combination gemcitabine and wt1 peptide vaccination ...reported that gem plus a wt1-targeting cancer...

Research Article

Combination Gemcitabine and WT1 PeptideVaccination Improves Progression-Free Survivalin Advanced Pancreatic Ductal Adenocarcinoma:A Phase II Randomized StudySumiyuki Nishida1, Takeshi Ishikawa2, Shinichi Egawa3, Shigeo Koido4,Hiroaki Yanagimoto5, Jun Ishii6, Yoshihide Kanno7, Satoshi Kokura2, Hiroaki Yasuda2,Mari Saito Oba8, Maho Sato8, Soyoko Morimoto9, Fumihiro Fujiki10, Hidetoshi Eguchi11,Hiroaki Nagano11, Atsushi Kumanogoh1,12, Michiaki Unno13, Masanori Kon5,Hideaki Shimada6, Kei Ito7, Sadamu Homma14, Yoshihiro Oka12,15, Satoshi Morita16, andHaruo Sugiyama10

Abstract

We investigated the efficacy of a Wilms' tumor gene 1 (WT1)vaccine combined with gemcitabine (GEMWT1) and compared itwith gemcitabine (GEM) monotherapy for advanced pancreaticductal adenocarcinoma (PDAC) in a randomized phase II study.We randomly assigned HLA-A�02:01– or HLA-A�24:02–positivepatients with advanced PDAC to receive GEMWT1 or GEM. Weassessed WT1-specific immune responses via delayed-type hyper-sensitivity (DTH) to the WT1 peptide and a tetramer assay todetect WT1-specific cytotoxic T lymphocytes (WT1-CTL). Of 91patients enrolled, 85 were evaluable (GEMWT1: n ¼ 42; GEM:n ¼ 43). GEMWT1 prolonged progression-free survival [PFS;hazard ratio (HR), 0.66; P¼ 0.084] and improved overall survivalrate at 1 year (1-year OS%; GEMWT1: 35.7%; GEM: 20.9%).However, the difference in OS was not significant (HR: 0.82;

P ¼ 0.363). These effects were particularly evident in meta-static PDAC (PFS: HR 0.51, P ¼ 0.0017; 1-year OS%: GEMWT127.3%; GEM 11.8%). The combination was well tolerated,with no unexpected serious adverse events. In patients withmetastatic PDAC, PFS in the DTH-positive GEMWT1 groupwas significantly prolonged, with a better HR of 0.27 com-pared with the GEM group, whereas PFS in the DTH-negativeGEMWT1 group was similar to that in the GEM group(HR 0.86; P ¼ 0.001). DTH positivity was associated with anincrease in WT1-CTLs induced by the WT1 vaccine. GEM plusthe WT1 vaccine prolonged PFS and may improve 1-yearOS% in advanced PDAC. These clinical effects were associatedwith the induction of WT1-specific immune responses.Cancer Immunol Res; 6(3); 320–31. �2018 AACR.

IntroductionPancreatic cancer remains one of the most lethal malignancies.

Despite the development of novel diagnostic methods and ther-apeutic agents,most pancreatic cancer patients are diagnosedwith

unresectable advanced disease and succumb to disease within1 year (1). Since 1997, gemcitabine (GEM) monotherapy hasbeen the first-line therapy for unresectable, locally advanced andmetastatic pancreatic ductal adenocarcinoma (PDAC; ref. 2). The

1Department of Respiratory Medicine and Clinical Immunology, Osaka Uni-versity Graduate School of Medicine, Osaka, Japan. 2Department of Molec-ular Gastroenterology and Hepatology, Graduate School of Medical Science,Kyoto Prefectural University of Medicine, Kyoto, Japan. 3Division of Inter-national Cooperation for Disaster Medicine, International Research Instituteof Disaster Science, Tohoku University, Sendai, Japan. 4Division of Gastro-enterology and Hepatology, Department of Internal Medicine, The JikeiUniversity School of Medicine, Tokyo, Japan. 5Department of Surgery, KansaiMedical University, Hirakata, Japan. 6Division of General and Gastroentero-logical Surgery, Department of Surgery, Toho University Faculty of Medicine,Tokyo, Japan. 7Department of Gastroenterology, Sendai City Medical Center,Sendai, Japan. 8Department of Biostatistics, Yokohama City University,Yokohama, Japan. 9Department of Cancer Immunotherapy, Osaka UniversityGraduate School of Medicine, Osaka, Japan. 10Department of Cancer Immu-nology, Osaka University Graduate School of Medicine, Osaka, Japan.11Department of Gastroenterological Surgery, Osaka University GraduateSchool of Medicine, Osaka, Japan. 12Department of Immunopathology, WPIImmunology Frontier Research Center, Osaka University, Osaka, Japan.

13Department of Surgery, Tohoku University Graduate School of Medicine,Sendai, Japan. 14Division of Oncology, Research Center for Medical Science,The Jikei University School of Medicine, Tokyo, Japan. 15Department ofCancer Stem Cell Biology, Osaka University Graduate School of Medicine,Osaka, Japan. 16Department of Biomedical Statistics and Bioinformatics,Kyoto University Graduate School of Medicine, Kyoto, Japan.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Trial registration: University Hospital Medical Information Network (UMIN)UMIN000005248

Corresponding Author: Sumiyuki Nishida, Osaka University Graduate School ofMedicine, 2-2, Yamada-oka, Suita, 565-0871, Japan. Phone: 81-6-6879-3831; Fax:81-6-6879-3839; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-17-0386

�2018 American Association for Cancer Research.

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combination of GEM with a variety of conventional drugs ornovel molecular targeted agents has generally shown no substan-tial survival advantage compared with GEM alone (3). Twochemotherapeutic regimens of fluorouracil, leucovorin, irinote-can, and oxaliplatin (FOLFIRINOX) and GEM plus nanoparticlealbumin-bound paclitaxel (nab-PTX), both of which showedlonger survival benefit than GEM alone, changed the standardof care against advanced PDAC (4, 5). Survival of patients treatedwith these regimens, however, is marginal and has remainedextremely poor over the past two decades, with 5-year survivalunder 10%. Novel therapeutic strategies are urgently needed toimprove prognosis.

Cancer immunotherapy induces or enhances the preexistinghost antitumor immune response. Since the discovery of the firsttumor-associated antigen (TAA), MAGE-A1, many TAAs havebeen identified and used as targets for cancer immunotherapy.One of the most promising TAAs is the Wilms' tumor gene 1(WT1), which was ranked as the top antigen among 75 TAAs (6).The WT1 gene was first isolated as a tumor suppressor generesponsible for Wilms' tumor (7). Subsequent studies, however,indicated that WT1 can play oncogenic roles during tumorigen-esis, such as promotion of growth, inhibition of differentiation,resistance to cell death, and promotion of tumor angiogenesis(8–10, 11). Further, wild-type WT1 is overexpressed in varioushuman cancers including PDAC and is a poor prognostic marker(12–15). In addition to these oncogenic functions, WT1 is immu-nogenic. The WT1 protein elicits cellular and humoral immuneresponses in vitro and in vivo (16, 17). BecauseWT1 is expressed innot only cancer cells but also vascular endothelial cells in thetumor microenvironment (10), WT1-expressing tumor vessels,as well as WT1-expressing cancer cells, can be targeted byWT1-specific immunity.We and others have demonstrated prom-ising clinical effects of targeting WT1 for immunotherapy inadvanced malignancies (11, 17–21).

GEM, a cytotoxic agent, also possesses immune-modulatingfunctions, such as increase in antigen cross-presentation andselective inhibition of myeloid-derived suppressor cells(22–24), and enhances the expression of WT1 in pancreaticcancer cells, sensitizing them to WT1-specific cytotoxicT lymphocytes (WT1-CTL; ref. 25). We and others previouslyreported that GEM plus a WT1-targeting cancer vaccine is welltolerated and has promising and synergistic clinical effects inadvanced pancreatic cancer (26–28).

In this study, we designed an open-label, randomized phaseII study to investigate the clinical efficacy of GEM plus a WT1peptide vaccine (WT1 vaccine) compared with GEM mono-therapy as first-line therapy for patients with unresectableadvanced PDAC. This study also aimed to demonstrate boththe immunogenicity of the WT1 vaccine in pancreatic cancerpatients and the synergistic effects of GEM plus the WT1vaccine.

Materials and MethodsStudy design

This open-label, randomized phase II study was conductedat seven medical centers in Japan. Randomization was centrallyperformed at a 1:1 ratio using the minimization method withthe following balancing factors: extent of disease [UICC-stageIII (locally advanced), stage IV (metastatic), or recurrent disease

after surgery], primary tumor localization (head or body/tail),liver metastasis (yes or no), HLA-A locus typing (HLA-A�02:01or HLA-A�24:02), and institution. The primary endpoint wasoverall survival (OS). Secondary endpoints were progression-free survival (PFS), disease control rate, safety, quality of life(QOL), and WT1-specific immune response. The study wasapproved by the independent ethics committee or institutionalreview board of each center, was conducted in accordance withthe ethical principles of the Declaration of Helsinki, and wasregistered in the University Hospital Medical InformationNetwork Clinical Trials Registry as UMIN000005248.

PatientsHuman leukocyte antigen (HLA)-A�02:01– or A�24:02–

positive patients with histologically or cytologically confirmedlocally advanced or metastatic PDAC, or recurrent disease aftersurgery, were eligible. Patients were ages � 20 years with aKarnofsky performance status (KPS) of 80% to 100%; had ade-quate hematologic, hepatic, and renal function; and had a lifeexpectancy of at least 3 months. Patients with a KPS of 70% werealso included but only if KPS was decreased by poorly controlledcancerous pain at enrollment. All patients provided writteninformed consent.

TreatmentsAll patients received GEM at a dose of 1,000 mg/m2 intra-

venously over 30 minutes on days 1, 8, and 15 of a 28-day cycle.Patients allocated to GEM plus WT1 vaccine (GEMWT1) wereintradermally administered WT1 vaccine at six different sites(bilateral upper arms, lower abdomen, and femoral regions) ondays 1 and 15 of a 28-day cycle. Patients received the studytreatment until any of the following occurred: disease progres-sion, discontinuation due to toxicity, withdrawal of consent, orloss to follow-up. Patients in the GEMWT1 group were per-mitted to continue the study treatment beyond initial progres-sive disease if they were considered to demonstrate clinicalbenefit by the investigators (for example, continuing symptomor disease control despite radiological progression). Patients inthe GEM group were permitted to receive WT1 vaccine with orwithout GEM as the second-line or later treatment, but onlyafter disease progression.

The WT1 vaccine was a water-in-emulsion product composedof 3 mg of HLA class I–restricted WT1 peptide and MontanideISA51VG immune adjuvant (SEPPIC). TheWT1 peptide sequencewas as follows: HLA-A�02:01–restricted 9-mer WT1126 peptideRMFPNAPYL (np126) and HLA-A�24:02–restricted modified9-mer WT1235 peptide CYTWNQMNL (mp235). Patients typedas HLA-A�02:01/any (including A�24:02) and HLA-A�24:02/any(except for A�02:01) were administered np126 and mp235,respectively. These peptides were produced according to theGoodManufacturing Practice Guidelines at Peptide Institute (Osaka,Japan). We previously reported details regarding the preparationof WT1 vaccine (17). For the preparation of np126 peptidesolution, 3 mg of np126 was dissolved in 350 mL of 5% glucose.For the preparation of mp235 solution, 3 mg of mp235 peptidewas dissolved in a small volume of dimethyl sulfoxide (DMSO;Sigma) and then diluted with 350 mL of 5% glucose. This peptidesolution was finally emulsified with an equal weight of Monta-nide ISA51 adjuvant. The total volume of the WT1 vaccine was700 mL.

WT1 Vaccine Plus GEM in Pancreatic Cancer

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AssessmentsPatients were assessed by physical examinations, complete

blood count, and blood chemistry at each administration ofGEM. All adverse events (AEs) were assessed according to theCommon Terminology Criteria for Adverse Events (CTCAE) ver-sion 4.0. Computed tomography was performed every 6 weeksuntil disease progression. Tumor response was defined by inves-tigator assessments according to the response evaluation criteriain solid tumors (RECIST) version 1.1. QOLwas assessed using theFunctional Assessment of Cancer Therapy-General (FACT-G)measurement system at baseline and after the second, third, andfourth treatment courses.

Assessments of the WT1-specific immune responseThe WT1-specific immune response was assessed by (i)

delayed-type hypersensitivity (DTH) to theWT1peptide (assessedfor patients in the GEMWT1 group) and (ii) WT1-specific CD8þ

cytotoxic T lymphocytes (WT1-CTL). WT1-CTLs, which weredefined as WT1-tetramerþ CD3þ CD8þ T lymphocytes, wereassessed by an HLA-peptide tetramer staining assay (WT1-tetramer assay) with phycoerythrin (PE)-labeled WT1 tetramerfor patients in both groups. DTH was assessed every courseuntil discontinuation of study treatment. Ten micrograms ofWT1 peptide (np126 for HLA-A�02:01 or mp235 for HLA-A�24:02) diluted by saline or saline alone were intradermallyinjected at the forearm, and the maximum diameter of erythe-

ma and other skin reactions were measured after 48 hours. DTHpositivity was defined as a diameter of visible erythema of 1mm or longer.

For the WT1-tetramer assay, peripheral blood mononuclearcells (PBMC) were collected on day 1 of each course andcryopreserved until use. Frozen PBMCs were thawed and incu-bated for 1 hour at 37�C in X-VIVO 15 medium (Lonza)supplemented with 10% AB serum (Gemini Bio-Products).Half of the PBMCs were used for the WT1-tetramer assay, andthe other half were used for fluorescence minus one to deter-mine background levels of tetramer staining. Thawed PBMCswere incubated with Clear Back (MBL) in phosphate-bufferedsaline containing 2% FBS and 0.02% sodium azide (FACSbuffer) at room temperature for 5 minutes, then stained withWT1-tetramer for 1 hour at 4�C. These cells were then stainedwith monoclonal antibodies (mAB) against CD3, CD8, andCD4 for 25 minutes at 4�C in the dark, washed three times, andfinally resuspended in appropriate quantities of FACS bufferand incubated with 7-AAD (eBioscience) for 5 minutes beforeanalysis. The cells were analyzed on a FACSAria (BD Bio-sciences). The data were analyzed with FlowJo software (Tree-Star). For the WT1-tetramer assay, the following PE-labeledWT1-tetramer and mAbs were used: WT1126 peptide/HLA-A�02:01 tetramer or modified WT1235 peptide/HLA-A�24:02tetramer (MBL), anti-CD3-Pacific Blue, and anti-CD4-V500(BD Biosciences), anti-CD8-FITC (Beckman Coulter).

Figure 1.

CONSORT diagram of the randomized phase II study of GEM plus WT1 vaccination versus GEM monotherapy for the treatment of PDAC.

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Statistical analysisOS was defined as the time from the date of randomization

to the date of death by any cause. PFS was defined as thetime from randomization to documented disease progressionor death by any cause. Patients who started a second-linetreatment before disease progression were censored at the lastassessment. OS and PFS were analyzed with the use of theKaplan–Meier method and the log-rank test. The a level for thisstudy was set at 10% (two-sided). Adverse events were com-pared by Fisher exact test. FACT-G scores were summarized asmean and standard deviation and compared between groupsusing t tests. We used the intention-to-treat population, exceptfor patients who were ineligible based on our criteria. Thecomparison of GEM dose intensity and the number of treat-ment courses between the two treatment arms was performedusing the Mann–Whitney U test.

The studywas projected to include 150patients. Approximately142 patients enrolled during the 3-year accrual and 1.5-yearfollow-up period provided 80% power to detect a hazard ratio(HR) of 0.65 compared with a 1-year survival rate of 25% inthe GEM group. Interim analysis was prespecified 2 years after theinitiation of the study or when 100 patients were enrolled. Theobjective of interim analysis was to assess the futility of the studyusing Bayesian posterior probability.

ResultsPatient characteristics

We performed HLA typing in 162 patients to screen for eligi-bility and identified 123 patients (74.5%) who had HLA-A�02:01orHLA-A�24:02. A total of 91 patientswere enrolled betweenMay31, 2011, and June 23, 2015. Interim analysis for futility was

Table 1. Patient demographics and clinical characteristics at baseline

GEM plus WT1 vaccine n ¼ 42 (%) GEM n ¼ 43 (%) Total n ¼ 85 (%)

Age, yMedian (range) 66.0 (37–77) 65.0 (43–77) 66.0 (37–77)

GenderMale 26 (61.9) 25 (58.1) 51 (60.0)Female 16 (38.1) 18 (41.9) 34 (40.0)

KPS100% 9 (21.4) 8 (18.6) 17 (20.0)90% 20 (47.6) 24 (55.8) 44 (51.8)80% 11 (26.2) 8 (18.6) 19 (22.4)�70% 2 (4.8) 3 (7.0) 5 (5.9)

HLA-A locus�02:01/X 13 (31.0) 15 (34.9) 28 (32.9)�02:01/�24:02 3 9 12�24:02/Y 29 (69.0) 28 (65.1) 57 (67.1)

UICC stageIII (locally advanced) 7 (16.7) 9 (20.9) 16 (18.8)IV (metastatic) 33 (78.6) 34 (79.1) 67 (78.8)Recurrence 2 (4.8) 0 (0.0) 2 (2.4)

Pancreatic tumor locationHead 24 [1]� (57.1) 22 (51.2) 46 (54.1)Body/tail 18 [1]� (42.9) 21 (48.8) 39 (45.9)

MetastasisLiver 26 [1]� (61.9) 27 (62.8) 53 (62.4)Distal LN 13 [1]� (31.0) 18 (41.9) 31 (36.5)Lung 7 [1]� (16.7) 3 (7.0) 10 (11.8)Peritoneum 6 (14.3) 5 (11.6) 11 (12.9)

Ascites (mild) 10 [1]� (23.8) 12 (27.9) 22 (25.9)CA19-9 (U/mL)Range 2–3, 114, 349 5–13, 010, 000 2–13, 010, 000Median [10%, 90%] 1,261 [11.8, 51,333] 924 [9.6, 43,267] 1,261 [11.3, 45,010]0–100 11 (26.2) 18 (41.9) 29 (34.1)101–1,000 8 (19.0) 3 (7.0) 11 (12.9)1,001–10,000 16 (38.1) 10 (23.3) 26 (31.7)10,001–100,000 5 (11.9) 9 (20.9) 14 (16.5)>100,001 2 (4.8) 3 (7.0) 5 (5.9)

CRP (mg/dL)Range 0.03–11.5 0.20–3.6 0.20–11.5Median [10%, 90%] 0.27 [0.08, 2.84] 0.22 [0.04, 1.69] 0.23 [0.04, 2.10]<1.2 mg/dL 31 (73.8) 35 (81.4) 66 (77.6)>¼1.2 mg/dL 11 (26.2) 8 (18.6) 19 (22.4)

Serum albumin (g/dL)Range 2.3–4.6 3.2–4.6 2.3–4.6Median [10%, 90%] 3.8 [3.4–4.2] 4.0 [3.3–4.3] 3.9 [3.4–4.3]<3.5 g/dL 7 (16.7) 8 (18.6) 15 (17.6)>¼3.5 g/dL 35 (83.3) 35 (81.4) 70 (82.4)

NOTE: HLA, X include �24:02, and Y not include �02:01; � [ ], number of patients with recurrence.Abbreviations: CRP, C-reactive protein; GEM, gemcitabine; KPS, Karnofsky performance status; LN, lymph node; PC, pancreatic cancer.






Figure 2.

Clinical effects. A, Kaplan–Meier curves for OS of patients in all stages (GEMWT1: n ¼ 42; GEM: n ¼ 43) or B, those with metastatic disease (GEMWT1:n ¼ 33; GEM: n ¼ 34). C, PFS of patients in all stages (GEMWT1: n ¼ 42; GEM: n ¼ 43) or D, those with metastatic disease (GEMWT1: n ¼ 33; GEM: n ¼ 34).Black lines: GEMWT1 group; red dashed lines: GEM group. (Continued on the following page.)

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performed in September 2014, and the independent data mon-itoring committee (IDMC) recommended continuing the study.However, patient accrual was terminated according to the IDMC'srecommendation before accrual of the planned number ofpatients because enrollment was slow. Forty-five patients wererandomly allocated to receive GEMWT1, while the remaining 46received GEM monotherapy. Eighty-five patients (GEMWT1:n ¼ 42; GEM: n ¼ 43) were finally included in the main efficacyand safety analyses (Fig. 1). Baseline characteristics were wellbalanced between the two groups, except for recurrent disease,which was not observed in the GEM group (Table 1).

Study treatmentAlthough the relative dose intensity of GEM was not different

between the two groups, themedian number of treatment courses

in the GEMWT1 group and the GEM group were 6 (range, 1–19)and 3 (range, 1–18), respectively (P¼ 0.0040). The main reasonsfor treatment discontinuation were either disease progression[GEMWT1: 37 patients (88.1%); GEM: 34 (79.1%)] or AEs[GEMWT1: 4 (11.9%); GEM: 6 (13.9%)]. One patient in theGEMWT1 group (2.4%) underwent surgery for the completeresection, and three in the GEM group (7.0%) dropped out forreasons unrelated to AEs.

Overall survivalThe analysis of OS was based on 85 patients. The median OS

was 9.6 months in the GEMWT1 group and 8.9 months in theGEM group (HR, 0.82; 90% CI, 0.57–1.18, P ¼ 0.363; Fig. 2A).GEM plus the WT1 vaccine, however, improved the 1-year OS%comparedwithGEMmonotherapy (35.7%vs. 20.9%). Inpatients

Figure 2.

(Continued. ) E, Representative waterfall plots for patients in all stages, showing the maximum percentage change in target lesions compared with baselinemeasurements. Top, GEMWT1 group; bottom, GEM group. Dashed lines: 20% progression and 30% shrinkage relative to baseline. Double-arrowed lines:disease stabilization from baseline. F, Representative spider plots showing changes from baseline in the tumor burden, measured as the sum of the maximaldiameters of all target lesions. Top, GEMWT1 group; bottom: GEM. Y-axis: relative changes in target lesions; X-axis: time from baseline radiologicalassessment (weeks). PD: progressive disease; SD: stable disease; PR: partial response.






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with metastatic disease, the 1-year OS% in the GEMWT1 groupwas 27.3%, approximately 2.5 times higher than that in the GEMgroup (11.8%), although the median OS was similar in bothgroups (GEMWT1: 9.3months; GEM: 8.7months; HR, 0.93; 90%CI, 0.61–1.40, P ¼ 0.759; Fig. 2B).

Progression-free survivalGEM plus the WT1 vaccine improved PFS significantly. The

median PFS was 5.2 months in the GEMWT1 group and 3.3months in the GEM group (HR, 0.66; 90% CI, 0.44–0.98, P ¼0.084; Fig. 2C). The PFS rate at 6 months (6-mo PFS%) in theGEMWT1 group was 40.5%, three times higher than that in theGEM group (12.4%). PFS was significantly improved in patientswith metastatic disease in the GEMWT1 group. The median PFSand 6-mo PFS% were 3.7 months and 37.8% in the GEMWT1group, and 2.2 months and 3.9% in the GEM group, respectively(HR, 0.51; 90% CI, 0.32–0.82, P ¼ 0.017; Fig. 2D).

Response to therapyThe disease control rate was 52.4% [partial response (PR):

n ¼ 6; stable disease (SD): n ¼ 16) in the GEMWT1 group and37.2% (PR: n¼ 5; SD: n¼ 11) in the GEM group (P¼ 0.194). Inmost patients with SD, the tumor burden was lower in theGEMWT1 group than in the GEM group (the latter showingslightly increased tumor burden but not reaching progressivedisease). However, these differences were not significant (P ¼0.0725; Fig. 2E). The response or disease stabilization in theGEMWT1 group was more durable compared with that in theGEM group (Fig. 2F).

Adverse events and QOLIn theGEMWT1group, themost commonly reported (�50%of

patients) AEs of any grade were hematologic toxicities, fatigue,fever, gastrointestinal symptoms, elevation of hepatic enzymes,and hypoalbuminemia (Supplementary Table S1), and the fre-quently reported (�10% of patients) grade 3 to 4 clinicallysignificant AEs were leukocytopenia, neutropenia, hepatic-and-biliary tract infection, nausea, and increased AST (Table 2). Notreatment-related deaths were reported, and no significant differ-ences in the frequencies of any AEs between the two groups wereobserved (Table 2). Themost frequently reported AE related to theWT1 vaccine was local skin reactions at vaccine injection sites(erythema: 92.9%; induration: 78.6%; pruritus: 54.7%). Most ofthese were grade 1 or 2 and easily managed, except for a grade 3ulceration in one case.

We assessed QOL using the FACT-G scale (SupplementaryTable S2). At baseline, scores were similar between the two groups(mean total score: GEMWT1 73.1; GEM 72.4; P ¼ 0.830). Aftertreatment, the total score in the GEMWT1 group increased. At thefourth course, the mean total score in the GEMWT1 group was80.2, whichwas higher than that in theGEMgroup (70.4), but thedifference was not significant (P ¼ 0.063).

WT1-specific immune responses: DTHDTH to theWT1 peptide was evaluated in the GEMWT1 group.

No patients showed DTH before treatment. Of 42 patients, 38(HLA-A�02:01: n ¼ 12; HLA-A�24:02: n¼ 26) were evaluable forDTH at least once after treatment. The remaining four patientscould not be assessed because they discontinued the study treat-ment within the first course due to rapid disease progression.About 50% of patients (HLA-A�02:01: 5/12; HLA-A�24:02:13/26) showed DTH after treatment. All of the 18 DTH-positivepatients showed DTH within four courses of treatment, and 12(80.0%; HLA-A�02:01: 3/5; HLA-A�24:02: 9/13) exhibited DTHafter at least one course of treatment.

Table 2. Grade 3 or higher grade clinically significant adverse events

GEM plus WT1vaccine (N ¼ 42)n (%)

GEM(N ¼ 43)n (%) P

WBC decreased 7 (16.7) 5 (11.6) 0.549Neutropenia 15 (35.7) 17 (39.5) 0.824Lymphopenia 3 (7.1) 5 (11.6) 0.713Anemia 4 (9.5) 8 (18.6) 0.351Thrombocytopenia 2 (4.8) 1 (2.3) 0.616Hepatic and biliary tract infection 8 (19.1) 7 (16.3) 0.783Febrile neutropenia 2 (4.8) 0 (0.0) 0.241Fatigue 3 (7.1) 2 (4.7) 0.676Anorexia 3 (7.1) 3 (7.0) 1.0Nausea 5 (11.9) 7 (16.3) 0.757Vomiting 3 (7.1) 3 (7.0) 1.0Diarrhea 1 (2.4) 0 (0.0) 0.494Constipation 1 (2.4) 1 (2.3) 1.0Ileus 1 (2.4) 1 (2.3) 1.0AST increased 6 (14.3) 5 (11.6) 0.757ALT increased 3 (7.1) 2 (4.7) 0.676ALP increased 4 (9.5) 2 (4.7) 0.433Gastric–duodenal hemorrhage 3 (0.0) 0 (0.0) 0.055Sepsis 1 (2.4) 2 (4.7) 1.0Hyponatremia 1 (2.4) 2 (4.7) 1.0Hyperkalemia 1 (2.4) 1 (2.3) 1.0Hypokalemia 2 (4.8) 0 (0.0) 0.241Hyperglycemia 1 (2.4) 3 (7.0) 0.616Thromboembolic event 1 (2.4) 0 (0.0) 0.494Interstitial pneumonitis 0 (0.0) 1 (2.3) 1.0

NOTE: Adverse events were graded according to the National Cancer InstituteCommon Toxicity Criteria of Adverse Events (NCI CTCAE) version 4.0.Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST,aspartate aminotransferase; GEM, gemcitabine; WBC, white blood cell.

Figure 3.WT1-specific immune responses and clinical effects. A, Kaplan–Meier curves for PFS (DTH-positive GEMWT1: n ¼ 15; DTH-negative GEMWT1: n ¼ 15, GEM: n ¼ 30)and B, OS (DTH-positive GEMWT1: n ¼ 15; DTH-negative GEMWT1: n ¼ 15, GEM: n ¼ 30) in patients with metastatic disease who received at least twocourses of the study treatments. GEMWT1 patients who received GEM plus WT1 vaccination were classified into two groups according to the presence or absenceof DTH. Black lines: DTH-positive GEMWT1; black dashed lines: DTH-negative GEMWT1; red dashed lines: GEM. C, Tetramer assay for WT1-CTLs. Top, data foreach subgroup at baseline; bottom, data after treatment. WT1-CTLs: WT1-tetramerþ CD3þ CD8þ T cells (quadrilateral area). D, Dynamic change in WT1-CTLs. Left,HLA-A�02:01; right, HLA-A�24:02. Red boxplots: DTH-positive GEMWT1; green boxplots: DTH-negative GEMWT1; blue boxplots: GEM. Line graphs below boxplots:Each line graph represents the dynamic change in WT1-CTLs for individual cases. Left, DTH-positive GEMWT1; center, DTH-negative GEMWT1; right, GEM. Y-and X-axes represent percentage of WT1-CTLs out of CD3þ CD8þ T cells and the treatment course, respectively. Experiments were performed one time for eachsample. E, Kaplan–Meier curves for PFS in patients with metastatic disease who were evaluated for WT1-CTLs before and after treatment (GEMWT1 >5 times:n¼ 14; GEMWT1 < 5 times: n¼ 14; GEM: n¼ 25). Patients in theGEMWT1 group are classified into two groups according to the extent ofWT1-CTL increase. Black lines:5 times or higher increase; black dashed lines: Less than 5 times increase/no increase; red dashed lines: GEM.






Association between clinical effect and WT1-specific immuneresponses

Significant prolongation of PFS by GEM plus the WT1vaccine suggested that WT1-specific immune effector cellsinduced by the WT1 vaccine affected the clinical outcome. Weevaluated the association between the clinical effect, in terms ofPFS, and the elicitation of WT1-specific immune responses. Thisanalysis was performed in patients with metastatic disease whoreceived at least two courses of a study treatment (GEMWT1:n ¼ 30; GEM: n ¼ 30) because posttreatment DTH couldnot be assessed in patients in the GEMWT1 group who hadnot completed the first course. Half of the patients in theGEMWT1 group (HLA-A�02:01: 5/10; HLA-A�24:02: 10/20)showed DTH.

The PFS curve in the GEMWT1 group could clearly be dividedinto two separate curves according to the presence or absence ofDTH (Fig. 3A). Themedian PFS and 6-mo PFS%were 6.5monthsand 60.0% in DTH-positive patients, and 2.9 months and 17.8%in DTH-negative patients, respectively. PFS in DTH-positivepatients was significantly prolonged compared with that inthe GEM group (median PFS: 2.8 months; 6-mo PFS%: 4.2%;HR, 0.27; 90% CI, 0.14–0.51), whereas PFS in DTH-negativepatients was similar to that in the GEM group (HR, 0.86; 90%CI, 0.49–1.51) (P ¼ 0.001).

We next analyzed the association between PFS and both DTHand well-known prognostic factors in PDAC (29). In the uni-variate analysis, DTH-positivity in the GEMWT1 group wasassociated with longer PFS than in the GEM group (HR,0.25; 95% CI, 0.10–0.62, P ¼ 0.003). These prognostic factorswere, therefore, not confounders of DTH, suggesting thatDTH-positivity was an independent predictive marker for betterPFS following treatment with GEM plus the WT1 vaccine.

We also evaluated the association between OS and DTH(Fig. 3B). The median OS and 1-year OS% were 11.2 monthsand 40.0% in DTH-positive patients (HR, 0.69; 90% CI, 0.41–1.18), 7.8months and20.0% inDTH-negative patients (HR, 1.29;90% CI, 0.76–2.19), and 8.9 months and 13.3% in the GEMgroup, respectively (P ¼ 0.229).

WT1-specific immune responses: WT1 tetramer assayWe used a WT1 tetramer assay to analyze dynamic changes in

WT1-CTLs in patients with metastatic disease. Before treat-ment, the median percentages of WT1-CTLs in the GEMWT1and GEM groups were 0.011% and 0.014%, respectively, inHLA-A�02:01 patients (P ¼ 0.880), and 0.035% and 0.044%,respectively in HLA-A�24:02 patients (P ¼ 0.403). Most of theDTH-positive patients in the GEMWT1 group (n ¼ 15) showedan increased percentage of WT1-CTLs after treatment (Fig. 3Cand D), and 11 patients (73.3%; HLA-A�02:01: 3/5; HLA-A�24:02: 8/10) showed an increase of five-fold or higher,peaking at the second to fourth course of treatment (Supple-mentary Table S3). In contrast, in the 14 DTH-negative patients(HLA-A�02:01: 5; HLA-A�24:02: 9) and the 29 patients in theGEM group (HLA-A�02:01: 10; HLA-A�24:02: 19), almost nochange in WT1-CTL percentages were seen (Fig. 3C and D),except for three DTH-negative patients who exhibited increasesof 5-fold or higher (Supplementary Table S3). DTH positivitywas statistically associated with the increase in WT1-CTLsinduced by WT1 vaccination (Fisher exact test, P ¼ 0.0092).We further evaluated the association between PFS andincreased WT1-CTLs. The PFS curve in the GEMWT1 group

was divided into two separate curves according to whetherWT1-CTLs exhibited a 5 times or higher increase and less than 5times increase (including no increase; Fig. 3E). In the patientswith increased WT1-CTLs, PFS was significantly prolongedcompared with others (P < 0.0001).

DiscussionThe goal of this study was to evaluate the efficacy of GEM

plus a WT1 vaccine compared with GEMmonotherapy, with OSas the primary endpoint. Unfortunately, the trial failed to meetthe primary endpoint of a statistically significant improvementin OS. The 1-year OS% in the GEMWT1 group, however, wasabout 1.7 times higher than that in the GEM group. PFS in theGEMWT1 group was also longer (HR, 0.66) and 6-mo PFS%was 3.3 times higher than those in the GEM group. The tumorburdens in the GEMWT1 group were reduced, with a longerduration of disease stabilization. This study, however, shouldbe considered underpowered as the sample size was statisticallyinsufficient to evaluate some endpoints. The major reason forthe delayed subject recruitment was the success of two novelregimens, FOLFIRINOX and GEM plus nab-PTX (4, 5).Although GEM monotherapy was the standard chemotherapywhen this study started, approval of these new treatments inJapan resulted in a rapid change in first-line chemotherapy foradvanced PDAC.

Cancer immunotherapy is a promising new strategy forpancreatic cancer. Unfortunately, immunotherapies in mid-to large-sized or late-phase clinical trials failed to show favor-able clinical outcomes, which had been expected in someearly-phase studies (30, 31). Immune checkpoint inhibitorshave also failed to meet expectations thus far (32). Consid-ering the difficulty in developing cancer immunotherapy forpancreatic cancer, our results in this midsized randomizedstudy provide a way to increase antitumor responses inpatients.

Compared with GEM monotherapy, GEM plus the WT1vaccine was well tolerated without any additional serioustreatment-related AEs, even in elderly individuals with poorperformance status (PS), and did not impair QOL in patientswith advanced PDAC. These results are consistent with those ofprevious studies in which various cancer vaccines were com-bined with GEM-based chemotherapies (33–35). According tothe 2016 guidelines for pancreatic cancer (36), intensive regi-mens using FOLFIRINOX and GEM plus nab-PTX are recom-mended for patients with good PS, while GEMmonotherapy isstill recommended for those with poor PS. However, manypatients might not tolerate these toxic multidrug chemothera-pies because of multiple prior comorbidities and diseaseprogression. Hence, developing alternative regimens, such asGEM plus the WT1 vaccine, are needed.

In the subgroup analysis of patients with metastatic disease,GEM plus WT1 vaccination significantly prolonged PFS, withthe improvement of 1-year OS%, compared with GEM,although it failed to show a significant survival benefit. There-fore, we have to consider its failure mechanism. First, it ispossible that the GEMWT1 group contained two subpopula-tions. In fact, patients in the GEMWT1 group could be dividedinto two groups based on DTH positivity. Compared with theGEM group, the DTH-positive subgroup demonstrated thesignificantly prolonged PFS with a much better HR of 0.27.

Nishida et al.





The OS was improved with a HR of 0.69, although this was notstatistically significant, probably due to the low power sec-ondary to the small sample size. On the other hand, the PFS inthe DTH-negative subgroup was identical to that in the GEMgroup, and the OS was relatively poor with an HR of 1.29.These results suggested that WT1-specific immune responseswere required for prolonging PFS and, thus, improving OS,and that WT1-specific immunogenicity was not achieved insome patients with advanced disease. Another possibility is thecrossover effect of WT1 vaccination. Approximately 45% ofpatients in the GEM group received GEM plus the WT1 vaccineas second-line therapy, while another 45% received otherchemotherapy regimens, including S-1 and GEMþS-1 (Sup-plementary Table S4-1). In 5 patients who survived for 1 yearor longer, 4 received GEM plus WT1 vaccination (Supplemen-tary Table S4-2). These results suggest that the addition of theWT1 vaccine to GEM as second-line therapy could also elicitWT1-specific immune responses and contribute to improvedsurvival. As a result, the OS benefit derived from prolongedPFS in the GEMWT1 group could be blurred by the crossovereffect of GEM plus the WT1 vaccine as second-line therapy inthe GEM group.

Considering the mechanism of action of cancer vaccines, ourresults are reasonable because the clinical effects of cancervaccines, which do not directly affect cancer cells, can beexpected only after vaccines induce and augment TAA-specificCTLs sufficient to eliminate cancer cells. To assess the T-cellresponses elicited by the WT1 vaccine (37), we used thefollowing two assays: (i) DTH to the WT1 peptide, whichreflects the in vivo immunologic response and (ii) the tetramerassay, which reflects the frequency of WT1-specific T cells.Approximately 50% of patients in this study were DTH positive,which is consistent with other reports, as well as our ownprevious phase I study (19–21). In the clinical setting of GEMplus WT1 vaccination, DTH was a useful predictive marker forPFS, independent of other prognostic factors associated withPDAC. DTH positivity was significantly correlated with anincrease in WT1-CTLs. These results suggest that DTH is asimple and useful method that qualitatively reflects the induc-tion of functional WT1-CTLs. We did not, however, identify anyimmunologic biomarkers suitable for the pretreatment deter-mination of either the clinical benefit of the WT1 vaccine orWT1-specific immune responses.

This study had three main limitations. First, patients wereselected according to HLA-A loci. HLA molecules affect T cell–mediated immune responses, but the association betweenHLA-type and PDAC prognosis remains unclear. We selectedthe patients with specific HLA-A loci because np126 and mp235of the WT1 peptide are restricted to HLA-A�02:01 and HLA-A�24:02, respectively. Hence, the clinical results of patientswith other HLA-types are unknown. Second, observer andsubject biases should be considered when interpreting ourresults because this was an open-label study with investigatorassessment. Third, as already discussed, was the lack of statis-tical power.

Our findings show that WT1-specific immunity can improvethe prognosis of PDAC patients. If WT1-specific immunity canbe induced more effectively, PFS and OS can be improved inmore patients. One method is to coadminister HLA-class IIepitopes derived from WT1 (27, 38). Another option is thedevelopment of safe and highly effective immune adjuvants to

enhance TAA-specific immunity. For example, beneficial effectson the survival of pancreatic cancer patients resulted from boththe addition of IMM-101 to GEM (39), as well as the combi-nation of GVAX pancreas and CRS-207 (40). Novel therapeuticstrategies to overcome the desmoplastic and immunosuppres-sive microenvironment of PDAC, which prevents the infiltra-tion of CTLs, are also important (41). Ultimately, the devel-opment and clinical application of multimodal cancer therapythat fully mobilizes both conventional cancer treatments andnovel immunotherapies will improve the prognosis of morePDAC patients in the future.

In conclusion, the combination therapy of GEM plus WT1vaccination significantly prolonged PFS and improved 1-yearOS%, although not significantly, compared with GEM mono-therapy in patients with advanced PDAC, especially those withmetastatic disease. These clinical effects were associated withWT1-specific immune responses, providing a proof of conceptfor addition of WT1 vaccination to GEM. This combination wasgenerally well tolerated without unexpected toxicities and didnot impair QOL. These results encourage us to conduct furtherclinical trials of the WT1 vaccine containing class I and class IIpeptides in combination with a standard chemotherapy, suchas GEM plus nab-PTX.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

DisclaimerMasanori Kon was not available to confirm coauthorship, but the corre-

sponding author, Sumiyuki Nishida, affirms that Masanori Kon contributed tothe article and thus confirms his coauthorship status.

Authors' ContributionsConception and design: S. Nishida, S. Egawa, S. Koido, S. Homma, Y. Oka,S. Morita, H. SugiyamaDevelopment of methodology: S. Nishida, F. Fujiki, S. MoritaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Nishida, T. Ishikawa, S. Egawa, S. Koido,H. Yanagimoto, J. Ishii, Y. Kanno, S. Kokura, H. Yasuda, M. Sato, S. Morimoto,F. Fujiki, H. Eguchi, H. Nagano, H. Shimada, K. ItoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Nishida, T. Ishikawa, S. Egawa, M.S. Oba,S. Morimoto, F. Fujiki, Y. Oka, S. MoritaWriting, review, and/or revision of the manuscript: S. Nishida, S. Egawa,S. Koido, H. Yanagimoto, M.S. Oba, S. Morimoto, Y. Oka, S. MoritaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Nishida, T. Ishikawa, S. Koido, M. Sato,S. Morimoto, A. Kumanogoh, M. UnnoStudy supervision: M. Unno, S. Homma

AcknowledgmentsThis study was supported, in part, by the Japanese Ministries of Education,

Culture, Sports, Science and Technology (grant numbers 21790665 and15K09050) and the Japanese Foundation for Multidisciplinary Treatment ofCancer.

We thank all the patients who participated in this study, their supportingfamilies, and all referring physicians and the supporting medical staffs at allclinical sites. We also thank S. Tada (Osaka University) for her data manage-ment; S. Kobayashi, Y. Oji, A. Tsuboi, N. Hosen, and H. Nakajima (OsakaUniversity); T. Okayama (Kyoto Prefectural University); Y. Otsuka (TohoUniversity); M. Ueno and S. Ohkawa (Kanagawa Cancer Center); S. Ohno,and K. Sano (Teikyo University) for their kind professional support; S. Umeda(Osaka University); S. Tsuchiya (Kyoto Prefectural University); and S. Kuramae,






E. Shibuya, and K. Kawamura (Tohoku University) for their kind technicalsupport.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 20, 2017; revised October 17, 2017; accepted January 9, 2018;published OnlineFirst January 22, 2018.

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2018;6:320-331. Published OnlineFirst January 22, 2018.Cancer Immunol Res Sumiyuki Nishida, Takeshi Ishikawa, Shinichi Egawa, et al. Adenocarcinoma: A Phase II Randomized StudyProgression-Free Survival in Advanced Pancreatic Ductal Combination Gemcitabine and WT1 Peptide Vaccination Improves

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