Prognostic significance of plasma interleukin-6/-8 in pancreatic cancer patients receiving chemoimmunotherapy

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1 Submit Mnuscript: Help Desk: DOI: /wjg.v21.i World J Gstroenterol 21 October 21; 21(39): ISSN (print) ISSN (online) 21 Bishideng Publishing Group Inc. All rights reserved. Clinicl Trils Study ORIGINAL ARTICLE Prognostic significnce of plsm interleukin-6/-8 in pncretic cncer ptients receiving chemoimmunotherpy Shintro Tsuking, Mikio Kjihr, Kzuki Tkkur, Zensho Ito, Tomoy Kni, Keisuke Sito, Shinichiro Tkmi, Hiroko Kobyshi, Yoshihiro Mtsumoto, Shunichi Odhr, Kn Uchiym, Hiroshi Arkw, Msto Okmoto, Hruo Sugiym, Kzuki Sumiym, Toshifumi Ohkus, Shigeo Koido Shintro Tsuking, Mikio Kjihr, Kzuki Tkkur, Zensho Ito, Tomoy Kni, Keisuke Sito, Shinichiro Tkmi, Hiroko Kobyshi, Yoshihiro Mtsumoto, Shunichi Odhr, Kn Uchiym, Toshifumi Ohkus, Shigeo Koido, Division of Gstroenterology nd Heptology, Deprtment of Internl Medicine, The Jikei University School of Medicine, Kshiw City, Chib , Jpn Shintro Tsuking, Hiroshi Arkw, Kzuki Sumiym, Deprtment of Endoscopy, The Jikei University School of Medicine, Minto City, Tokyo , Jpn Msto Okmoto, Deprtment of Advnced Immunotherpeutics, Kitsto University School of Phrmcy, Tokyo , Jpn Toshifumi Ohkus, Shigeo Koido, Institute of Clinicl Medicine nd Reserch, The Jikei University School of Medicine, Kshiw City, Chib , Jpn Hruo Sugiym, Deprtment of Functionl Dignostic Science, Grdute School of Medicine, Osk University, Suit City, Osk 6-871, Jpn Author contributions: Koido S, Ohkus T, Sugiym H, Okmoto M, Sumiym K designed the reserch; Tsuking S, Kjihr M, Tkkur K, Ito Z, Kni T, Sito K, Tkmi S, Kobyshi H, Mtsumoto Y, Odhr S, Uchiym K, Arkw H performed the reserch, nlyzed the dt; nd Tsuking S, Koido S wrote the pper. Supported by Grnts-in-Aid for Scientific Reserch (C) from the Ministry of Eduction, Culture, Sports, Science nd Technology of Jpn. Institutionl review bord sttement: The study ws reviewed nd pproved by the ethics committee of the Jikei Institutionl Review Bord, Jikei University School of Medicine (Tokyo, Jpn), nd the clinicl study committee of Jikei University Kshiw Hospitl [No (329) nd No (682)]. Informed consent sttement: All study prticipnts, or their legl gurdin, provided informed written consent prior to study enrollment. The procedures were performed in ccordnce with the Helsinki Declrtion. Conflict-of-interest sttement: Okmoto M holds ownership interest in Tell, Inc. Sugiym H is the inventor of ptents PCT/JP2/2794 nd PCT/JP4/16336 which re held by the Interntionl Institute of Cncer Immunotherpy. No potentil conflicts of interest were disclosed by the other uthors. Dt shring sttement: No dditionl dt re vilble. Open-Access: This rticle is n open-ccess rticle which ws selected by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ccordnce with the Cretive Commons Attribution Non Commercil (CC BY-NC 4.) license, which permits others to distribute, remix, dpt, build upon this work non-commercilly, nd license their derivtive works on different terms, provided the originl work is properly cited nd the use is non-commercil. See: licenses/by-nc/4./ Correspondence to: Shigeo Koido, MD, Division of Gstroenterology nd Heptology, Deprtment of Internl Medicine, The Jikei University School of Medicine, Kshiw-shit, Kshiw, Chib , Jpn. shigeo_koido@jikei.c.jp Telephone: Fx: Received: July 2, 21 Peer-review strted: July 6, 21 First decision: July 19, 21 Revised: August 2, 21 Accepted: August 3, 21 Article in press: August 3, October 21, 21 Volume 21 Issue 39

2 Published online: October 21, 21 chemoimmunotherpy. Abstrct AIM: To investigte the ssocition of plsm levels of interleukin (IL)-6 nd -8 with Wilms tumor 1 (WT1)- specific immune responses nd clinicl outcomes in ptients with pncretic ductl denocrcinom (PDA) treted with dendritic cells (DCs) pulsed with three types of mjor histocomptibility complex clss Ⅰ nd Ⅱ-restricted WT1 peptides combined with chemotherpy. METHODS: During the entire tretment period, plsm levels of IL-6 nd -8 were nlyzed by ELISA. The induction of WT1-specific immune responses ws ssessed using the WT1 peptide-specific delyed-type hypersensitivity (DTH) test. RESULTS: Three of 7 ptients displyed strong WT1- DTH rections throughout long-term vccintion with significntly decresed levels of IL-6/-8 fter vccintions compred with the levels prior to tretment. Moreover, overll survivl (OS) ws significntly longer in PDA ptients with low plsm IL-6 levels (< 2 pg/ml) fter vccintions thn in ptients with high plsm IL-6 levels ( 2 pg/ml) (P =.2). After disese progression, WT1-DTH rections decresed severely nd were ultimtely negtive t the terminl stge of cncer. The decresed levels of IL-6/-8 observed throughout long-term vccintion were ssocited with WT1-specific DTH rections nd long-term OS. CONCLUSION: Prolonged low levels of plsm IL-6/-8 in PDA ptients my be prognostic mrker for the clinicl outcomes of chemoimmunotherpy. Key words: Chemoimmunotherpy; Dendritic cell; Delyed-type hypersensitivity; Interleukin-6; Interleukin-8; Pncretic cncer; Wilms tumor 1 The Author(s) 21. Published by Bishideng Publishing Group Inc. All rights reserved. Core tip: We recently reported phse 1 clinicl study in pncretic cncer ptients using dendritic cells (DCs) pulsed with multiple mjor histocomptibility complex clss Ⅰ nd Ⅱ-restricted Wilms tumor 1 (WT1) epitopes (DC/WT1-Ⅰ/Ⅱ) in combintion with chemotherpy. Little is known bout the prognostic mrkers for the clinicl outcomes of chemoimmunotherpy. We exmined the ssocition of plsm levels of interleukin (IL)-6/-8 with WT1-specific immune responses nd clinicl outcomes in pncretic cncer ptients treted with chemotherpy combined with DC/WT1-Ⅰ/Ⅱ. The study demonstrtes tht prolonged low levels of plsm IL-6/-8 in pncretic ductl denocrcinom ptients my be prognostic mrker for the clinicl outcomes of Tsuking S, Kjihr M, Tkkur K, Ito Z, Kni T, Sito K, Tkmi S, Kobyshi H, Mtsumoto Y, Odhr S, Uchiym K, Arkw H, Okmoto M, Sugiym H, Sumiym K, Ohkus T, Koido S. Prognostic significnce of plsm interleukin-6/-8 in pncretic cncer ptients receiving chemoimmunotherpy. World J Gstroenterol 21; 21(39): Avilble from: URL: DOI: INTRODUCTION Ptients with pncretic ductl denocrcinom (PDA) hve prticulrly poor prognosis, with -yer survivl rte of < 1% [1]. Thus, PDA remins one of the dedliest humn tumors, chrcterized by high mortlity, rpid progression, nd resistnce to chemotherpy nd rdition therpy. Compred with single chemotherpy with stndrd gents such s gemcitbine, multi chemotherpy regimens such s FOLFIRINOX (consisting of -fluorourcil/folinic cid/ oxlipltin/irinotecn) nd gemcitbine/nb-pclitxel hve been ssocited with significnt improvement in medin overll survivl (OS) from 6 to 11 mo [2,3]. New therpeutic pproches for PDA re urgently needed. PDA cells express tumor-ssocited ntigens (TAAs), including Wilms Tumor gene 1 (WT1) [4]. Therefore, immunotherpy trgeting PDA-ssocited ntigens my be n lterntive pproch in ptients with PDA. Dendritic cells (DCs) re potent ntigen-presenting cells (APCs) extensively used for the development of nticncer immunotherpies [,6]. DCs cpture nd process TAAs into peptides nd present these frgments through mjor histocomptibility complex (MHC) clss Ⅰ nd Ⅱ pthwys, thus simultneously stimulting both CD4 + nd CD8 + T cells [,6]. TAAs re recognized by CD8 + cytotoxic T-lymphocytes (CTLs) in the context of MHC clss Ⅰ molecules, wheres CD4 + T cells recognize ntigenic peptides in ssocition with MHC clss Ⅱ molecules. CD8 + CTLs recognize MHC clss Ⅰ-peptide complexes on cncer cells nd destroy these cells through effector molecules such s grnzyme B nd perforin [7]. DCs hve been pulsed with vrious MHC clss Ⅰ-restricted ntigenic peptides for the tretment of ptients with PDA in clinicl studies; however, the ntitumor effects of these vccines trgeting only CD8 + CTLs re not s vigorous in clinicl trils [7]. Incresing evidence hs suggested tht CD4 + T cells prime nd mintin ntigen-specific CD8 + CTLs [8] nd ply direct role in the destruction of tumor cells [9]. Therefore, the stimultion of both CD4 + nd CD8 + T cells is n efficient strtegy for treting ptients with dvnced cncer. We recently conducted phse 1 clinicl study in ptients with PDA to exmine the clinicl nd immunologicl responses to DCs pulsed with October 21, 21 Volume 21 Issue 39

3 Apheresis GEM multiple MHC clss Ⅰ nd Ⅱ-restricted WT1 epitopes (DC/WT1-Ⅰ/Ⅱ) in combintion with chemotherpy [1,11]. The vccintion of PDA ptients with DC/WT1-Ⅰ /Ⅱ simultneously induced WT1-specific CD4 + nd CD8 + T cell responses in vivo nd in vitro [1,11]. WT1- specific delyed-type hypersensitivity (DTH) induced by combintion therpy ws ssocited with mintennce of WT1-specific memory CTLs, resulting in long-term clinicl responses [1]. Moreover, we previously reported tht the post-tretment neutrophil to lymphocyte (N/L) rtio is tretment-relted prognostic fctor for improved survivl of PDA ptients fter DC/WT1-Ⅰ/Ⅱ tretment [11]. In the present study, we nlyzed the ssocition of plsm interleukin (IL)-6 nd IL-8 levels nd WT1 peptide-specific DTH in PDA ptients during long-term chemoimmunotherpy using DC/WT1-Ⅰ/Ⅱ. The vlue of plsm IL-6 nd IL-8 levels s prognostic mrkers ws lso ssessed. MATERIALS AND METHODS 1 st 2 nd 3 rd cycle GEM DC/WT1-Ⅰ/Ⅱ Figure 1 Tretment schedule for ptients with pncretic ductl denocrcinom. The ptients were treted with gemcitbine lone, followed by dendritic cells pulsed with Wilms tumor 1 (WT1)-specific peptide mixture restricted by multiple mjor histocomptibility complex (MHC) clss Ⅰ nd Ⅱ molecules (DC/WT1-Ⅰ/Ⅱ) in combintion with gemcitbine or S-1, n orl -fluorourcil (-FU). GEM: Gemcitbine. Study design The study ws reviewed nd pproved by the ethics committee of the Jikei Institutionl Review Bord, Jikei University School of Medicine (Tokyo, Jpn), nd the clinicl study committee of Jikei University Kshiw Hospitl [No (329) nd (682)]. All 7 PDA ptients provided written informed consent nd underwent chemoimmunotherpy with DC/WT1-Ⅰ/Ⅱ vccintion nd chemotherpy [1]. All procedures were performed in ccordnce with the Helsinki Declrtion. DC/WT1-Ⅰ/Ⅱ vccine Autologous peripherl blood mononucler cells (PBMCs) were isolted from peripherl blood by Ficoll-Plque Premium (GE Helthcre Bio-Sciences, Pisctwy, NJ, United Sttes) density grdient solution. Adherent PBMCs were cultured for d in AIM-V medium (Gibco Life Technologies, New York, United Sttes) contining grnulocyte mcrophge colony-stimulting fctor ( ng/ml, Primmune Corp. Kobe, Jpn) nd IL-4 ( ng/ml, R&D Systems, Minnepolis, MN, United Sttes) to generte immture DCs. The immture DCs were ctivted with penicillin-killed nd lyophilized preprtions of low-virulence strin (Su) of Streptococcus pyogenes (OK-432; 1 mg/ ml, Chugi Phrmceuticl Co, Ltd, Tokyo, Jpn) nd prostglndin E2 (PGE2; ng/ml, Diichi Fine Chemicl Co, Ltd, Toym, Jpn) for n dditionl 24 h. The mture DCs were pulsed with mixture of three WT1 peptide types restricted to HLA-A*2:1, A*2:6 ( : RMFPNAPYL), A*24:2 (23-243: CYTWNQMNL) nd MHC-clss Ⅱ ( : KRYF- KLSHLQMHSRKH; NeoMPS Inc., Sn Diego, CA, United Sttes) s previously described [1]. Chemoimmunotherpy Gemcitbine ws intrvenously dministered t dose of 1 mg/m 2 on dys 1, 8, nd 1 of 28-d cycle. After the first cycle of gemcitbine dministrtion, the ptients were treted with combintion of gemcitbine nd DC/WT1-Ⅰ/Ⅱ. The DC/WT1-Ⅰ /Ⅱ vccine (usully cells/dose) ws intrdermlly dministered biweekly, regrdless of the chemotherpy regimen (Figure 1). In Jpn, the orl -fluorourcil (FU) S-1 is used to tret ptients with gemcitbine-refrctory PDA [12]. Therefore, some ptients with gemcitbine-refrctory PDA received S-1 during chemoimmunotherpy. Ptients without erly progressive disese received chemoimmunotherpy until the occurrence of disese progression, uncceptble dverse events, or withdrwl of ptient consent. Clinicl responses Computed tomogrphy ws performed every 4 to 8 wk during tretment until disese progression. The clinicl response ws determined ccording to Response Evlution Criteri in Solid Tumors. Stble disese (SD) ws defined s disese tht ws stble for more thn 8 wk fter the strt of tretment. Overll survivl (OS) nd progression-free survivl (PFS) were clculted from the dte of tretment to the dte of deth or finl follow-up nd the dte of disese progression, respectively. WT1 peptide-specific immune responses To determine the induction of WT1-specific immune responses during chemoimmunotherpy, the WT1 peptide-specific DTH test ws performed before tretment nd fter every vccintion. Briefly, 3 μg of the three types of WT1 peptides in sline or sline lone ws intrdermlly injected seprtely into the forerm. The mximum dimeter of erythem nd indurtion were mesured 48 h fter WT1 peptide injection. WT1-specific DTH positivity ws defined s erythem nd indurtion greter thn 2 mm in the mximum dimeter. We selected the vlue of -mm erythem nd indurtion to discriminte between wek (2- mm) nd strong (> mm) WT1-specific DTH rections. The DTH test ws performed prior to tretment nd during long-term tretment October 21, 21 Volume 21 Issue 39

4 Tble 1 Ptient chrcteristics No. Sex Age (yr) Loction Size (mm) Metstses UICC stge Vccine OS (d) PFS (times) (d) HLA type HLA-A DRB1 DPB1 Best overll tumor response 1 M 7 Body 22 Peritonitis Ⅳ :1 24:2 4: 1:2 :1 9:1 Stble disese 2 M 68 Body 1 Liver, lymph Ⅳ :2 33:3 8:3 13:2 2:2 4:1 Stble disese nodes 3 F 49 Hed 18 Liver, peritonitis, lymph nodes 4 M 3 Body 2 Liver, lymph nodes F 72 Body 22 Peritonitis, lymph nodes Ⅳ :1 24:2 4: 9:1 2:2 :1 Progressive disese Ⅳ :1-9:1 1:1 2:1 :1 Stble disese Ⅳ :6 24:2 8:2 12:1 2:1 :1 Stble disese 6 F 69 Body-til 4 Lymph nodes Ⅳ :2 33:3 13:2 1:1 4:1 13:1 Stble disese 7 M 39 Hed-body 3 Peritonitis Ⅳ :1 24:2 1:1 1:2 2:2 9:1 Stble disese Stble disese conformtion is determined t lest for more thn 2 mo. Ope: Opertion; Cx: Chemotherpy; OS: Overll survivl; PFS: Progression-free survivl; HLA: Humn leukocyte ntigen. Enzyme-linked immunosorbent ssy Throughout the vccintion period, plsm ws collected nd immeditely frozen t -14 until further use. To investigte the quntittive reltionship between IL-6 or IL-8 plsm levels nd WT1-specific DTH rections during vccintions, the stored plsm ws tested for IL-6 or IL-8 every vccintions using Enzyme-linked immunosorbent ssy (ELISA) kits (R&D Systems) ccording to the mnufcturer s instructions. Bckground cytokine levels were subtrcted from ech smple. Sttisticl nlysis Sttisticl nlyses of prognostic fctors for OS nd PFS were performed ccording to the Kpln-Meier method nd evluted using the log-rnk test. IL-6 or IL-8 levels were evluted in t-test nlysis. A P vlue of <. ws considered sttisticlly significnt. RESULTS Clinicl chrcteristics All 7 ptients with PDA received gemcitbine followed by combintion of gemcitbine nd biweekly vccintions with DC/WT1-Ⅰ/Ⅱ (Figure 1). The clinicl chrcteristics of ll PDA ptients re presented in Tble 1. All ptients hd disese stge Ⅳ nd HLA types of A (A*2:1, A*2:6, or A*24:2), DR (DRB1*4:, DRB1*8:3, DRB1*1:1, or DRB1*1:2) or DP (DPB1*:1, or DPB1*9:1). Tretment with the DC/WT1-Ⅰ/Ⅱ vccine ws non-toxic nd sfe [1]. None of the 7 PDA ptients chieved complete or prtil response, nd 6 (8.7%) exhibited SD. As OS of 1 yer in dvnced PDA ptients generlly indictes tht tretment hs been beneficil [13], the treted PDA ptients were clssified into 2 groups: OS 1 nd < 1 yer. Three of 7 ptients (No. 1, 2, nd 6) exhibited OS of 1 yer, nd the remining 4 ptients (No. 3, 4,, 7) exhibited OS of < 1 yer (Tble 1). These 3 ptients (No. 1, 2, nd 6) hd long-term SD, resulting in longterm survivl (OS 1 yer). From the beginning of tretment, one ptient (No. 6) received biweekly 1 mg/m 2 gemcitbine combined with DC/WT1-Ⅰ/Ⅱ vccintion becuse of neutropeni. Despite receiving insufficient doses of gemcitbine, the locl pncretic lesions in the ptient were stble for more thn 1 yer (Figure 2A-C, left pnel); however, we identified liver metstses t 4 d fter the first tretment (Figure 2C, right pnel). Therefore, the ptient continued tretment with S-1, n orl fluoropyrimidine, or gemcitbine/nb-pclitxel combined with the DC/WT1- Ⅰ/Ⅱ vccine. At 4 d fter the first tretment, the ptient mintined stble primry pncretic cncer (Figure 2D, left pnel) with slightly enlrged liver metstses (Figure 2D, right pnel), nd survived for more thn 1 d with 1% Krnofsky Performnce Sttus (KPS). WT1-specific DTH rections WT1-specific DTH rections were not detected prior to tretment in ll ptients. Chemoimmunotherpy induced strong WT1-specific DTH rections in ll 3 ptients (No. 1, 2, nd 6) with long-term OS ( 1 yer) fter receiving only one dose of the DC/WT1- Ⅰ/Ⅱ vccine (Tble 2). Moreover, the strong WT1- specific DTH rections in the 3 super-responders were efficiently mintined during the entire tretment period with t lest 3 DC/WT1-Ⅰ/Ⅱ vccintions (Tble 2). However, the DTH rections in 2 (No. 1 nd 2) of the super-responders were severely decresed nd becme negtive t the terminl stge of cncer (3 nd 4 vccintions, respectively). Interestingly, one super-responder (No. 6) remined live more thn 1 d nd received more thn 71 vccintions, resulting in the induction of strong WT1-specific DTH rections throughout the vccintion period (Tble 2). Moreover, clinicl response in terms of SD ws chieved in ll 3 ptients (No. 1, 2, nd 6) with strong WT1-specific DTH responses. These ptients lso mintined 1% KPS during tretment. By contrst, October 21, 21 Volume 21 Issue 39

5 A Prior-to tretment B 398 d C 4 d 4 d D 963 d 963 d Figure 2 Response ssessment in super-responder, cse 6. A: Computed tomogrphy (CT) imging of cse 6 reveling pncretic body cncer before tretment nd no liver metstses; B: CT imging fter chemoimmunotherpy reveled tht locl pncretic lesions were stble; C: Locl pncretic lesions were stble (left pnel), multiple liver metstses ppered t 4 d fter the first tretment (right pnel); D: Locl pncretic lesions were stble (left pnel), the sizes of multiple liver metstses were mrkedly incresed t 963 d fter the first tretment (right pnel). strong WT1-specific DTH rections were not observed in ll 4 nonsuper-responders with OS < 1 yer (No. 3, 4,, nd 7). In the 4 nonsuper-responders, one ptient (No. 7) exhibited stble disese nd wekly positive DTH rections ginst WT1 peptides fter 1 to 1 vccintions; however, the DTH rections becme negtive fter 2 vccintions, nd the ptient died t 32 d fter the first tretment. Importntly, ll 4 PDA ptients with WT1-specific DTH rections (No. 1, 2, 6, nd 7) displyed significntly improved OS compred with the negtive control ptients (No. 3, 4, nd ) (P =.18) (Figure 3A). In prticulr, ll 3 PDA ptients with strong DTH rections (No. 1, 2, nd 6) survived more thn 1 yer, with significntly longer OS compred to the negtive control ptients (No. 1, 2, nd 6) (P =.39) (Figure 3B). In ddition, the WT1- specific DTH rections observed in this clinicl tril were HLA restricted (Tble 2). Plsm IL-6 level chnges in PDA ptients fter DC/WT1- Ⅰ/Ⅱ vccintion To ssess the prognostic significnce of plsm IL-6 levels in PDA ptients receiving chemoimmunotherpy using DC/WT1-Ⅰ/Ⅱ vccines, we nlyzed plsm IL-6 levels by quntittive ELISA during tretment (Figure 4A). Prior to tretment, there ws no difference in plsm IL-6 levels between super-responders (OS 1 yer) nd nonsuper-responders (OS < 1 yer) (Figure 4B). In one nonsuper-responder (No. ), plsm IL-6 levels were extremely high prior to tretment nd October 21, 21 Volume 21 Issue 39

6 Tble 2 Wilms tumor 1-specific delyed-type hypersensitivity rections Ptient Gemcitbine HLA-A*2:1 HLA-A*24:2 HLA-DRB1/DPB1 No. Vccine times Vccine times Vccine times Pre Post ND ND ND ND ND ND ND - - ND - - ND ND - - ND - - ND ND ND ND ND ND ND Delyed-type hypersensitivity (DTH) erythem < 1 mm, +; DTH erythem 2- mm, ++; DTH erythem > mm, not done (ND). continuously incresed during tretment ( pg/ml), nd we excluded these dt from the ssessment of the reltionship between plsm IL-6 levels nd OS. After 1 course of gemcitbine lone, plsm IL-6 levels decresed in some ptients but not significntly compred with prior to tretment (Figure 4A). All 3 superresponders (No. 1, 2, nd 6) hd long-term SD (44, 28, nd 4 d, respectively), nd plsm IL-6 levels in these ptients were significntly decresed compred with the levels prior to tretment fter DC/WT1-Ⅰ/Ⅱ vccintions. This significnt decrese continued for 1 vccintions (Figure 4C). Plsm IL-6 levels in 2 superresponders (No. 1 nd 2) were incresed fter 3 nd 2 vccintions, respectively. However, 1 super-responder (No. 6) mintined low levels of IL-6 for t lest 4 vccintions, resulting in long-term OS. Becuse of erly disese progression in nonsuper-responders (No. 3, 4,, nd 7), these ptients received t lest 6 (7, 6, 14, nd 2 times, respectively) DC/WT1-Ⅰ/Ⅱ vccintions. Therefore, we lso compred plsm IL-6 levels in super-responders nd nonsuper-responders fter DC/WT1- Ⅰ/Ⅱ vccintions. Compred with nonsuper-responders, plsm IL-6 levels in super-responders were significntly lower fter vccintions (Figure 4D), indicting tht plsm IL-6 levels decresed in super-responders fter the initil vccintions. In ddition, IL-6 levels were remrkbly incresed t the terminl stge of cncer. Moreover, we ssessed the ssocition of IL-6 levels nd OS nd PFS fter DC/WT1-Ⅰ/Ⅱ vccintions. The PDA ptients with low levels of plsm IL-6 (< 2 pg/ml) fter vccintions displyed significntly improved OS (P =.2) but not PFS (P =.11) compred with those ptients with plsm IL-6 2 pg/ml (Figure A nd B). Chnges in IL-8 plsm levels in PDA ptients fter DC/WT1-Ⅰ/Ⅱ vccintion Plsm IL-8 levels in PDA ptients fter chemoimmunotherpy using DC/WT1-Ⅰ/Ⅱ vccines were lso nlyzed by quntittive ELISA (Figure 6A). Prior to tretment, there ws no difference in plsm IL-8 levels between super-responders (No. 1, 2, nd 6) nd nonsuper-responders (No. 3, 4,, 7) (Figure 6B). Importntly, plsm IL-8 levels in ll 3 super-responders with long-term SD were significntly decresed fter 1 vccintions compred with the levels observed prior to tretment, nd this decrese continued fter 2 vccintions (Figure 6C). However, plsm IL-8 levels in 2 super-responders (No. 1 nd 2) were incresed fter 3 nd 4 vccintions, respectively. The ptients died 82 nd 717 d, respectively (Tble 1). However, one super-responder (No. 6) mintined low levels of IL-8 until t lest 4 vccintions nd survived more thn 1 d. All 3 ptients (No. 2, 4, nd ) with negtive-dth rections ginst WT1 peptides received less thn 1 DC/WT1-Ⅰ /Ⅱ vccintions (7, 6, nd 14 vccintions, respectively) due to disese progression. Therefore, we lso compred plsm IL-8 levels in super-responders t vrious vccintion periods nd nonsuper-responders fter DC/WT1-Ⅰ/Ⅱ vccintions. As shown in Figure 6D, plsm IL-8 levels were significntly higher in nonsuperresponders fter DC/WT1-Ⅰ/Ⅱ vccintions thn in super-responders fter 1, 2 nd 2 vccintions. In ddition, plsm IL-8 levels were remrkbly incresed t the terminl stge of cncer. DISCUSSION In the present study, we nlyzed plsm IL-6 nd IL-8 levels nd ssessed WT1-specific DTH rections in PDA ptients receiving DC/WT1-Ⅰ/Ⅱ vccines. The dt October 21, 21 Volume 21 Issue 39

7 A 1 B OS (%) 6 4 P =.18 OS (%) 6 4 P = Dys fter first tretment Dys fter first tretment WT1-specific DTH rections Positive (n = 4) Negtive (n = 3) WT1-specific DTH rections Strongly positive (n = 3) Negtive (n = 3) Figure 3 Assocition of Wilms tumor 1-specific delyed-type hypersensitivity rections with overll survivl. Kpln-Meier estimtes of overll survivl (OS) for ptients with PDA treted with dendritic cells (DCs) pulsed with Wilms tumor 1 (WT1) MHC clss Ⅰ nd -Ⅱ peptides (DC/WT1-Ⅰ/Ⅱ) combined with chemotherpy. A: OS in WT1-specific delyed type hypersensitivity (DTH)-positive (n = 4) or -negtive (n = 3) responders; B: OS in strongly WT1-specific DTHpositive (n = 3) or -negtive (n = 3) responders. A OS 1 yer IL-6 (pg/ml) OS < 1 yer (vccine times) DC/WT1-Ⅰ/Ⅱ vccines Chemotherpy Tretment B C D Pre-tretment 1 1 OS 1 yer 1 OS < 1 yer OS 1 yer IL-6 (pg/ml) IL-6 (pg/ml) IL-6 (pg/ml) < OS (yer) (vccine times) (vccine times) Figure 4 Plsm interleukin-6 levels in pncretic ductl denocrcinom ptients receiving chemoimmunotherpy. A: Plsm interleukin (IL)-6 levels in 6 ptients during tretment; B: Comprison of plsm IL-6 levels prior to tretment between super-responders [overll survivl (OS) 1 yer] nd nonsuperresponders (OS < 1 yer); C: Comprison of plsm IL-6 levels in super-responders prior to tretment nd post-vccintion; D: Comprison of plsm IL-6 levels fter vccintions between nonsuper-responders nd super-responders fter, 1, 1, 2, nd 2 vccintions. The vlues re expressed s men ± SD. P < October 21, 21 Volume 21 Issue 39

8 A DC/WT1-Ⅰ/Ⅱ ( vccines) B DC/WT1-Ⅰ/Ⅱ ( vccines) 1 8 IL-6 (pg/ml) < IL-6 (pg/ml) < 2 2 OS (%) 6 4 P =.2 PFS (%) 6 4 P = Dys fter first tretment Dys fter first tretment Figure Assocition of plsm interleukin-6 levels with overll survivl nd progression-free survivl. The plsm interleukin (IL)-6 levels of 7 pncretic ductl denocrcinom ptients receiving chemoimmunotherpy were nlyzed fter vccintions. Kpln-Meier estimtes of OS (A) nd PFS (B) in these ptients. A OS 1 yer 2 6 IL-8 (pg/ml) OS < 1 yer (vccine times) DC/WT1-Ⅰ/Ⅱ vccines Chemotherpy Tretment B 1 Pre-tretment C 1 OS 1 yer D 1 OS < 1 yer OS 1 yer IL-8 (pg/ml) IL-8 (pg/ml) IL-8 (pg/ml) < OS (yer) (vccine times) (vccine times) Figure 6 Plsm interleukin-8 levels in pncretic ductl denocrcinom ptients receiving chemoimmunotherpy. A: Plsm interleukin (IL)-8 levels in 7 ptients during tretment; B: Comprison of plsm IL-8 levels prior to tretment between super-responders [overll survivl (OS) 1 yer] nd nonsuperresponders (OS < 1 yer); C: Comprison of plsm IL-8 levels in super-responders prior to tretment nd post-vccintion; D: Comprison of plsm IL-8 levels fter vccintions in nonsuper-responders nd super-responders fter, 1, 1, 2, nd 2 vccintions. The vlues re expressed s mens ± SD. P < October 21, 21 Volume 21 Issue 39

9 presented herein suggest tht long-term decreses in plsm IL-6 nd IL-8 levels compred to the erly tretment period re ssocited with the induction of WT1-specific DTH rections. Significntly prolonged OS ws observed in PAD ptients with strong WT1-specific DTH rections. Thus, long-term low levels of plsm IL-6 nd IL-8 during chemoimmunotherpy my be prognostic mrkers of clinicl outcomes. In recent clinicl tril, to simultneously ctivte WT1-specific CD4 + nd CD8 + T cells, mture DCs were pulsed with mixture of three types of WT1 peptides, including MHC clss Ⅰ nd Ⅱ-restricted epitopes, nd injected into one site biweekly s cncer vccine [1]. DC/WT1-Ⅰ/Ⅱ vccintions not only induced WT1- specific CD4 + nd CD8 T cells but lso mintined long-term WT1-specific memory CD8 + T cells [1]. The phse 1 study indicted tht WT1-specific DTH rections induced fter tretment with DC/WT1-Ⅰ /Ⅱ combined with chemotherpy were ssocited with long-term disese stbility in dvnced PDA. Moreover, the WT1-specific CTL responses continued throughout long-term vccintion nd were ssocited with long-term OS [1]. With chemotherpy lone, SD is often trnsient nd not considered indictive of true ntitumor ctivity. Becuse, cncer vccines re not s rpidly effective s cytotoxic gents, SD is considered n indictor of meningful therpeutic effect [14]. Long-term SD in ptients receiving immunotherpy my be unique to cncer vccines nd is considered evidence of ctivity [1]. In the present study, we ssessed immunologicl responses in 7 ptients with stge Ⅳ PDA who received DC/WT1-Ⅰ/Ⅱ combined with chemotherpy. Three ptients displyed strong WT1-specific DTH rections in long-term chemoimmunotherpy, resulting in long-term OS 1 yer. Antigenic peptide-specific DTH is n inflmmtory rection tht is primrily medited through CD4 + effector-memory T cells primed by the cncer vccines [1]. A significnt correltion between fvorble clinicl outcomes nd the induction of cncer vccine-relted ntigen-specific DTH rection hs been observed [1]. Therefore, the long-term mintennce of strong WT1-specific DTH rections in these 3 ptients might be ssocited with long-term SD, resulting in prolonged survivl. Indeed, one ptient hd longterm SD nd continuously strong WT1-specific DTH, surviving t lest 1 d with 1% KPS. The plsm levels of circulting IL-6/-8 were lso nlyzed by ELISA, more quntittive nlysis thn the multiplexed mesurement system of flow cytometry, nd the cytokines levels were compred with the clinicl outcomes. In the present study, none of the 7 PDA ptients chieved complete or prtil response, nd 6 (8.7%) exhibited SD. Three ptients (No. 1, 2, nd 6) exhibited long-term SD, resulting in long-term survivl. These 3 ptients displyed significntly decresed levels of plsm IL-6 fter to 1 vccintions. IL-6 is secreted from vriety of cells, primrily lymphocytes, mcrophges, monocytes, fibroblsts, endothelil cells, nd kertinocytes [16]. Some tumor cells lso secrete IL-6 [16]. One histologicl hllmrk of PDA is the presence of highly desmoplstic strom, including severl inflmmtory cell popultions, such s fibroblst, stellte, endothelil, endocrine, nd immune cells, ll of which produce different inflmmtory cytokines [17]. The multifunctionl inflmmtory cytokine IL-6 plys role in the development nd progression of PDA by directly ffecting tumorigenesis [18]. Moreover, IL-6 regultes the secretion of vsculr endothelil growth fctor in PDA cells, thereby stimulting ngiogenesis nd tumor vsculriztion resulting in lymphtic nd distnt metstsis nd disese progression [18-2]. Therefore, the mintennce of low plsm IL-6 levels in 3 ptients receiving chemoimmunotherpy using DC/WT-1-I/ Ⅱ t erly vccintion periods ( vccines) nd its continution for 1 vccintions my be responsible for the prolonged survivl of the PDA ptients. Importntly, these ptients lso mintined longterm strong WT1-specific DTH rections. By contrst, extremely high levels of IL-6 nd IFN-γ (dt not shown) were detected in nonsuper-responder (No. ). The expression of IL-6 is potentited by IFN-γ vi prolonged NF-κB ctivtion [21]. Three super-responders exhibited slightly incresed IFN-γ production from CD4 + nd CD8 + T cells upon stimultion with WT1 peptides in vitro [1]. The low levels of plsm IL-6 in the superresponders ptients did not interfere with the induction of WT1-specific ntitumor immune responses nd were ssocited with prolonged survivl period. After chemoimmunotherpy, significntly decresed plsm IL-8 levels were lso obvious in ll 3 superresponders with strong WT1-specific DTH rections. Compred with IL-6, significntly decresed levels of plsm IL-8 were observed in the ptients fter longer periods of tretment (from 1 to 2 vccintions). Although tumor-ssocited mcrophges nd monocytes re the most likely source of IL-8, this cytokine is lso overexpressed in most humn PDA cells under inflmmtory conditions [22]. Severl cell types within the tumor microenvironment produce vriety of cytokines. Differences in cells producing inflmmtory cytokines such s IL-6 nd IL-8 my underlie periods of decresed plsm IL-6 nd IL-8 levels. In the tumor environment, these cytokines interct with other cell types in complex network. Importntly, IL-8 plys mjor role in the progression of PDA by promoting prolifertion, migrtion, ngiogenesis nd metstsis [23]. Elevted levels of circulting IL-8 re ssocited with poor clinicl outcome in ptients with PDA nd hve been suggested s prognosis mrker [24,2]. Therefore, significnt decrese in plsm IL-8 levels might lso be ssocited with good prognosis in PDA ptients receiving the chemoimmunotherpy using DC/WT1-Ⅰ/Ⅱ. Indeed, ll 3 super-responders with strong WT1- DTH rections mintined low levels of IL-8 fter 2 vccintions nd exhibited prolonged SD. However, plsm IL-8 nd October 21, 21 Volume 21 Issue 39

10 IL-6 levels in 2 super-responders (No. 1 nd 2) were incresed t the terminl stge of cncer, nd these ptients hd WT1-negtive DTH rections. However, one super-responder (No. 6) mintined low levels of IL-8 nd IL-6 nd displyed strong WT1-specific DTH rections fter t lest 4 vccintions. Recently, we reported tht IL-8 secretion from tumor cells enhnces the genertion nd ctivtion of CD163- positive M2 mcrophges producing IL-1, leding to poor clinicl outcomes in ptients with cncer [26]. Tht finding is consistent with the results of the present study demonstrting tht long-term low levels of circulting IL-8 in PDA ptients receiving DC/WT1- Ⅰ/Ⅱ is ssocited with the mintennce of strong WT1-specific DTH rections, resulting in good clinicl outcomes. In conclusion, both IL-6 nd IL-8 were mintined t low levels in ll 3 PDA ptients with strong WT1- specific DTH rections who received DC/WT1-Ⅰ/Ⅱ combined with chemotherpy. All 3 PDA ptients exhibited long-term SD nd prolonged survivl times (82 to more thn 1 d). One ptient with longterm strong WT1-specific DTH rections mintined decresed levels of plsm IL-6 nd IL-8 during therpy nd mintined long-term SD, resulting in survivl for more thn 1 d. Therefore, the mintennce of low plsm IL-6 nd IL-8 levels my be ssocited with immunogenic chnges in the desmoplstic strom. Low levels of plsm IL-6 nd IL-8 with strong WT1- DTH rections my be prognostic fctor for PDA ptients following chemoimmunotherpy using DC/ WT1-Ⅰ/Ⅱ. These cytokine interctions re ssocited with tumor growth nd progression, invsion nd metstsis, ngiogenesis nd immune evsion. Although trgeting IL-6 nd IL-8 my improve not only clinicl outcome but lso the response to tretment in PDA ptients, it is not cler if IL-6/-8-signling inhibitors will trnslte into clinicl benefits for PDA [19]. A limittion of the present study is the reltively smll smple size. Further studies re needed to evlute the clinicl significnce of circulting IL-6/-8 levels in PDA ptients treted with DC/WT1-Ⅰ/Ⅱ combined with chemotherpy. COMMENTS Bckground CD4 + T cells prime nd mintin ntigen-specific CD8 + CTLs nd ply direct role in the destruction of tumor cells. Therefore, the stimultion of both CD4 + nd CD8 + T cells is n efficient strtegy for treting ptients with pncretic ductl denocrcinom (PDA). The uthors hd conducted phse 1 clinicl study in PAD ptients to exmine the clinicl nd immunologicl responses to dendritic cells (DCs) pulsed with multiple mjor histocomptibility complex clss Ⅰ nd Ⅱ-restricted Wilms tumor 1 (WT1) epitopes (DC/WT1-Ⅰ/Ⅱ) in combintion with chemotherpy. The combintion of DC/WT1-Ⅰ/Ⅱ nd chemotherpy were ssocited with disese stbility in PAD ptients. Reserch frontiers Authors investigte the ssocition of plsm levels of interleukin (IL)-6 nd -8 with WT1-specific immune responses nd clinicl outcomes in ptients with PDA treted with DC/WT1-Ⅰ/Ⅱ combined with stndrd chemotherpy. The uthors reported the ssocition of plsm IL-6 nd -8 levels nd WT1 peptidespecific DTH in PDA ptients during long-term chemoimmunotherpy. Innovtions nd brekthroughs Authors showed tht plsm IL-6/-8 levels in PDA ptients during long-term tretments. Prolonged low levels of plsm IL-6/-8 my be prognostic mrker for the clinicl outcomes of chemoimmunotherpy. Applictions The study s results suggest tht plsm IL-6/-8 in PDA ptients during chemoimmunotherpy using DC/WT1-Ⅰ/Ⅱ is prognostic biologicl mrker for ssessing the induction of WT1-specific immune responses nd long-term overll survivl. Terminology The WT1 is highly expressed in vrious types of cncers, including PDA nd hs been found to be highly immunogenic. Thus, WT1 is one of the excellent tumor-ssocited ntigens for the trget of cncer immunotherpy. Peer-review The uthors reported tht prolonged low levels of plsm IL-6/-8 in PDA ptients my be prognostic mrker for the clinicl outcomes of chemoimmunotherpy. The dt re well represented, orgnized, nd cler. REFERENCES 1 Jeml A, Siegel R, Xu J, Wrd E. Cncer sttistics, 21. CA Cncer J Clin 21; 6: [PMID: DOI: / cc.273] 2 Conroy T, Desseigne F, Ychou M, Bouché O, Guimbud R, Bécourn Y, Adenis A, Roul JL, Gourgou-Bourgde S, de l Fouchrdière C, Bennoun J, Bchet JB, Khemiss-Akouz F, Péré- Vergé D, Delbldo C, Assent E, Chuffert B, Michel P, Montoto- Grillot C, Ducreux M. FOLFIRINOX versus gemcitbine for metsttic pncretic cncer. 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11 S, Tsuking S, Odhr S, Koym S, Imzu H, Uchiym K, Kjihr M, Arkw H, Misw T, Toym Y, Yngisw S, Ikegmi M, Kn S, Hyshi K, Komit H, Kmt Y, Ito M, Ishido T, Yus S, Shimodir S, Gong J, Sugiym H, Ohkus T, Tjiri H. Tretment with chemotherpy nd dendritic cells pulsed with multiple Wilms tumor 1 (WT1)-specific MHC clss I/II-restricted epitopes for pncretic cncer. Clin Cncer Res 214; 2: [PMID: DOI: 1.118/ CCR ] 11 Tkkur K, Koido S, Kn S, Yoshid K, Mori M, Hirno Y, Ito Z, Kobyshi H, Tkmi S, Mtsumoto Y, Kjihr M, Misw T, Okmoto M, Sugiym H, Homm S, Ohkus T, Tjiri H. Prognostic mrkers for ptient outcome following vccintion with multiple MHC Clss I/II-restricted WT1 peptide-pulsed dendritic cells plus chemotherpy for pncretic cncer. Anticncer Res 21; 3: -62 [PMID: 262] 12 Ueno H, Iok T, Iked M, Ohkw S, Yngimoto H, Boku N, Fukutomi A, Sugimori K, Bb H, Ymo K, Shimmur T, Sho M, Kitno M, Cheng AL, Mizumoto K, Chen JS, Furuse J, Funkoshi A, Htori T, Ymguchi T, Egw S, Sto A, Ohshi Y, Okusk T, Tnk M. Rndomized phse III study of gemcitbine plus S-1, S-1 lone, or gemcitbine lone in ptients with loclly dvnced nd metsttic pncretic cncer in Jpn nd Tiwn: GEST study. J Clin Oncol 213; 31: [PMID: DOI: 1.12/jco ] 13 Stthis A, Moore MJ. Advnced pncretic crcinom: current tretment nd future chllenges. Nt Rev Clin Oncol 21; 7: [PMID: DOI: 1.138/nrclinonc ] 14 Wolchok JD, Hoos A, O Dy S, Weber JS, Hmid O, Lebbé C, Mio M, Binder M, Bohnsck O, Nichol G, Humphrey R, Hodi FS. Guidelines for the evlution of immune therpy ctivity in solid tumors: immune-relted response criteri. Clin Cncer Res 29; 1: [PMID: ] 1 Arntzen EH, Figdor CG, Adem GJ, Punt CJ, de Vries IJ. Dendritic cell vccintion nd immune monitoring. Cncer Immunol Immunother 28; 7: [PMID: DOI: 1.17/s y] 16 Yo X, Hung J, Zhong H, Shen N, Fggioni R, Fung M, Yo Y. Trgeting interleukin-6 in inflmmtory utoimmune diseses nd cncers. Phrmcol Ther 214; 141: [PMID: DOI: 1.116/j.phrmther ] 17 Lippitz BE. Cytokine ptterns in ptients with cncer: systemtic review. Lncet Oncol 213; 14: e218-e228 [PMID: DOI: 1.116/s147-24(12)782-x] 18 Roshni R, McCrthy F, Hgemnn T. Inflmmtory cytokines in humn pncretic cncer. Cncer Lett 214; 34: [PMID: DOI: 1.116/j.cnlet ] 19 Codony-Servt J, Mrín-Aguiler M, Vis L, Grcí-Albéniz X, Pined E, Fernández PL, Filell X, Gscón P, Melldo B. Nucler fctor-kpp B nd interleukin-6 relted docetxel resistnce in cstrtion-resistnt prostte cncer. Prostte 213; 73: [PMID: DOI: 1.12/pros.2291] 2 Tng RF, Wng SX, Zhng FR, Peng L, Wng SX, Xio Y, Zhng M. Interleukin-1lph, 6 regulte the secretion of vsculr endothelil growth fctor A, C in pncretic cncer. Heptobiliry Pncret Dis Int 2; 4: [PMID: ] 21 McLoughlin RM, Witowski J, Robson RL, Wilkinson TS, Hurst SM, Willims AS, Willims JD, Rose-John S, Jones SA, Topley N. Interply between IFN-gmm nd IL-6 signling governs neutrophil trfficking nd poptosis during cute inflmmtion. J Clin Invest 23; 112: [PMID: DOI: / jci17129] 22 Kuwd Y, Sski T, Morink K, Kitdi Y, Mukid N, Chym K. Potentil involvement of IL-8 nd its receptors in the invsiveness of pncretic cncer cells. Int J Oncol 23; 22: [PMID: ] 23 Li M, Zhng Y, Feurino LW, Wng H, Fisher WE, Brunicrdi FC, Chen C, Yo Q. Interleukin-8 increses vsculr endothelil growth fctor nd neuropilin expression nd stimultes ERK ctivtion in humn pncretic cncer. Cncer Sci 28; 99: [PMID: DOI: /j x] 24 Bellone G, Smirne C, Muri FA, Tonel E, Crbone A, Buffolino A, Dugher L, Robecchi A, Pirisi M, Emnuelli G. Cytokine expression profile in humn pncretic crcinom cells nd in surgicl specimens: implictions for survivl. Cncer Immunol Immunother 26; : [PMID: DOI: 1.17/ s ] 2 Chen Y, Shi M, Yu GZ, Qin XR, Jin G, Chen P, Zhu MH. Interleukin-8, promising predictor for prognosis of pncretic cncer. World J Gstroenterol 212; 18: [PMID: DOI: /wjg.v18.i1.1123] 26 Fujit Y, Okmoto M, God H, Tno T, Nkshiro K, Sugit A, Fujit T, Koido S, Homm S, Kwkmi Y, Hmkw H. Prognostic significnce of interleukin-8 nd CD163-positive cellinfiltrtion in tumor tissues in ptients with orl squmous cell crcinom. PLoS One 214; 9: e11378 [PMID: DOI: /journl.pone.11378] P- Reviewer: Celio G Freire-De-Lim, Seong SY S- Editor: Yu J L- Editor: A E- Editor: Liu XM October 21, 21 Volume 21 Issue 39

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