Nivolumab in Platinum-Resistant Ovarian Cancer - JCO 2015

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
O R I G I N A L
R E P O R T
Safety and Antitumor Activity of AntiPD-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
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 Takafumi Ikeda, Manabu Minami, Atsushi
Kawaguchi, Toshinori Murayama,
Satoshi Morita, Masayuki Yokode, and
Akira Shimizu, Kyoto University Hospital, Kyoto, Japan.
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 antiPD-1 antibody that blocks
PD-1 signaling. We assessed the safety and antitumor activity of nivolumab in patients with
platinum-resistant ovarian cancer.
Published online ahead of print at

www.jco.org on September 8, 2015.
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.
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 Technology of Japan Grant No. 2013037 (to
J.H. and I.K.); and by Kyoto University
grants.
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.
Both J.H. and M.M. contributed nearly

equally to this work.
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 platinumresistant ovarian cancer indicate the merit of additional large-scale investigations (UMIN Clinical
Trials Registry UMIN000005714).
Terms in blue are defined in the glossary, 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
J Clin Oncol 33. 2015 by American Society of Clinical Oncology
INTRODUCTION
Ovarian cancer is the leading cause of death among

patients with gynecologic malignancies. Greater
than 50% of patients with ovarian cancer are diagnosed at an advanced stage.1,2 Despite cytoreductive surgery and platinum- and taxane-based
chemotherapies, greater than 70% of patients
with advanced ovarian cancer who achieve remission ultimately experience relapse. Because there
are few effective treatments for these patients,3 the
development of new treatment strategies is urgently required.3,4
Studies have demonstrated that ovarian cancers express cancer-specific antigens and elicit
spontaneous antitumor immune responses from
immunotherapy.4-7 As we and others have shown,
the number of tumor-infiltrating lymphocytes is a
significant prognostic factor,5-10 which suggests
that host immunity plays a pivotal role in the
clinical course of ovarian cancer. However, it has
recently been shown that ovarian cancer cells acquire the potential to escape from host immunity
in the tumor microenvironment, and new treatment strategies to overcome this phenomenon
are needed.3,11-13
2015 by American Society of Clinical Oncology
Information downloaded from jco.ascopubs.org and provided by at HOFFMANN-LA ROCHE AG on September 15, 2015
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Hamanishi et al
Programmed cell death-1 (PD-1), an immune checkpoint receptor 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 melanoma, renal cancer, and ovarian cancer, express PD-L1 and have the
ability to escape from the host immune system via PD-1/PD-L1 signaling.7, 17-19 We previously reported that PD-L1 expression was associated with poor prognosis in ovarian cancer5 and that PD-L1
promoted progression of ovarian cancer by inducing host immunosuppression 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 antiPD-1 receptor blocking monoclonal antibody.
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 nivolumab has been studied for ovarian cancer, two dose cohorts were implemented. 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-toevent end points were used to estimate median survival times with the KaplanMeier method. The two-sided 95% CIs were calculated with the Brookmeyer
and Crowley method for the median survival time. These analyses were implemented for the pooled cohort and for each cohort individually.
PATIENTS AND METHODS

Study Design and Patients
This single-center, open-label, phase II trial was conducted in two cohorts of patients with advanced or relapsed, platinum-resistant ovarian cancer.
Eligible patients were characterized by the following: documented platinumresistant (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 following: complications of autoimmune disease; previous systemic corticosteroid therapy or immune suppression; or a history of antiPD-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 (nivolumab 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 evaluated 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, respectively (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 evaluated 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 prelim2
Drug and Drug Supply

Nivolumab is a fully human immunoglobulin G4 antiPD-1 monoclonal 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, platinumresistant ovarian cancer were eligible for the protocol and were treated
with antiPD-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). Histologic tumor subtypes were as follows: serous (n 15; 75%), endometrioid (n 3; 15%), and clear cell (n 2; 10%; Table 1;
International Federation of Gynecology and Obstetrics (FIGO) staging 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 [ 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).
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AntiPD-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

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

AST increased
Hypothyroidism
Lymphocytopenia
Albumin decreased
Fever
ALT increased
Maculopapular rash
Arthralgia
Arrhythmia
Fatigue
Anemia
19 (95)
8 (40)
8 (40)
7 (35)
6 (30)
6 (30)
5 (25)
5 (25)
5 (25)
6 (30)
4 (20)
4 (20)
8 (40)
0 (0)
0 (0)
3 (15)
2 (10)
1 (5)
1 (5)
1 (5)
0 (0)
0 (0)
0 (0)
3 (15)
9 (90)
6 (60)
4 (40)
4 (40)
2 (20)
2 (20)
4 (40)
3 (30)
5 (50)
4 (40)
2 (20)
3 (30)
4 (40)
0 (0)
0 (0)
3 (30)
1 (10)
1 (10)
1 (10)
0 (0)
0 (0)
0 (0)
0 (0)
2 (20)
10 (100)
2 (20)
4 (40)
3 (30)
4 (40)
4 (40)
1 (10)
2 (20)
0 (0)
2 (20)
2 (20)
1 (10)
4 (40)
0 (0)
0 (0)
0 (0)
1 (10)
0 (0)
0 (0)
1 (10)
0 (0)
0 (0)
0 (0)
1 (10)
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Hamanishi et al
Table 3. Clinical Effect of AntiPD-1 Antibody Treatment

No. (%) of Patients [95% CI]
Response
Dose of
Nivolumab
No. of
Patients
CR
PR
SD
PD
NE
1 mg/kg
3 mg/kg
Total
10
10
20
0
2
2
1
0
1
4
2
6
4
6
10
1
0
1
RR
Survival, Months (95% CI)
DCR
1 (10) [0.3 to 44.5]

2 (20) [2.5 to 55.6]
3/20 (15%) [3.2 to 37.9]
PFS
5 (50) [18.7 to 81.3]

4 (40) [12.2 to 73.8]
9/20 (45%) [23.1 to 68.5]
OS
3.5 (0.7 to 3.9)

3.0 (1.5 to 3.9)
3.5 (1.7 to 3.9)
16.1 (2.6 to NR)

NR (2.9 to NR)
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.
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 independent 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
CA-125 (U/mL)
150
111
100
50
28
9
Baseline
4 Months
250
500
Time (days)
CA-125 (U/mL)
300
316
200
100
16
Baseline
4 Months
10
10
200
400
Time (days)
Fig 1. Activity of antiprogrammed 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 antiPD-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
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200
Change in Target Lesions

From Baseline (%)
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 lesions 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).
150
1 mg/kg (n = 10)
3 mg/kg (n = 10)
100
50
0
50
100
150
200
250
300
-50
-100
350
400
PR
CR*
SD*
CR
Time (days)
Change in Target Lesions
From Baseline (%)
B
150
1 mg/kg (n = 10)
3 mg/kg (n = 10)
PD
100
50
PD PD PD PD
PD PD SD SD PD* PD* SD SD SD NE PD*
0
-50
-100
PR
SD* CR
*
CR
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 antiPD-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 antiPD-1 antibody treatment [Fig 1A]; []
another patient with peritoneal dissemination evaluated [Fig 1B]) were still
receiving treatment with antiPD-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.
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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 significantly correlated with objective response (P .509; Fig 4E).
DISCUSSION
This study, to our knowledge, is the first investigator-initiated, phase II

clinical trial to test the safety and efficacy of nivolumab against
platinum-resistant ovarian cancer. The 3-mg/kg dose of nivolumab
may be more favorable than the 1-mg/kg dose for ovarian cancer,
because this dose showed better efficacy without a significant increase
in toxicity. A previous clinical trial of nivolumab in other solid tumors
showed the same finding.21 According to the National Comprehensive Cancer Network guidelines, the current standard treatment for
platinum- and taxane-resistant ovarian cancer is single-agent chemotherapy with pegylated liposomal doxorubicin, topotecan, or gemcitabine; however, these agents did not demonstrate a satisfactory clinical
antitumor effect.22-24 In an early phase II trial in 44 patients with
platinum-resistant ovarian cancer, monotherapy with bevacizumab, a
vascular endothelial growth factor inhibitor, no patients experienced a
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 nivolumab 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 recurrent 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.
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Hamanishi et al
50
10
20
3 mg/kg
100
Progression-Free
Survival (%)
1 mg/kg
100
Progression-Free
Survival (%)
Progression-Free
Survival (%)
50
30
10
Time (months)
20
30
Total
100
50
Time (months)
10
20
30
Time (months)
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
50
3 mg/kg
100
10
20
30
50
10
Time (months)
20
30
Time (months)
No. at risk 10 10 9 8 7 6 6 5 5 5 5 2 1 1 1 1 0
Total
100
Overall Survival (%)
1 mg/kg
100
No. at risk 10 9 2 2 2 2 0 0 0 0 0 0 0 0 0 0 0
No. at risk 10 9 8 7 5 5 2 1 0 0 0 0 0 0 0 0 0
50
10
20
30
Time (months)
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 antiPD-L1 antibody

(BMS-936559) in 17 patients with ovarian cancers,28 one patient experienced a PR (RR, 5.9%), and three had stable disease, which yielded
a disease control rate of 23.5%. These findings, as well as those of this
study, indicate that a certain patient population may demonstrate an
antitumor effect in response to PD-1 signal blocking. Thus, targeting
PD-1 signaling with antiPD-L1 or antiPD-1 treatment is a potential
treatment strategy in patients with platinum-resistant ovarian cancer.
No. of Patients
15
14 of 16
10
Responder
Nonresponder
P = .509
5
2 of 16
3 of 4
1 of 4
0
PD-L1 high
(n = 16)
In this study, treatment-related adverse events of grade 3 or 4

occurred in eight (40%) of 20 patients, although treatment could be
completed in most (19 [95%] of 20) patients. The frequencies of
arrhythmias and elevated AST were relatively higher in this study than
in larger previous studies of nivolumab for other solid tumors,21 but
all of these events were grade 1 or 2 and were controllable. Greater than
one half of the treatment-related adverse events in this study, such as
fever, maculopapular rash, hypothyroidism, and lymphocytopenia
Fig 4. The relationship between programmed death-1 ligand 1 (PD-L1) expression

on tumor cells and objective response to anti
programmed death-1 (PD-1) antibody treatment. Immunohistochemical analysis of
formalin-fixed paraffin-embedded ovarian cancer specimens before patients underwent
antiPD-1 antibody treatment with antiPD-1
monoclonal antibody. Representative (A) high
(3) and (B) low (1) staining patterns of
PD-L1 on ovarian cancers are shown. (C)
Positive-control staining of PD-L1 on placenta
(2) disappeared with the (D) absorption test.
Scale bars, 100 m. (E) The correlation between the expression of PD-L1 on tumor cells
and the objective response to antiPD-1 antibody in 20 patients with platinum-resistant
ovarian cancers. Expression of PD-L1 was not
significantly correlated with objective response (P .509).
PD-L1 low
(n = 4)
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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 immunerelated 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 antitumor 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 establishment of a biomarker to predict the antitumor response of nivolumab 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 expression 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 clusters, the immunoreactive phenotype, contains several immunerelated genes, but this phenotype composes only approximately 20%
of ovarian cancers. Therefore, this classification might have contributed to the low RR in our study, even in the presence of high expression 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 inflamREFERENCES
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investigation to evaluate the priority of nivolumab for platinumresistant 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
tumor-infiltrating lymphocytes recognize multiple

antigenic epitopes on autologous tumor cells. J
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8. Hamanishi J, Mandai M, Iwasaki M, et al:
Programmed cell death 1 ligand 1 and tumorinfiltrating CD8 T lymphocytes are prognostic factors of human ovarian cancer. Proc Natl Acad Sci
USA 104:3360-3365, 2007
9. Hamanishi J, Mandai M, Abiko K, et al: The
comprehensive assessment of local immune status
of ovarian cancer by the clustering of multiple immune factors. Clin Immunol 141:338-347, 2011
10. Hwang WT, Adams SF, Tahirovic E, et al:
Prognostic significance of tumor-infiltrating T cells in
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11. Dunn GP, Bruce AT, Ikeda H, et al: Cancer

immunoediting: From immunosurveillance to tumor
escape. Nat Immunol 3:991-998, 2002
12. Schreiber RD, Old LJ, Smyth MJ: Cancer
immunoediting: Integrating immunitys roles in cancer suppression and promotion. Science 331:15651570, 2011
13. Mellman I, Coukos G, Dranoff G: Cancer immunotherapy comes of age. Nature 480:480-489,
2011
14. Keir ME, Butte MJ, Freeman GJ, et al: PD-1
and its ligands in tolerance and immunity. Annu Rev
Immunol 26:677-704, 2008
15. Iwai Y, Ishida M, Tanaka Y, et al: Involvement
of PD-L1 on tumor cells in the escape from host
immune system and tumor immunotherapy by
PD-L1 blockade. Proc Natl Acad Sci USA 99:1229312297, 2002
16. Zou W, Chen L: Inhibitory B7-family molecules in the tumor microenvironment. Nat Rev Immunol 8:467-477, 2008
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17. Okazaki T, Chikuma S, Iwai Y, et al: A rheostat

for immune responses: The unique properties of
PD-1 and their advantages for clinical application.
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18. Thompson RH, Gillett MD, Cheville JC, et al:
Costimulatory B7H1 in renal cell carcinoma
patients: Indicator of tumor aggressiveness and
potential therapeutic target. Proc Natl Acad Sci USA
101:17174-17179, 2004
19. Hino R, Kabashima K, Kato Y, et al: Tumor cell
expression of programmed cell death-1 ligand 1 is a
prognostic factor for malignant melanoma. Cancer
116:1757-1766, 2010
20. Abiko K, Mandai M, Hamanishi J, et al: PD-L1
on tumor cells is induced in ascites and promotes
peritoneal dissemination of ovarian cancer through
CTL dysfunction. Clin Cancer Res 19:1363-1374,
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21. Topalian SL, Hodi FS, Brahmer JR, et al:
Safety, activity, and immune correlates of anti-PD-1
antibody in cancer. N Engl J Med 366:2443-2454,
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al: National comprehensive cancer networks: Ovarian cancer, version 2.2013. J Natl Compr Canc Netw
11:1199-1209, 2013
23. Mutch DG, Orlando M, Goss T, et al: Randomized phase III trial of gemcitabine compared with
pegylated liposomal doxorubicin in patients with
platinum-resistant ovarian cancer. J Clin Oncol 25:
2811-2818, 2007
24. Gordon AN, Fleagle JT, Guthrie D, et al:
Recurrent epithelial ovarian carcinoma: A randomized phase III study of pegylated liposomal doxorubicin versus topotecan. J Clin Oncol 19:3312-3322,
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25. Cannistra SA, Matulonis UA, Penson RT, et al:
Phase II study of bevacizumab in patients with
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expression profiles of serous, endometrioid, and
clear cell subtypes of ovarian and endometrial cancer. Clin Cancer Res 11:6422-6430, 2005
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Safety and activity of antiPD-L1 antibody in patients
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Nivolumab plus ipilimumab in advanced melanoma.
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5064-5074, 2014
GLOSSARY TERMS
CA-125 (cancer antigen 125): a protein produced by the

fallopian tubes, the endometrium, and the lining of the abdominal 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 determine whether these tumors are responding to therapy. The normal 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.
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 negatively modulates the duration and amplitude of immune responses. 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 monoclonal) specific to the antigen (primary reaction). The antigen-antibody
signal is then amplified using a second antibody conjugated to a complex of peroxidase-antiperoxidase, avidin-biotin-peroxidase, or avidinbiotin alkaline phosphatase. In the presence of substrate and
chromogen, the enzyme forms a colored deposit at the sites of antibodyantigen binding. Immunofluorescence is an alternate approach to visualize 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 Tumors): 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 extrapolate an overall response to treatment.
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AUTHORS DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Safety and Antitumor Activity of AntiPD-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 ASCOs conflict of interest policy, please refer to www.asco.org/rwc or jco.ascopubs.org/site/ifc.
Junzo Hamanishi
No relationship to disclose
Kaoru Abiko
Masaki Mandai
Tsukasa Baba
Takafumi Ikeda
Ken Yamaguchi
Manabu Minami
Akihiko Ueda
Atsushi Kawaguchi
Yuko Hosoe
Toshinori Murayama
Honoraria: Kowa Pharmaceuticals America
Masashi Kanai
Satoshi Morita
Honoraria: Ono Pharmaceutical, Bristol-Myers Squibb
Masayuki Yokode
Yukiko Mori
Speakers Bureau: Bayer
Research Funding: Taiho Pharmaceutical (Inst), Yakult Honsha (Inst)
Akira Shimizu
Shigemi Matsumoto
Tasuku Honjo
Research Funding: Ono Pharmaceutical
Shunsuke Chikuma
Ikuo Konishi
Noriomi Matsumura
Research Funding: Daiichi Sankyo (Inst)
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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 Greenwoods
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 Greenwoods 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 60C.
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 120C.
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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 (SuperBioChips, 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 antiB7-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
Study treatment status
On study
Off study
Follow-up period, months
Median
SD (range)
Duration of treatment, months
Median
SD (range)
Key reason for study discontinuation
PD
Adverse effect (treatment-related)
Total
1 mg/kg
3 mg/kg
2 (10)
18 (90)
0 (0)
10 (100)
2 (20)
8 (80)
8.0
5.80 (2-22)
12.5
5.83 (3-22)
5.0
3.96 (2-15)
4.0
3.7 (1-12)
3.5
3.1 (1-12)
4.0
4.2 (1-12)
16 (89)
2 (11)
8 (80)
2 (20)
8 (100)
0 (0)
Abbreviations: PD, progressive disease; SD, standard deviation.
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
1 mg/kg
3 mg/kg
Event
Grade
Grade
Grade
Grade
Grade
3
3
3
3
3
disorientation
gait disorder
fever
fever
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

AST increased
Hypothyroidism
Lymphocytopenia
Albumin decreased
Fever
Arrhythmia
ALT increased
Cough
Maculopapular rash
Arthralgia
Itching
Sore throat
Fatigue
Anemia
CRP increased
20 (100)
8 (40)
8 (40)
8 (40)
8 (40)
6 (30)
6 (30)
5 (25)
5 (25)
5 (25)
5 (25)
4 (20)
4 (20)
4 (20)
4 (20)
4 (20)
13 (65)
0 (0)
0 (0)
3 (15)
3 (15)
1 (5)
0 (0)
1 (5)
0 (0)
1 (5)
0 (0)
0 (0)
0 (0)
0 (0)
3 (15)
0 (0)
10 (100)
6 (60)
4 (40)
5 (50)
2 (20)
2 (20)
4 (40)
4 (40)
3 (30)
3 (30)
5 (50)
4 (40)
0 (0)
2 (20)
3 (30)
1 (10)
6 (60)
0 (0)
0 (0)
3 (30)
1 (10)
1 (10)
0 (0)
1 (10)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
2 (20)
0 (0)
10 (100)
2 (20)
4 (40)
3 (30)
6 (60)
4 (40)
2 (20)
1 (10)
2 (20)
2 (20)
0 (0)
0 (0)
4 (40)
2 (20)
1 (10)
3 (30)
7 (70)
0 (0)
0 (0)
0 (0)
2 (20)
0 (0)
0 (0)
0 (0)
0 (0)
1 (10)
0 (0)
0 (0)
0 (0)
0 (0)
1 (10)
0 (0)
Abbreviation: CRP, C-reactive protein.
Society
211.144.221.1
Baseline
Nivolumab
1 mg/kg, n = 10
3 mg/kg, n = 10
PD or
progression
Off study
CR, PR, SD
0
2w
CT
4w
6w
8w
Follow-up
48 w
1 cycle
Approx. 6 cycles
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
Did not meet inclusion criteria
Refused to participate
(n = 114)
(n = 114)
(n = 0)
Registration
(n = 21)
Cohort transition
1 mg/kg cohort
(n = 11)
Received allocated intervention
(n = 10)
Did not receive allocated intervention
(n = 1)
(disease progression after registration)
3 mg/kg cohort
Received allocated intervention
Did not receive allocated intervention
Lost to follow-up
Discontinued intervention
Progressive disease
Adverse effect (treatment related)
Lost to follow-up
Discontinued intervention
Progressive disease
Adverse effect (treatment related)
Analyzed
Excluded from analysis
(n = 0)
(n = 8)
(n = 2)
(n = 10)
(n = 0)
Analyzed
Excluded from analysis
(n = 10)
(n = 10)
(n = 0)
(n = 0)
(n = 8)
(n = 0)
(n = 10)
(n = 0)
Fig A2. Flow diagram of patient progress through the steps of this trial.
www.jco.org
Society
211.144.221.1

Nivolumab in Platinum-Resistant Ovarian Cancer - JCO 2015

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Published Ahead of Print on September 8, 2015 as 10.1200/JCO.2015.62.

JOURNAL OF CLINICAL ONCOLOGY

Safety and Antitumor Activity of AntiPD-1 Antibody,

Published online ahead of print at

Patients and Methods

Supported by research funds from

Both J.H. and M.M. contributed nearly

J Clin Oncol 33. 2015 by American Society of Clinical Oncology

Ovarian cancer is the leading cause of death among

Copyright 2015 by American Society of Clinical Oncology

PATIENTS AND METHODS

2015 by American Society of Clinical Oncology

Drug and Drug Supply

Baseline Patient Characteristics

AntiPD-1 Antibody for Ovarian Cancer

Table 1. Patient Characteristics

Median (range) age, years

Treatment-related adverse events in this study included several events

in this patient. Nivolumab treatment was discontinued in two

Any adverse event

2015 by American Society of Clinical Oncology

Table 3. Clinical Effect of AntiPD-1 Antibody Treatment

Survival, Months (95% CI)

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)

No. of patients of the total listed in the same row.

patient data, therefore, were classified as not evaluated. In another

2015 by American Society of Clinical Oncology

JOURNAL OF CLINICAL ONCOLOGY

AntiPD-1 Antibody for Ovarian Cancer

Change in Target Lesions

In the 3-mg/kg cohort, two patients had a complete response

PD PD SD SD PD* PD* SD SD SD NE PD*

The median progression-free survival times were 3.5 months

This study, to our knowledge, is the first investigator-initiated, phase II

Overall Survival (%)

Overall Survival (%)

Overall Survival (%)

In a previous phase I clinical trial of an antiPD-L1 antibody

In this study, treatment-related adverse events of grade 3 or 4

2015 by American Society of Clinical Oncology

Fig 4. The relationship between programmed death-1 ligand 1 (PD-L1) expression

JOURNAL OF CLINICAL ONCOLOGY

AntiPD-1 Antibody for Ovarian Cancer

without neutropenia, were reported in a previous clinical trial of

matory alterations at the tumor site.32 A recent trial of combination

AUTHORS DISCLOSURES OF POTENTIAL CONFLICTS

tumor-infiltrating lymphocytes recognize multiple

11. Dunn GP, Bruce AT, Ikeda H, et al: Cancer

2015 by American Society of Clinical Oncology

17. Okazaki T, Chikuma S, Iwai Y, et al: A rheostat

22. Morgan RJ Jr, Alvarez RD, Armstrong DK, et

27. Zorn KK, Bonome T, Gangi L, et al: Gene

CA-125 (cancer antigen 125): a protein produced by the

CD8 T cell: cytotoxic T cell that is the primary effector cells

FIGO staging: a tumor staging system established and revised

2015 by American Society of Clinical Oncology

Immunohistochemistry: the application of antigen-antibody

PD-1: programmed cell death protein 1 (CD279), a receptor expressed

PD-L1: programmed cell death 1 ligand 1 (CD274; also known as B7-H1),

JOURNAL OF CLINICAL ONCOLOGY