carcinoma. Ovarian clear cell
carcinoma has a worse prognosis than the more common serous
adenocarcinoma.26 There are few effective chemotherapies for recur-
rent clear cell carcinoma; the overall RR for this malignancy was
reported to be approximately 2% (ie, one of 51 patients).26 The gene
expression profiles are similar between renal and ovarian clear cell
carcinomas,27 so nivolumab may show potential benefit in treating
clear cell carcinoma of the ovary.
© 2015 by American Society of Clinical Oncology 5
 Hamanishi et al

A 100
1 mg/kg B 3 mg/kg C Total
100 100
Progression-Free

Progression-Free

Progression-Free
Survival (%)

Survival (%)

Survival (%)
50 50 50

0 10 20 30 0 10 20 30 0 10 20 30
Time (months) Time (months) Time (months)
No. at risk 10 9 2 2 2 2 0 0 0 0 0 0 0 0 0 0 0 No. at risk 10 6 3 3 3 3 3 1 0 0 0 0 0 0 0 0 0 No. at risk 20 15 5 5 5 5 2 1 0 0 0 0 0 0 0 0 0

D 100
1 mg/kg E 3 mg/kg F Total
100 100
Overall Survival (%)

Overall Survival (%)

Overall Survival (%)

50 50 50

0 10 20 30 0 10 20 30 0 10 20 30
Time (months) Time (months) Time (months)
No. at risk 10 10 9 8 7 6 6 5 5 5 5 2 1 1 1 1 0 No. at risk 10 9 8 7 5 5 2 1 0 0 0 0 0 0 0 0 0 No. at risk 20 19 17 15 12 11 8 6 5 5 5 2 1 1 1 1 0

Fig 3. Progression-free survival and overall survival. Progression-free survival in the (A) 1-mg/kg cohort and (B) 3-mg/kg cohort and (C) in all patients; bars indicate
censoring events. Overall survival in the (D) 1-mg/kg cohort and (E) 3-mg/kg cohort and (F) in all patients; bars indicate censoring events.

In a previous phase I clinical trial of an anti–PD-L1 antibody In this study, treatment-related adverse events of grade 3 or 4
(BMS-936559) in 17 patients with ovarian cancers,28 one patient ex- occurred in eight (40%) of 20 patients, although treatment could be
perienced a PR (RR, 5.9%), and three had stable disease, which yielded completed in most (19 [95%] of 20) patients. The frequencies of
a disease control rate of 23.5%. These findings, as well as those of this arrhythmias and elevated AST were relatively higher in this study than
study, indicate that a certain patient population may demonstrate an in larger previous studies of nivolumab for other solid tumors,21 but
antitumor effect in response to PD-1 signal blocking. Thus, targeting all of these events were grade 1 or 2 and were controllable. Greater than
PD-1 signaling with anti–PD-L1 or anti–PD-1 treatment is a potential one half of the treatment-related adverse events in this study, such as
treatment strategy in patients with platinum-resistant ovarian cancer. fever, maculopapular rash, hypothyroidism, and lymphocytopenia

A B
E 15
No. of Patients
C D
Fig 4. The relationship between pro-
grammed death-1 ligand 1 (PD-L1) expression
on tumor cells and objective response to anti–
programmed death-1 (PD-1) antibody treat-
ment. Immunohistochemical analysis of
formalin-fixed paraffin-embedded ovarian can-
cer specimens before patients underwent
anti–PD-1 antibody treatment with anti–PD-1
monoclonal antibody. Representative (A) high
(⫹3) and (B) low (⫹1) staining patterns of
14 of 16
PD-L1 on ovarian cancers are shown. (C)
Responder Positive-control staining of PD-L1 on placenta

Nonresponder (⫹2) disappeared with the (D) absorption test.

10 P = .509 Scale bars, 100 ␮m. (E) The correlation be-
tween the expression of PD-L1 on tumor cells
and the objective response to anti–PD-1 anti-
5 body in 20 patients with platinum-resistant
3 of 4 ovarian cancers. Expression of PD-L1 was not
2 of 16 significantly correlated with objective re-
1 of 4
sponse (P ⫽ .509).
0
PD-L1 high PD-L1 low
(n = 16) (n = 4)

6 © 2015 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

 Anti–PD-1 Antibody for Ovarian Cancer

without neutropenia, were reported in a previous clinical trial of
nivolumab for other solid tumors.21 Neither pneumonitis nor colitis,
previously reported as severe immune-related adverse events,21 was
observed in this study.
Thyroiditis was observed in two patients in the 1-mg/kg cohort.
We revised the protocol to allow corticosteroid treatment for
immune-related adverse events by referring to a previous phase I
trial.21 After this protocol revision, one patient experienced immune-
related adverse events of eosinophilia and rash during the third course
of treatment; after corticosteroid administration, the patient was able
to tolerate the subsequent four courses of nivolumab.
Compared with a previous report about nivolumab,21 a late an-
titumor response pattern was not found in our series; rather, a quick
antitumor response was demonstrated (Figs 1 and 2). Interestingly,
few patients had stable disease or experienced a PR in this study,
whereas two patients experienced a CR. This discrete difference in the
effectiveness of nivolumab was not observed in other solid tumors21
and may be specific to patients with ovarian cancer. Thus, the estab-
lishment of a biomarker to predict the antitumor response of niv-
olumab in ovarian cancer is promising.
To identify predictive biomarkers for the antitumor effect of
nivolumab, as done in previous reports,21,29 we evaluated the expres-
sion of PD-L1 in ovarian cancer tissues but did not find any significant
correlation between clinical response and PD-L1 expression (Fig 4).
The discrepancy between this study and previous reports might be a
result of the following two points: First, ovarian cancers are classified
into several clusters on the basis of the gene expression profile data,
including data from the Cancer Genome Atlas.30,31 One of these clus-
ters, the immunoreactive phenotype, contains several immune-
related genes, but this phenotype composes only approximately 20%
of ovarian cancers. Therefore, this classification might have contrib-
uted to the low RR in our study, even in the presence of high expres-
sion of PD-L1 on tumor cells.
Second, our study may have had several problems related to
materials or methods, including the timing of tumor sampling. We
acquired most tumor samples during primary operations of patients,
whereas most samples in previous reports were obtained via biopsy
before nivolumab treatment for solid tumors.21,29 The expression
of PD-L1 might be altered by the therapeutic process or by inflam-
matory alterations at the tumor site.32 A recent trial of combination
therapy with anti–PD-1 antibody and anti-CTLA4 (cytotoxic T-
lymphocyte–associated protein 4) antibody for advanced mela-
noma showed no significant correlation between objective
response and PD-L1 expression in tumor tissues.29 A methodo-
logic problem may have been the use of different antibody clones
or unstable staining of formalin-fixed, paraffin-embedded
samples.21,29,32 Therefore, this study could not determine whether
the expression of PD-L1 is a true biomarker for the antitumor
effect against ovarian cancer, and additional investigation will be
needed to resolve this issue.
In conclusion, nivolumab demonstrated encouraging clinical ef-
ficacy and tolerability in patients with platinum-resistant ovarian can-
cer. Therefore, this study indicates the merit of a large-scale
investigation to evaluate the priority of nivolumab for platinum-
resistant ovarian cancer, and we plan to perform such a trial with a
pharmaceutical company.
AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS
OF INTEREST
Disclosures provided by the authors are available with this article at
www.jco.org.
AUTHOR CONTRIBUTIONS
Conception and design: Junzo Hamanishi, Masaki Mandai, Takafumi
Ikeda, Manabu Minami, Toshinori Murayama, Satoshi Morita, Akira
Shimizu
Administrative support: Junzo Hamanishi, Takafumi Ikeda
Collection and assembly of data: Junzo Hamanishi, Manabu Minami,
Toshinori Murayama, Masashi Kanai, Yukiko Mori, Shigemi
Matsumoto, Shunsuke Chikuma, Noriomi Matsumura, Kaoru Abiko,
Tsukasa Baba, Ken Yamaguchi, Akihiko Ueda, Yuko Hosoe, Masayuki
Yokode, Tasuku Honjo, Ikuo Konishi
Data analysis and interpretation: Junzo Hamanishi, Atsushi Kawaguchi,
Satoshi Morita, Akira Shimizu
Manuscript writing: All authors
Final approval of manuscript: All authors

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■ ■ ■

GLOSSARY TERMS

CA-125 (cancer antigen 125): a protein produced by the
fallopian tubes, the endometrium, and the lining of the abdomi-
nal cavity (peritoneum). CA-125 is a tumor marker present in
higher than normal amounts in the blood and urine of patients
with certain cancers. Typically, women with ovarian cancer have
high levels of CA-125. Other conditions associated with elevated
levels of CA-125 include endometriosis, pancreatitis, pregnancy,
normal menstruation, and pelvic inflammatory disease. CA-125
levels may be used to help diagnose ovarian cancer and to deter-
mine whether these tumors are responding to therapy. The nor-
mal range for CA-125 is less than 35 U/mL and less than 20
U/mL for women who have been treated for ovarian cancer.
Women with ovarian cancer may show values higher than 65
U/mL.
ⴙ
CD8 T cell: cytotoxic T cell that is the primary effector cells
of the immune system. They are characterized by the presence of
the CD8 cell-surface marker.
Computed tomography (CT) scan: a series of pictures
created by a computer linked to an x-ray machine taken of the
inside of the body from different angles.
FIGO staging: a tumor staging system established and revised
by the International Federation of Gynecology and Obstetrics
(FIGO) that takes into account the postoperative histopathologic
evaluation of the specimen. The FIGO stage classification has
prognostic value.
Immune checkpoint: immune inhibitory pathway that neg-
atively modulates the duration and amplitude of immune re-
sponses. Examples include the CTLA-4:B7.1/B7.2 pathway and
the PD-1:PD-L1/PD-L2 pathway.
Immunohistochemistry: the application of antigen-antibody
interactions to histochemical techniques. Typically, a tissue section is
mounted on a slide and incubated with antibodies (polyclonal or mono-
clonal) specific to the antigen (primary reaction). The antigen-antibody
signal is then amplified using a second antibody conjugated to a com-
plex of peroxidase-antiperoxidase, avidin-biotin-peroxidase, or avidin-
biotin alkaline phosphatase. In the presence of substrate and
chromogen, the enzyme forms a colored deposit at the sites of antibody-
antigen binding. Immunofluorescence is an alternate approach to visu-
alize antigens. In this technique, the primary antigen-antibody signal is
amplified using a second antibody conjugated to a fluorochrome. On
ultraviolet light absorption, the fluorochrome emits its own light at a
longer wavelength (fluorescence), thus allowing localization of
antibody-antigen complexes.
Immunotherapy: a therapeutic approach that uses cellular and/or
humoral elements of the immune system to fight a disease.
PD-1: programmed cell death protein 1 (CD279), a receptor expressed
on the surface of activated T, B, and NK cells that negatively regulates
immune responses, including autoimmune and antitumor responses.
PD-L1: programmed cell death 1 ligand 1 (CD274; also known as B7-H1),
the major binding partner (ligand) for the PD-1 inhibitory immune receptor.
PD-L1 is expressed on the surface of activated antigen presenting cells, such as
dendritic cells, and by many types of cancer cells. Its expression is induced by the
inflammatory cytokine interferon-alfa
RECIST (Response Evaluation Criteria in Solid Tu-
mors): a model proposed by the Response Evaluation Criteria Group
by which a combined assessment of all existing lesions, characterized by
target lesions (to be measured) and nontarget lesions, is used to extrapo-
late an overall response to treatment.

8 © 2015 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

 Anti–PD-1 Antibody for Ovarian Cancer

AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Safety and Antitumor Activity of Anti–PD-1 Antibody, Nivolumab, in Patients With Platinum-Resistant Ovarian Cancer
The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are
self-held unless noted. I ⫽ Immediate Family Member, Inst ⫽ My Institution. Relationships may not relate to the subject matter of this manuscript. For more
information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or jco.ascopubs.org/site/ifc.
Junzo Hamanishi Kaoru Abiko
No relationship to disclose No relationship to disclose
Masaki Mandai Tsukasa Baba
No relationship to disclose No relationship to disclose
Takafumi Ikeda Ken Yamaguchi
No relationship to disclose No relationship to disclose
Manabu Minami Akihiko Ueda
No relationship to disclose No relationship to disclose
Atsushi Kawaguchi Yuko Hosoe
No relationship to disclose
No relationship to disclose
Toshinori Murayama
Satoshi Morita
Honoraria: Kowa Pharmaceuticals America
Honoraria: Ono Pharmaceutical, Bristol-Myers Squibb
Masashi Kanai
No relationship to disclose Masayuki Yokode
No relationship to disclose
Yukiko Mori
Speakers’ Bureau: Bayer Akira Shimizu
Research Funding: Taiho Pharmaceutical (Inst), Yakult Honsha (Inst) No relationship to disclose

Shigemi Matsumoto Tasuku Honjo

No relationship to disclose Research Funding: Ono Pharmaceutical

Shunsuke Chikuma Ikuo Konishi

No relationship to disclose No relationship to disclose
Noriomi Matsumura
Research Funding: Daiichi Sankyo (Inst)

www.jco.org © 2015 by American Society of Clinical Oncology

 Hamanishi et al

Acknowledgment
We thank Hiroyuki Yoshikawa, MD, Hiroshi Date, MD, and Kaichiro Yamamoto, MD, for support on the Data and Safety Monitoring
Board; Kazuhiko Ino, MD, Kimihiko Ito, MD, and Shigeaki Umeoka, MD, for support as central reviewers; and Shingo Fujii, MD, for
supervision.

Appendix

Appendix Materials and Methods

Sample Size Estimation Details. The sample size was based on the precision of the estimate of the primary end point (the response
rate), because the main purpose of this trial was to provide preliminary evidence of drug efficacy before embarking on a larger trial. For
study sample sizes of 3, 6, 10, 15, and 20, the corresponding upper limits of the one-sided 97.5% (95%) CI calculated on the basis of the
binomial distribution if no patient had a response were 70.7% (63.2%), 45.9% (39.3%), 30.9% (25.9%), 21.8% (18.1%), and 16.8%
(13.9%), respectively; if one patient had a response, the respective values were 90.6% (86.5%), 64.1% (58.2%), 44.5% (39.4%), 32.0%
(27.9%), and 24.9% (21.6%). The next phase would be considered worth undertaking if more than one patient had a response.
Conversely, if none of the sample of 20 patients was to demonstrate a response, the response rate would have an upper one-sided 97.5%
confidence limit of 16.8%; 16.8% or lower would be unacceptable, and the treatment would not be promising in the target population.
Neither the precisions of the estimates for each cohort nor a comparison between cohorts was evaluated in this trial, but the precision for
10 patients would be considered guaranteed on the basis of the upper-limit values presented above. Thus, for this trial with two cohorts,
10 patients were included per cohort, for a total sample size of 20.

Definition of Disease Control Rate, Progression-Free Survival, and Overall Survival

The disease control rate was the proportion of patients whose best overall response was either complete response or partial response.
Progression-free survival (PFS) was defined as the period from the day of registration to recurrence, progression, or death as a result of any
cause, whichever came first. For patients who did not experience recurrence or progression, PFS was censored on the last known date free
of recurrence or progression on the basis of radiologic criteria. For reference and exploratory comparisons, PFS was censored on the last
known date free of recurrence or progression on the basis of clinical evaluation (radiologic confirmation not necessary). PFS was not
censored at the start of follow-up therapy. The occurrence of heterochronous multiple cancers was not regarded as a PFS event. Therefore,
PFS was not censored at the discovery of heterochronous multiple cancers. The study site (not the central evaluation committee) evaluated
clinical progression. Overall survival (OS) was defined as the period from the date of registration to the date of death as a result of any cause.
For patients who remained alive, OS was censored at the time the patient was last known to be alive. For patients whose data were lost to
follow-up, OS was censored on the date that the patients were last known to be alive before they became unreachable.

Efficacy End Points

Tumor response was the primary end point of this study. By using the exact binomial test, we estimated the tumor response rate and
its two-sided 95% CI (equivalent to the one-sided 97.5% CI) for the full analysis set (FAS; ie, the two dose cohorts combined). In addition,
we repeated the procedure separately for each cohort.
The disease control rate was a secondary end point of this study. By using the exact binomial test, we estimated the disease control rate
and its two-sided 95% CI for the FAS. In addition, we repeated the procedure separately for each cohort. PFS was an additional secondary
end point of this study. We used the Kaplan-Meier method to estimate the median PFS and the N-year PFS rates (N ⫽ 1, 2, etc) for the FAS.
We used the Brookmeyer and Crowley method to estimate the two-sided 95% CI for the median PFS, and we employed Greenwood’s
formula to determine the two-sided 95% CI for the N-year PFS rates. In addition, we repeated the procedure separately for each cohort.
OS was the final secondary end point of this study. We used the Kaplan-Meier method to estimate the median OS and the N-year OS rates
(N ⫽ 1, 2, etc) for the FAS. We used the Brookmeyer and Crowley method to estimate the two-sided 95% CI for median OS, and we
employed Greenwood’s formula to determine the two-sided 95% CI for the N-year OS rates. In addition, we repeated the procedure
separately for each cohort.

Immunohistochemical Analysis of Programmed Death-1 Ligand 1

Immunohistochemical analysis of programmed death-1 ligand 1 (PD-L1) was performed with formalin-fixed, paraffin-embedded
tumor specimens and with murine anti– human PD-L1 monoclonal antibody by LSI Medience (Tokyo, Japan). Briefly, LSI Medience
conducted immunohistochemistry of PD-L1 as follows:
● Heat the sections for 1 hour at 60°C.
● Deparaffinize the sections with xylene three times for 5 minutes each.
● Wash the slides with ethanol for 5 minutes. Wash the slides with 90% ethanol and 80% ethanol for 30 seconds each. Then,
wash the slides with 70% ethanol for 5 minutes.
● Wash the slides with flowing water for 5 minutes, and wash the slides with purified water.
● Place the slides on a staining basket in a 500-mL beaker with 500 mL of 10 mmol/L citrate buffer (pH 6.0). Cover the beaker
with plastic wrap; then process the slides in the autoclave for 10 minutes at 120°C.

© 2015 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

 Anti–PD-1 Antibody for Ovarian Cancer

● Let the slides cool down in the beaker at room temperature for approximately 1 hour.
● Remove the slides from the citrate buffer and wash the slides with phosphate-buffered saline (PBS) –T (0.3% Tween-20 in
PBS) two times for 5 minutes each with a magnetic stirrer.
● Cover each section with 0.3% H2O2 in MeOH for 15 minutes at room temperature to block endogenous peroxidase activity.
Wash two times in PBS-T for 5 minutes each with a magnetic stirrer.
● Remove the slides from PBS-T, wipe gently around each section, and cover tissues with blocking solution II (blue bottle,
Histofine SAB-PO (M) kit; Nichirei, Tokyo, Japan) for 30 minutes to block nonspecific staining. Do not wash.
● Tip off the blocking buffer, wipe gently around each section, and cover tissues with anti– human CD274/PD-L1 antibody
(27A2) diluted with PBS that contains 1% bovine serum albumin at 10 ␮g/mL. Incubate the sections for 1 hour at room
temperature.
● Wash the slides twice in PBS-T for 5 minutes each with a magnetic stirrer.
● Wipe gently around each section, and cover tissues with biotinylated anti-mouse antibody (yellow bottle, Histofine SAB-PO
kit; Nichirei). Incubate for 30 minutes at room temperature. Wash the slides twice in PBS-T (0.3% Tween-20 in PBS) for 5
minutes each time with a magnetic stirrer.
● Wipe gently around each section, and cover tissues with streptavidin-peroxidase (pink bottle, Histofine SAB-PO kit;
Nichirei). Incubate for 30 minutes at room temperature. Wash the slides two times in PBS-T (0.3% Tween-20 in PBS) for
5 minutes each with a magnetic stirrer.
● Visualize by reacting for 3 minutes with DAB solution.
● Wash the slides with flowing water for 5 minutes, and wash the slide with purified water.
● Counterstain with hematoxylin for 3 to 30 seconds, wash the slides with flowing water for 5 minutes, and wash the slide with
purified water.
● Dehydrate by immersing in 70% ethanol and 95% ethanol for 3 minutes and in ethanol three times for 3 minutes each
followed by immersion in xylene three times for 3 minutes each.
● Ready for mounting.
LSI Medience used an isotype control antibody (mouse immunoglobulin G2b) as a negative control and conducted an antibody
absorption test with recombinant human B7-H1/PD-L1 Fc chimera (R&D Systems; Minneapolis, MN). Normal placenta (Super-
BioChips, Seoul, South Korea, and US BioMax, Rockville, MD) was used as a positive control. The expression of PD-L1 was evaluated
according to the intensity of the staining and was scored as follows: 0, negative; 1, very weak expression; 2, moderate expression but weaker
than placenta; and 3, equivalent to or stronger expression than placenta. Occurrences with scores of 0 or 1 were defined as the
low-expression group (low), and occurrences with scores of 2 or 3 were the high-expression group (high; Appendix Fig A2). Validation,
assessment, and scoring of PD-L1 expression were performed in a third-party review (LSI Medience) by two independent pathologists
who were unaware of the outcomes.
Validation Study of PD-L1 Staining
To validate PD-L1 expression, four primary antibodies and three detection agents were tested to determine the optimal conditions for
immunohistochemistry labeling for the detection of PD-L1.
The primary antibodies were anti– human CD274/PD-L1 antibody (27A2; MBL, Nagoya, Japan), purified anti– human CD274
(B7-H1, PD-L1) antibody (BioLegend, San Diego, CA), PDL-1 antibody (ProSci, Poway, CA), and anti–B7-H1/PD-L1/CD274 antibody
(LifeSpan, Seattle, WA). Two kinds of ovarian cancer tissue microarrays were evaluated to confirm the reproducibility of staining for
PD-L1. Consequently, of the four primary antibodies, 27A2 was the best in terms of staining of PD-L1 compared with human normal
placenta. Furthermore, the specific staining of PD-L1 was acquired by incubation with 50 ␮L/mL of primary antibody (27A2) for 1 hour
at room temperature after heat-induced epitope retrieval at pH 9. The antigen absorption test was performed with recombinant PD-L1.
There was no positive reaction of PD-L1 at placental epithelial cells. Thus, these results confirmed the specificity of 27A2 antibodies.
Of the 108 ovarian cancer tissues, 83.3% (n ⫽ 45 , SBC; n ⫽ 45, UBM) demonstrated specific staining of PD-L1 (score, ⫹1 to ⫹3) in
two kinds of tissue microarrays, whereas 11.1% (n ⫽ 10, SBC; n ⫽ 2, UBM) were negative (score, 0). Finally, 4.6% (n ⫽ 4, SBC; n ⫽ 1,
UBM) were not evaluated.
Trial Steering Committee and Data Monitoring Committee Monitoring Committee
The Trial Steering Committee and Data Monitoring Committee were located at the Department of Data Science and Clinical
Innovative Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan. Every month, the management team monitored the
trial progress.
Ethical Considerations and Registration
The study protocol complied with the Declaration of Helsinki and the Ethics Guidelines for Clinical Research published by the
Ministry of Health, Labor, and Welfare of Japan. We obtained approval for this study from the institutional review board of Kyoto
University Hospital on June 23, 2011. The protocol and informed consent forms were approved by the ethics committee.

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 Hamanishi et al

Table A1. Summary of Exposure to Nivolumab in All Treated Patients

No. (%) of Patients by Nivolumab Dose

Factor Total 1 mg/kg 3 mg/kg

Study treatment status
On study 2 (10) 0 (0) 2 (20)
Off study 18 (90) 10 (100) 8 (80)
Follow-up period, months
Median 8.0 12.5 5.0
SD (range) 5.80 (2-22) 5.83 (3-22) 3.96 (2-15)
Duration of treatment, months
Median 4.0 3.5 4.0
SD (range) 3.7 (1-12) 3.1 (1-12) 4.2 (1-12)
Key reason for study discontinuationⴱ
PD 16 (89) 8 (80) 8 (100)
Adverse effect (treatment-related) 2 (11) † 2 (20) 0 (0)

Abbreviations: PD, progressive disease; SD, standard deviation.

ⴱ
Total number of patients was 18 (10 patients at 1 mg/kg; eight patients at 3 mg/kg). The other two patients continued to receive nivolumab (3 mg/kg) in key reason
for study discontinuation, each at the trial end date.
†Key reason for study discontinuation of the two patients was thyroiditis.

Table A2. Treatment-Related Serious Adverse Events

Dose No. of Patients Event

1 mg/kg 1 Grade 3 disorientation
Grade 3 gait disorder
Grade 3 fever
3 mg/kg 1 Grade 3 fever
Grade 3 deep vein thrombosis

Table A3. All Adverse Events Regardless of Causality That Occurred in at Least 20% of All Treated Patients

No. (%) of Patients With Events by Grade and Treatment Cohort

Total No. (%) of Patients With
Event by Grade (N ⫽ 20) 1 mg/kg (n ⫽ 10) 3 mg/kg (n ⫽ 10)

Event All Grades Grade 3 or 4 All Grades Grade 3 or 4 All Grades Grade 3 or 4
Any adverse event 20 (100) 13 (65) 10 (100) 6 (60) 10 (100) 7 (70)
AST increased 8 (40) 0 (0) 6 (60) 0 (0) 2 (20) 0 (0)
Hypothyroidism 8 (40) 0 (0) 4 (40) 0 (0) 4 (40) 0 (0)
Lymphocytopenia 8 (40) 3 (15) 5 (50) 3 (30) 3 (30) 0 (0)
Albumin decreased 8 (40) 3 (15) 2 (20) 1 (10) 6 (60) 2 (20)
Fever 6 (30) 1 (5) 2 (20) 1 (10) 4 (40) 0 (0)
Arrhythmia 6 (30) 0 (0) 4 (40) 0 (0) 2 (20) 0 (0)
ALT increased 5 (25) 1 (5) 4 (40) 1 (10) 1 (10) 0 (0)
Cough 5 (25) 0 (0) 3 (30) 0 (0) 2 (20) 0 (0)
Maculopapular rash 5 (25) 1 (5) 3 (30) 0 (0) 2 (20) 1 (10)
Arthralgia 5 (25) 0 (0) 5 (50) 0 (0) 0 (0) 0 (0)
Itching 4 (20) 0 (0) 4 (40) 0 (0) 0 (0) 0 (0)
Sore throat 4 (20) 0 (0) 0 (0) 0 (0) 4 (40) 0 (0)
Fatigue 4 (20) 0 (0) 2 (20) 0 (0) 2 (20) 0 (0)
Anemia 4 (20) 3 (15) 3 (30) 2 (20) 1 (10) 1 (10)
CRP increased 4 (20) 0 (0) 1 (10) 0 (0) 3 (30) 0 (0)

Abbreviation: CRP, C-reactive protein.

© 2015 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY

 Anti–PD-1 Antibody for Ovarian Cancer

Baseline

Nivolumab
1 mg/kg, n = 10
3 mg/kg, n = 10 PD or
Off study
progression

CR, PR, SD Follow-up

0 2w 4w 6w 8w 48 w

1 cycle Approx. 6 cycles

CT CT CT

Fig A1. The trial scheme. Eligible patients were allocated sequentially to the nivolumab low-dose (1 mg/kg; n ⫽ 10) or high-dose (3 mg/kg; n ⫽ 10) cohort. The phase
II efficacy trial defined a primary end point of response rate and a composite secondary end point of safety. Patients received nivolumab intravenously every 2 weeks,
and their lesions were evaluated by computed tomography (CT) every 2 months for up to 1 year or until progressive disease (PD; assessment of treatment effect on
the basis of RECIST version 1.1) or clinical disease progression (progression), defined as symptomatic deterioration. Adverse effects were assessed on basis of the
Common Terminology Criteria for Adverse Events version 4.0. CR, complete response; PR, partial response; SD, stable disease.

Assessed for eligibility

(N = 135)

Excluded (n = 114)
Did not meet inclusion criteria (n = 114)
Refused to participate (n = 0)

Registration
(n = 21)

Cohort transition

1 mg/kg cohort (n = 11) 3 mg/kg cohort (n = 10)

Received allocated intervention (n = 10) Received allocated intervention (n = 10)
Did not receive allocated intervention (n = 1) Did not receive allocated intervention (n = 0)
(disease progression after registration)

Lost to follow-up (n = 0) Lost to follow-up (n = 0)

Discontinued intervention Discontinued intervention
Progressive disease (n = 8) Progressive disease (n = 8)
Adverse effect (treatment related) (n = 2) Adverse effect (treatment related) (n = 0)

Analyzed (n = 10) Analyzed (n = 10)

Excluded from analysis (n = 0) Excluded from analysis (n = 0)

Fig A2. Flow diagram of patient progress through the steps of this trial.

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