Multicenter Randomized Phase 2 Clinical Trial of a Recombinant Human Endostatin Adenovirus in Patients with Advanced Head and Neck Carcinoma

Transcription

1 The Americn Society of Gene & Cell Therpy originl rticle Multicenter Rndomized Phse Clinicl Tril of Recombinnt Humn Endosttin Adenovirus in Ptients with Advnced Hed nd Neck Crcinom Wen Ye, Rnyi Liu, Chngchun Pn, Wenqi Jing, Li Zhng, Zhongzhen Gun, Jingxue Wu, Xiofng Ying, Lixi Li, Su Li, Wen Tn, Musheng Zeng, Tiebng Kng, Qing Liu, George R Thoms 4, Mnli Hung 5, Wuguo Deng nd Wenlin Hung,6 Stte Key Lbortory of Oncology in South Chin, Collbortive Innovtion Center of Cncer Medicine, Sun Yt-sen University Cncer Center, Gungzhou, Chin; Medicl Oncology, Sichun Cncer Hospitl nd Institute, Chengdu, Chin; School of Bioscience nd Bioengineering, South Chin University of Technology, Gungzhou, Chin; 4 Mrsl Biotech, Winnipeg, Cnd; 5 Gungdong Provincil Key Lbortory of Tumor-trgeted Drug, Gungzhou Enterprise Key Lbortory of Gene Medicine, Gungzhou Doublle Bioproducts, Gungzhou, Chin; 6 CAS Key Lbortory of Pthogenic Microbiology nd Immunology, Institute of Microbiology, Chinese Acdemy of Science, Beijing, Chin A rndomized, open-lbel, phse, multicenter clinicl tril ws conducted to evlute the efficcy nd sfety of the ddition of recombinnt humn endosttin denovirus (EA) to cispltin nd pclitxel in ptients with dvnced hed nd neck squmous cell crcinom or nsophryngel crcinom. Ptients with loclly dvnced or metsttic hed nd neck squmous cell crcinom or nsophryngel crcinom not suitble for opertion or rdiotherpy were rndomly ssigned to receive EA plus chemotherpy every weeks for mximum of six cycles or to receive chemotherpy only. One hundred nd thirty-six eligible ptients were rndomly ssigned. The ddition of EA did not significntly improve the objective response rte (9.9 versus 9.7%, P =.54). However, ptients who received endosttin hd longer progression-free survivl (7. versus.6 months, P =.6; hzrd rtio:.55). The combintion of EA with chemotherpy benefited prior chemotherpy-treted ptients nd those who received three to four tretment cycles (6.5 versus.4 months, P =.; 8.7 versus 4.7 months, P =.8; respectively). The overll disese control rte significntly incresed from 8.6% in the control group to 9.6% in the test group (P =.4). Except for fever, no dverse events were ssocited with the EA tretment. In summry, EA plus chemotherpy is sfe nd effective therpeutic pproch in ptients with dvnced hed nd neck squmous cell crcinom or nsophryngel crcinom. Received October ; ccepted 6 Mrch 4; dvnce online publiction April 4. doi:.8/mt.4.5 INTRODUCTION Angiogenesis, crucil for the development, invsion, nd metstsis of tumors, depends mostly on the blnce between ngiogenic stimultors nd inhibitors. The ntitumor ctivity of endosttin, n endogenous ngiogenic inhibitor derived from collgen XVIII, ws first demonstrted in the Folkmn Lbortory, where the dministrtion of insoluble endosttin ws found to suppress VEGF-stimulted endothelil cell prolifertion, migrtion, nd tumor ngiogenesis with low toxicity nd n bsence of drug resistnce. Gene therpy s mens of continuous endosttin delivery hs ttrcted our interest. Our previous studies showed tht EA (Gungzhou Doublle Bioproducts, Gungzhou, Chin), type 5 recombinnt repliction-deficient denovirus vector crrying the humn endosttin gene, cn directly introduce the gene into tumors nd continuously expresses the endogenous endosttin protein in host cells to limit vsculriztion nd tht this endosttin protein is metbolized in the liver., In vivo studies of multiple intrtumorl dministrtions of EA to nsophryngel crcinom (NPC), gstric cncer, nd heptocellulr crcinom xenogrfts in nude mice demonstrted high-level endogenous endosttin expression with reltively long hlf-life nd significnt inhibition of tumor growth. 4 6 EA in combintion with low-dose cispltin induced high levels of poptosis nd significntly enhnced the tumor growth inhibitory effect of cispltin in xenogrft mouse models of hed nd neck squmous cell crcinom (HNSCC) nd lung cncer, respectively. 7,8 Menwhile, EA combined with docetxel inhibited prostte cncer growth nd metstsis. 9 A long-term, high-dose intrmusculr dministrtion of EA in cnines ws proved not toxic nd might be sfe for clinicl therpeutic use. In 7, dose-escltion phse clinicl tril on 5 ptients with dvnced solid tumors showed tht no dose-limiting toxicity ws developed from weekly intrtumorl injections of EA t doses rnging from virus prticles to virus prticles. A distribution study detected EA in the ptients blood, throt, nd injection site, but rrely in the urine nd stool. Endosttin expression ws incresed throughout the period of tretment despite the presence of neutrlizing ntidenovirus ntibodies. Correspondence: Wenling Hung, Stte Key Lbortory of Oncology in South Chin, Collbortive Innovtion Center of Cncer Medicine, Sun Yt-sen University Cncer Center, 65 Dongfeng Rod Est, Gungzhou, Chin. E-mil: hungwl@sysucc.org.cn Moleculr Therpy vol. no. 6, 9 jun. 4

2 Efficcy nd Sfety of Recombinnt Endosttin The Americn Society of Gene & Cell Therpy Surgery, rdiotherpy, nd chemotherpy hve generlly been dopted s primry tretment modlities for dvnced HNSCC nd NPC in recent decdes. However, locoregionl recurrence nd metsttic disese in these dvnced cncer ptients remin the min cuses of deth, with poor overll survivl (OS; HNSCC: 7 months; NPC: 5 months). Therefore, therpeutic pproch tht cn effectively control hed nd neck cncers is urgently needed. 6 We ssumed EA could benefit dvnced cncer ptients, nd vsculr-rich solid tumors on the body surfce re pplicble for the intrtumorl dministrtion of EA nd re strightforwrd for tumor observtion/evlution. Therefore, we used HNSCC nd NPC ptients s the trget popultion of this tril. To define the efficcy nd sfety of EA, we performed phse, open-lbel, multicenter clinicl tril in hospitls of Chin in ptients with dvnced HNSCC or NPC using pclitxel nd cispltin with or without EA in rndomized, controlled mnner. RESULTS From Mrch 8 to December, 4 ptients from hospitls in Chin were consecutively enrolled. Three ptients meeting the exclusion criteri were excluded, nd one withdrew before rndomiztion. In totl, 6 ptients were rndomly ssigned to two groups. One ptient in the pclitxel-cispltin lone group (the control group) refused to receive the llocted tretment; thus, 67 ptients in the control group nd 68 ptients in the pclitxel-cispltin plus EA group (the EA group) were included in the sfety popultion nd in the nlysis set for efficcy nd survivl. The bseline chrcteristics were well blnced between the two groups (Tble ). Efficcy The primry end point ws the objective response rte (RR), defined s the proportion of ptients who hd complete response (CR) or prtil response (PR) t the trget tumor lesion. The secondry end points were the objective disese control rte (DCR, or stble disese (SD) + PR + CR t the trget tumor lesion), the overll RR, the overll DCR, OS, nd progression-free survivl (PFS). The primry end point of objective RR did not meet sttisticl significnce (9.9% in the control group, 9.7% in the EA group, P =.54; odds rtio (OR).65; Tble ). However, the dministrtion of EA benefited some subgroups of ptients. In the HNSCC ptients, the objective RR ws 6.5% (5/4) with EA dministrtion, exhibiting trend of exceeding the rte of.% (7/5) in the control group (P =.9; OR:.4), wheres the objective RR ws 44.4% (/7) versus 4.6% (/) in the NPC ptients (P =.487; OR:.86). Ptients who hd previously received chemotherpy in the EA group hd 44.8% (/9) objective RR, wheres ptients in the control group hd only.6% objective RR (7/; P =.6, OR:.6). In contrst, ptients without previous chemotherpy hd similr RR in both groups (4. versus 9.4%; P =.46, OR:.5). There ws no reltionship between prior chemotherpy nd the response of treted ptients (Supplementry Tble S). Moreover, in the ptients who received three to four tretment cycles, there were better Tble Bseline chrcteristics of the study ptients Chrcteristics Gender, no. Control group (N = 67) (%) Femle 4 (.9) (9.) Mle 5 (79.) 55 (8.9) Age, yers Medin 5 5 Rnge 75 7 Type of primry tumor, no. NPC (47.8) 7 (9.7) HNSCC 5 (5.) 4 (6.) ECOG sttus 8 (6.9) 4 (5.) 49 (7.) 44 (64.7) Extent of disese, no. Locoregionl dvnced 5 (76.) 5 (7.5) EA group (N = 68) (%) P vlue b Distnt metstsis 6 (.9) 8 (6.5) Prior tretment, no..7 b Yes 45 (67.) 4 (6.) Surgery 9 (8.4) 9 (7.9). Rdiotherpy 4 (5.7) (47.).86 Chemotherpy (46.) 9 (4.6).878 Txne-bsed 7(.) 9(.).5 Pltinum-bsed (7.) 8(6.).5 5-Fu-bsed (67.7) (44.8).6 No (9.9) (.9) Missing dt or unknown (.9) 4 (5.9) Finished tretment cycle, no..67 b 5(7.5) 6(8.8) (47.8) (45.6) (4.5) 9(.) 4 7(4.) (.4) Follow-up, months.94 b Medium.. Rnge ECOG, Estern Coopertive Oncology Group; HNSCC, neck squmous cell crcinom; NPC, nsophryngel crcinom. Two-sided Fisher s exct test; b Kruskl-Wllis H test. objective/overll responses of the EA group thn the control group (P =.4,.6, respectively, Tble ). The overll DCR ws 8.6% in the control group, significntly lower thn the rte of 9.6% in the EA group (P =.4; OR:.; Tble ). Typicl cse presenttions re shown in the Supplementry Figure S. As shown in Figure nd Tble, the difference in the Kpln Meier estimtes of PFS fvored chemotherpy plus EA, which resulted in.4-month improvement. With medin follow-up of.47 months, the medin PFS ws.6 months (interqurtile rnge:.6 7.6) in the control group nd 7. months (interqurtile rnge:.7.7) in the EA group. As vol. no. 6 jun. 4

3 The Americn Society of Gene & Cell Therpy Efficcy nd Sfety of Recombinnt Endosttin Tble Tumor responses to tretment Outcome Control group (N = 67) (%) EA group (N = 68) (%) P vlue Response of trget lesions, no. Complete response (CR) (.5) (4.4) - Prtil response (PR) 9 (8.4) 4 (5.) - Stble disese (SD) 4 (6.) 8 (55.9) - Progressive disese (PD) 6 (9.) (4.4) - Objective response (9.9) 7 (9.7).54 (CR+PR) tretment cycles (7.) 7(8.9).9 4 tretment cycles (.) (64.5).4 Disese control 6 (9.) 65 (95.6).9 (CR + PR + SD) Overll response, no. Complete response (CR) (.) (.5) - Prtil response (PR) 9 (8.4) 4 (5.) - Stble disese (SD) 5 (5.) 8 (55.9) - Progressive disese (PD) (9.4) 5 (7.4) - Overll response (CR + PR) 9 (8.4) 5 (6.8).95 tretment cycles 9(4.) 6(6.).8 4 tretment cycles (.) 9(6.).6 Disese control (CR + PR + SD) 54 (8.6) 6 (9.6).4 The P vlues provided in bold re sttisticlly significnt. Fisher s exct χ test (one-sided). compred with the control group, the hzrd rtio (HR) of progression ws significntly lower in the EA group (HR:.55, P =.6). Explortory strtified nlysis of PFS cross key clinicl subgroups supported the primry nlysis, demonstrting the beneficil effects of dding EA to cispltin-pclitxel chemotherpy significntly improved PFS in the subpopultions of mles, ptients older thn 55 yers, ptients hd received prior tretment, nd those who finished three to four tretment cycles (P =.7, HR:.54; P =.6, HR:.45; P =., HR:.45; nd P =.8, HR:.49; respectively), showing HRs tht consistently fvored the EA-conting groups. The OS of the EA group ws reltively prolonged in different subgroups compred with the controls (e.g.,.7 months versus 9.67 months in the HNSCC ptients,. months versus.5 months in those who hd received prior tretment; Figure b; Tble ), but these results did not trnslte into significntly superior survivl. Endosttin expression nd ntingiogenic effects The systemic endosttin level in serum showed tht the genetherpeutic denovirus ws ctively producing the endosttin protein endogenously in ptients. After the first dministrtion of EA, serum endosttin incresed from.48 ± 6.59 ng/ml (bseline) to pek of 5.69 ± 7.49 ng/ml (P <.) 48 hours nd then grdully decresed before the next EA dministrtion (dy 8). During the subsequent tretment, serum endosttin mintined t high level (77.69 ± 6.86 t dy, ± 4.48 No. t risk Time (months) EA group Control group b Progression-free survivl (proportion) Overll survivl (proportion) No. t risk EA group Control group Chemotherpy lone (n = 67) Chemotherpy lone (n = 67) HR =.55; 95% Cl =.5.84 Log-rnk P =.6 Chemotherpy plus EA (n = 68) Chemotherpy plus EA (n = 68) Figure Kpln Meier estimtes of survivl for ll ptients by tretment groups. () Progression-free survivl. (b) Overll survivl. CI, confidence intervl; HR, hzrd rtio; PFS, progression-free survivl; OS, overll survivl. t dy 4, ± 6.77 t dy 6, nd 85.5 ± 4. t dy 84) thnks to the continuously expressed endosttin protein produced by repeted dministrtion of EA (Figure ; Tble 4) Immunohistochemicl ssys of seven pirs of biopsy smples (before nd fter tretment of the sme ptient) showed tht ll of the ptients hd significnt incresed of endosttin expression in injection site fter tretment compred with before tretment (P =.4; Figure b,c). As result of EA tretment, the tumor microvessel density nd the tumor tissue blood flow significntly decresed (Figure d h). There ws no difference in the serum concentrtion of endosttin over time in the subgroups of HNSCC versus NPC nd chemotherpy-untreted versus -treted (P =.57,.57, respectively; Supplementry Figure S). We mesured eight moleculr biomrkers (svcam-, TSP-, se-selectin, VEGF, bfgf, ngiopoietin-, ngiopoietin-, nd EGF) relted to ngiogenesis in the serum of some EA ptients before nd fter the first dministrtion of EA nd grouped them ccording to their therpy responses into two ctegories: group CR + PR nd group SD + progressive disese (PD; Tble 4; Supplementry Figure S). By performing Generl Liner Model Anlysis for Repeted Mesures, we found tht endosttin, bfgf, svcam-, ngiopoietin-, nd se-selectin chnged with 6 4 HR =.79; 95% Cl =.47. Log-rnk P = Time (months) Moleculr Therpy vol. no. 6 jun. 4

4 Efficcy nd Sfety of Recombinnt Endosttin The Americn Society of Gene & Cell Therpy Tble Strtified progression-free survivl nd overll survivl nlysis Subgroup No. of ptients Medin PFS (months) (Control versus EA) HR for PFS (95% CI) P vlue b Medin OS (months) (Control versus EA) HR for OS (95% CI) P vlue b All 5.6 versus (.5.84) versus (.47.).66 Gender Femle 7.4 versus 9..5 (..7).44. versus 7..5 (.8.47).4 Mle 8.6 versus (..9) versus (.49.6).7 Age <55 yers versus (..4) versus 7..4 (.5.6) yers 59.6 versus (..88) versus.6.55 (.5.).4 Type of tumor NPC versus (.8.) versus.7.68 (.9.56).64 HNSCC 76.6 versus (.4.7) versus.7.76 (.9.48).45 Extent of disese 4.8 versus (.4.99) versus 9..9 (.49.7) versus (.5.8).9 5. versus 7..5 (..4).97 Prior tretment Yes 88.4 versus (.6.77)..5 versus..74 (.4.).8 No versus.7.59 (..55).69 ND versus.7.5 (..47).9 Finished tretment cycle, no versus (.8.5) versus 5..(.55.65) versus (.6.9).8 4. versus (.9.6). The P vlues provided in bold re sttisticlly significnt. HNSCC, hed nd neck squmous cell crcinom; HR, hzrd rtio; NPC, nsophryngel crcinom; ; OS, overll survivl; PFS, progression-free survivl., locoregionl recurrence;, distnt metstsis; b Log-rnk test (Mntel-Cox). time. VEGF rnged from 59 to 75 pg/ml in group CR + PR, which ws the only biomrker tht ws significntly lower thn in group SD + PD ( pg/ml; P =.45, Supplementry Tble S). An elevtion of CD + T cells nd CD9 + B cells infiltrtion in the posttretment biopsies of the EA group is shown in Figure. Sfety The overll incidence of dverse events ws 9.6% (6/68) in the EA group nd 89.6% (6/67) in the control group (P =.95; Tble 5). The most frequent dverse events in both groups were bone mrrow suppression (leukopeni, neutropeni, nd nemi), fever, nd nuse. Fever ws more frequent in the EA group thn in the control group (P =.; OR: 4.). Of the 68 ptients who received EA, 7 hd grde or trnsient locl rections (locl pyrexi, pin, swelling, hemtom, or hemorrhge). No significnt differences were found between the groups with respect to the incidence of other dverse events. Twelve serious dverse events occurred in eight ptients: four in the EA group nd four in the control group (Supplementry Tble S). With timely tretment, four ptients remitted without sequele, nd the other four died. Among the deths, only one in the control group ws considered likely to be chemotherpyrelted, wheres no deth ws considered to be EA-relted. DISCUSSION For ptients with locoregionl recurrence or metsttic HNSCC or NPC, extensive surgery nd rdiotherpy usully fil, nd chemotherpy is integrted into the stndrd therpy in n ttempt to improve disese control nd prolong survivl. 7 Rndomized clinicl studies of ptients with dvnced HNSCC nd NPC hve shown tht the combintion of cispltin nd pclitxel yields DCR of up to 8%, n overll RR of 6%, nd medin PFS of ~4 months. 8,9 These findings re consistent with our results in the control group (9.9% RR, 8.6% DCR, nd.6-month PFS) but re inferior to the outcomes of the EA group (9.7% RR, 9.6% DCR, nd 7.-month PFS). We filed to detect significnt difference in the primry end point between the groups, most likely due to our limited smple size to detect n objective RR rised from 9.9% in the control group nd to 9.7% in the EA group, which were higher thn the 5 5% in the smple size clcultion ssumption. However, the tril still reveled tendency for the dministrtion of EA to rise disese control in HNSCC ptients nd those who hd previously received chemotherpy, compred with NPC ptients nd those without prior chemotherpy, respectively. We noticed the lrgely extended PFS in the EA group by contrst to the control group in ssocition with the nonspecific difference in objective RR, suggesting tht the PFS difference ws not driven by tumor shrinkge ctivity. The 45% reduction in the risk of progression with EA s compred with the control group is cliniclly importnt, indicting tht EA cted s n djuvnt chemotherpy gent could enhnce the tumor growth inhibitory effect of pclitxel-cispltin nd postpone tumor progression despite its disbility to reverse disese. For n explortive phse tril of new drug, enrolling suitble ptients regrdless of their tretment history enbled us to locte trget popultion effectively. Bsed on the findings of 4 vol. no. 6 jun. 4

5 The Americn Society of Gene & Cell Therpy Efficcy nd Sfety of Recombinnt Endosttin b Pretretment c Posttretment 4 Endosttin H score 8 Endosttin Endosttin in serum (ng/ml) PreEA Time (dy) d Pretretment Posttretment e f 5, Pek of Echo-power (.u) CD4 Microvessel density (MVD) 4 PreEA h 4,,,, PreEA Post tretment Before tretment After tretment Echo-power (.u),5 One cycle fter EA tretment Post tretment 4, g Pretretment Post tretment,,5,,5, Time (g) 6 7 Figure Endosttin expression nd ntingiogenic ssessments. () Serum endosttin concentrtion during tretment; Pretretment nd posttretment ssessments of (b) immunohistochemicl stining of endosttin in the biopsies of the EA group. Br = μm; (c) H score of endosttin in the biopsies of the EA group; (d) immunohistochemicl stining of CD4 (mrker of vsculr formtion). Br = μm; (e) microvessel density of tumors; (f) blood flow volume in tumors (evluted by dynmic contrst-enhnced ultrsound); (g) echo-power of blood flow volume in the tumor of ptient No. C75; (h) echo-power peks of blood flow volume in eight tumors. this phse tril, our phse tril, which lunched in November, hs selected previously treted HNSCC ptients s the trget popultion. Moreover, with more EA tretments, ptients hd better objective/overll RR compred with the control group. This finding encourged us to further confirm this correltion in future pplictions, exploring the possibilities of incresed dministrtion frequency nd prolonged mediction period without significnt dverse events. The serum endosttin level peked on the third dy fter the first injection, t level tht ws ~ ng/ml higher thn the bseline, nd then grdully dropped before the initition of the next EA dministrtion. During the subsequent tretment, serum endosttin mintined t high level (bout 5 ng/ml higher thn the bseline) thnks to the continuously expressed endosttin Moleculr Therpy vol. no. 6 jun. 4 protein produced by EA. The evidence bout sustined nd repeted cycles of endosttin expression proved tht redministrtion of EA ws efficcious. Despite the intrtumorl dministrtion, the expression of long hlf-life endogenous endosttin with systemiclly sustined levels (insted of rising nd dropping drsticlly within short time) showed the dvntge of this novel therpeutic denovirus compred with exogenous endosttin. Some clinicl trils with exogenous endosttin hve been conducted, but they were disdvntges due to its very low yield nd short bioctive hlf-life, which necessitte dily repeted dministrtion. 4 Gene therpy with EA cn produce endogenous, stble, nd lsting endosttin with the sme or even better tumor inhibitory effect thn endosttin exogenously produced in prokryotic cells.4,5 Our tril not only substntites tht EA hs 5

6 Efficcy nd Sfety of Recombinnt Endosttin The Americn Society of Gene & Cell Therpy Tble 4 Moleculr mrkers in the EA group serum Time Mrker Dy Dy Dy 5 Dy 7 Endosttin b 7.58 ± ± ± ± 7.5 c.5 ± ± ± ±.96 VEGF 77.4 ± ± ± ± ± ± ± ± 48. bfgf 8.85 ± ± ±.58.6 ± ± ± ± ± 6.68 svcam-,65.8 ±,8.5,747.4 ± 98.79,8.5 ±,48.7,.95 ±,7.6,76.58 ± 49.9,66.8 ± 694.8,65.4 ± 56.95,88.9 ± Ang. ±..76 ±.87. ±.6.88 ±.9. ± ± ±.54.5 ±.48 Ang 9. ± 5.7. ± ± 8.. ± ± ± ± ± 8.9 TSP- 5.8 ±.5. ±.8.89 ±.9 5. ±.84. ±..97 ± ± ±.9 se-selectin 45. ± ± ± ± ± ±..6 ± ± 6.97 EGF ± ± ±. 9.8 ± ± ± ± ± 4.96 Men ± stndrd devition, ng/ml; b represents PR + CR ptients; c represents PD + SD ptients. CD b CD9 Pretretment Posttretment μm μm μm μm Figure Leukocyte infiltrtion in the EA group tumor smples. Pretretment nd posttretment immunohistochemicl stining of () CD + T cells nd (b) CD9 + B cells. Br = μm. significnt inhibitory effect on tumors but lso provides simplified therpeutic schedule. We ssessed eight moleculr biomrkers relted to ngiogenesis, including the inhibitors TSP- nd ngiopoietin- nd the ctivtors VEGF, bfgf, se-selectin, svcam-, ngiopoietin-, nd EGF, in the ptients serum before nd fter the first dministrtion of EA. 6 8 The serum VEGF in the EA ptients ws significntly lower in the CR + PR group thn in the SD + PD group, which supports the hypothesis tht endosttin suppresses VEGF-stimulted endothelil cell prolifertion, migrtion, nd tumor ngiogenesis. Therefore, serum VEGF could be considered predictive mrker for tretment outcomes in future clinicl pplictions, but this possibility needs further vlidtion. Among the four biomrkers tht chnged with time, it is noteworthy tht the prongiogenic fctors were upregulted (bfgf, svcam-, nd se-selectin) nd ngiopoietin- ws downregulted. We my suppose tht the inhibitory effect of endosttin cused systemic negtive feedbck of ngiogenesis-relted pthwys. As result, the ctivtors were upregulted, nd ngiopoietin- ws downregulted. Knzw et l. 7 reported tht in the presence of high levels of VEGF, ngiopoietin- promotes remodeling of the bsl lmin, prolifertion/migrtion of endothelil cells, nd sprouting of new blood vessels. However, in the bsence of VEGF, ngiopoietin- induces vsculr regression. This finding helps explin how ngiopoietin- cn inhibit ngiogenesis when VEGF is downregulted, s occurred in our tril. Except for fever, which ws more frequent, but mild nd trnsient, in the EA group, the tril provided convincing sfety profile, with no other significnt systemic toxicity. We suppose tht in ddition to the symptoms cused by chemotherpy, the systemic relese of denovirus cused fever, which hs been the most common dverse event, reported in denovirus-relted clinicl trils, with n incidence of 9%. In our phse clinicl tril, fever occurred in dose-dependent mnner.,9 The ntidenovirus immune response induced by multiple dministrtions of recombinnt denovirus is usully considered to hinder the therpeutic efficiency. The significntly incresed infiltrtion of CD + T cells nd CD9 + B cells in trget lesions posttretment further indicted tht EA ctivted the immune 6 vol. no. 6 jun. 4

7 The Americn Society of Gene & Cell Therpy Efficcy nd Sfety of Recombinnt Endosttin Tble 5 Common dverse events Events EA group (N = 68) (%) Control group (N = 67) (%) Grdes Grdes I II III IV I II III IV Any event (7.6) 7 (9.7) (.4) (.9) 5 (.4) 5 (7.) 8 (6.9) (.).95 Leukopeni (.9) 9 (7.9) 4 (5.9) (.5) 7 (5.4) (7.9) 6 (9.) (.5).4 Neutropeni (7.6) (6.) 5 (.) (.5) 9 (.4) (4.9) (9.4) (.).96 Anemi 7 (5.) 4 (5.) (4.4) (.5) 4 (5.8) 6 (.9) 5 (7.5) (.).46 Fever (9.4) 9 (.) (.5) (.) 7 (.4) (4.5) (.5) (.). Nuse 8 (6.5) (.9) (.) (.) 7 (.4) (4.5) (.) (.).55 Anorexi (9.) 4 (5.9) (.) (.) 8 (.9) 4 (6.) (.) (.).5 Vomiting 8 (.8) 5 (7.4) (.9) (.) 5 (7.5) 5 (7.5) (.) (.).887 Alopeci (.9) (4.4) (.) (.) 5 (7.5) 5 (7.5) (.5) (.).57 Serum ALT (4.4) (4.4) (.) (.) 7 (.4) (.) (.) (.). Serum AST (.5) (.9) (.5) (.) (.) (.) (.) (.).69 Locl responses 6 (8.8) (.5) (.) (.). Thrombocytopeni (.) (.9) (.5) (.) (4.5) (.) (.5) (.).5 Astheni (.9) (.5) (.) (.) (4.5) (.) (.) (.). Dirrhe (.) (.9) (.5) (.) (.5) (.) (.) (.).496 Plpittions (4.4) (.) (.) (.) (.) (.5) (.) (.).44 ALT, lnine minotrnsferse; AST, sprtte minotrnsferse. Locl responses include locl pyrexi, pin, swelling, hemtom, nd hemorrhge t the injection site. b -sided Fisher s exct test. P vlue b system. In our preclinicl experiments in n immune-competent mouse model, multiple intrtumorl dministrtions of EA did not led to continuous increses of ntidenovirus neutrlizing ntibodies. Serum endosttin ws slightly decresed but remined bove the efficient therpeutic concentrtion to chieve n ntingiogenic effect. 5 In our phse tril, of the 4 ptients showed n incresed IgG titer posttretment, nd three ptients hd n incresed IgM ntibody titer fter the dministrtion of EA. Despite the presence of neutrlizing ntibodies, endosttin trnsgene expression ws detectble in most ptients throughout the tretment. No correltion between ntibody titer nd dverse events or response ws observed. Another report showed tht the intrtumorl injection of recombinnt denovirus ctivted the direct nd indirect immune response to exert bystnder effects for tumor inhibition in immune-competent individuls. Thus, coupled with the potentil bystnder effects induced by immunostimultion, the tumor inhibition of EA ws improved. Moreover, considering tht cncer ptients, especilly dvnced cses, re often immune-inhibited, the negtive effects of neutrlizing ntibodies re miniscule compred with the benefits. Overll, bsed on this pilot phse study to ssess the ntingiogenic effects of EA, we hve shown tht EA in combintion with pclitxel-cispltin chemotherpy cn increse disese control nd prolong PFS in dvnced hed nd neck crcinom ptients nd tht it hs fvorble sfety profile. In prticulr, ptients with dvnced HNSCC, prior chemotherpy or more EA tretments hd better tumor response. These findings encourge us to conduct further studies to confirm the effects of this novel combintion therpy. MATERIALS AND METHODS Eligibility criteri. Ptients older thn 8 yers with histologiclly or cytologiclly proven locoregionlly dvnced or metsttic HNSCC/NPC not suitble for opertion or rdiotherpy were eligible to prticipte. Ptients were required to hve t lest one mesurble (by imging or photogrph) lesion with the lrgest dimeter cm nd suitble for the intrtumorl injection of EA, not received chemotherpy, rdiotherpy, or biotherpy within 4 weeks, life expectncy weeks, n Estern Coopertive Oncology Group performnce sttus score of, nd dequte bone mrrow, renl, nd liver functions. Known llergies to the study drug, the presence of importnt blood vessels/nerves or ulcertion in the trget lesion not suitble for injection, tumor relpse within 6 months fter pclitxel chemotherpy, severe cogultion disorders or bleeding tendency, severe uncontrolled medicl conditions, recent history of myocrdil infrction, cute infection, pregnncy or lcttion, or symptomtic brin metstses rendered ptients ineligible. From Mrch 8 to December, 4 ptients were consecutively enrolled in hospitls of Chin. All ptients provided written informed consent before enrollment. Study design. This multicenter, open-lbel phse clinicl tril ws pproved by the Stte Food nd Drug Administrtion of Chin nd the ethics committee t ech study hospitl, nd complied with the Declrtion of Helsinki protocols nd the provisions of the Good Clinicl Prctice guidelines. The rndomiztion ssignment ws prepred with SAS9. (SAS Institute, Cry, NC) softwre in subblock rndomiztion mnner by sttisticin not involved in the dt mngement. The 4 rndom numbers were ssigned to two groups in : rtio nd yielded into the ptient lloction letters. If ptient enrolled successfully, he/she would be sequentilly ssigned letter with rndom number. As our study ws n open-lbel tril, ll ptients nd physicins were wre of the study group ssignments once they unseled the rndom letters. Moleculr Therpy vol. no. 6 jun. 4 7

8 Efficcy nd Sfety of Recombinnt Endosttin The Americn Society of Gene & Cell Therpy According to the Response Evlution Criteri in Solid Tumors, ll therpeutic effects were clssified into 4 grdes s follows: CR, PR, SD, nd PD. The primry end point ws the objective RR (PR + CR). The secondry end points were the objective DCR (SD + PR + CR), the overll RR, the overll DCR, OS, nd PFS. The dt were collected t ech study hospitl by principl investigtors with the oversight of the project supervisor. Tretment schedule. In the EA group, ptients received n EA dose of. virus prticles intrtumorlly on dy nd 8, pclitxel 6 mg/m on dy, nd cispltin 5 mg/m from dy to dy 5. We chose the sme single trget lesion in which it ws esiest to perform repeted intrtumorl injection. If the trget lesion obtined CR, EA dministrtion would be stopped. In the control group, the ptients received the sme chemotherpy s the EA group but without EA. This tretment regimen ws repeted every dys for scheduled tretment of two to six cycles. For the ptients evluted s PR, the cycle number could be up to six, wheres the mximum for SD ptients ws four cycles. The principl investigtors from ech study hospitl were trined in the stndrdized EA dministrtion procedure. First, EA ws diluted with.9% sodium chloride to pproprite dose ccording to the longest dimeter of the trget lesion (< cm: ml; 4 cm: ml; 4 cm: ml). After locl nesthesi, we penetrted the syringe under norml skin subcutneously 5 mm into the tumor or verticlly into the lymph node under direct visuliztion nd withdrew it to confirm the bsence of blood. Then, EA ws injected with one ( cm ), two ( 5 cm ), or four ( 5 cm ) equl injections ccording to the re of the trget lesion. Lst, we pplied locl compression for minutes nd psted sterile sticker on the injection site to void bleeding (Supplementry Figure S4). Tumor progression, tretment intervl of more thn 4 dys, or request by the ptient to withdrw due to intolerble side effects or the inbility to comply with the tril protocol, the tretment would be terminted. Ptient ssessment. Physicl exmintion, ssessment of hemtologic/ chemicl vribles, nd monitoring of dverse events were routinely performed during the tretment. Adverse events were grded bsed on the NCI-CTC. guidelines. From the time of the first EA dministrtion to the ltest follow-up, ll dverse events were recorded regrdless of their relevnce to EA (Supplementry Tble S4). The tumor evlution ws bsed on the ssessment of trget nd nontrget lesions. At bseline, the end of every tretment cycles, nd every months during follow-up until disese progression, tumor mesurements were collected by computed tomogrphy, mgnetic resonnce imging, or photogrphy. The CR or PR ptients were reconfirmed fter 4 weeks. During the entire tril process, the ssessment of lesion size in ny single ptient hd to be performed using the sme method nd the sme investigtor to ensure the comprbility of the results. We dopted the best responses s effective end points during the period from cycle to cycle 4 nd clculted the survivl during follow-up. Angiogenesis-relted moleculr mrkers (svcam-, TSP-, seselectin, VEGF, bfgf, ngiopoietin-, ngiopoietin-, nd EGF) in the serum were quntittively evluted on dy,, 5, nd 7 during the first tretment cycle. We performed ELISA ssys to quntify the serum endosttin expression over time. The time points selected were dy,, 5, nd 7 of the first cycle, the fifth dy fter ech subsequent EA dministrtion, nd the lst dy of ech tretment cycle. Ptients in the EA group t Sun Yt-sen University Cncer Center were rndomly chosen to undergo biopsy of their trget sites t bseline nd weeks fter the finl injection. Intrtumorl expression of endosttin ws scored by the following four ctegories bsed on the stining intensity: none (), wek (), medium () nd strong (). We pplied histoscore (H score) for ech smple to clculte endosttin expression by multiplying the percentge of positively stined cells with ech ctegory of stining intensity. Sttisticl nlysis. The necessry smple size of 4 ptients ws clculted bsed on our hypothesis tht we would observe n increse in the objective RR from 5% in the control group to no less thn 5% in the EA group, with power of 8% to detect such n increse, provided with one-sided χ test t the 5% significnce level nd % drop-out rte. The estimted efficcy ws bsed on our previous preliminry test of smll smple in the sme popultion. We nlyzed the primry nd secondry end points with P vlue <.5 ws considered sttisticlly significnt. The Fisher s exct test or the Kruskl-Wllis H test ws used to compre the rtes between groups. The distributions of PFS nd OS were estimted with Kpln Meier nlysis nd compred with the Mntel-Cox log-rnk test. A Generl Liner Model Anlysis for Repeted Mesures ws performed to ssess the effects of EA between the subgroups over time. We ssumed tht the ddition of EA would benefit ptients on the bsis of chemotherpy, thus onesided tests were used when we clculted the smple size nd compred the response rtes in this tril of superiority test. SUPPLEMENTARY MATERIAL Figure S. Typicl cses. Figure S. Serum endosttin of the EA subgroups. Figure S. Moleculr mrkers of ngiogenesis in the serum of the EA ptients. Figure S4. Administrtion method of EA. Tble S. Reltionship of previous chemotherpy nd ptient responses. Tble S. Generl Liner Model Anlysis for Repeted Mesures of ngiogenesis mrkers. Tble S. Serious Adverse Events (SAE). Tble S4. Flow chrt of tril. ACKNOWLEDGMENTS We thnk the prticipting ptients nd stff of the study hospitls: Zhiming Li, Yuhong Li (Sun Yt-sen University Cncer Center, Gungzhou, Chin); Wenchun Zho (Tinjin Medicl University Cncer Institute nd Hospitl, Tinjin, Chin); Weimin Zhng (Generl Hospitl of Gungzhou Militry Commnd of PLA, Gungzhou, Chin); Qingqun Hu, Qibin Song (Renmin Hospitl of Wuhn University, Wuhn, Chin); Yigng Li (Yue Bei People s Hospitl, Shogun, Chin); Ping Li (West Chin Hospitl of Sichun University, Chengdu, Chin); Wencho Liu (Xi Jing Hospitl of the Fourth Militry Medicl University, Xi n, Chin); Chunhong Hu (The second Xingy Hospitl of Centrl- South University, Chngsh, Chin); Meizuo Zhong (The first Xingy Hospitl of Centrl-South University, Chngsh, Chin); Ynbing Zhu (Bethune Interntionl Pece Hospitl, H erbin, Chin); Wnki Deng (Hubei Cncer Hospitl, Wuhn, Chin). This work ws supported by the Ntionl High Technology Reserch nd Development Progrm of Chin (Project 86, No. AA8), Ntionl Mjor Scientific nd Technologicl Specil Project of Chin (No. ZX945), Ntionl Key Bsic Reserch Progrm of Chin (Project 97, No.CB5994), Gungdong Innovtive Reserch Tem Progrm (No. 958), Gungdong Provincil Science nd Technology Project (No. A85; A85), nd Gungzhou Doublle Bioproducts. This tril is registered with CliniclTrils.gov, number: NCT The uthors declre no conflict of interest. REFERENCES. Folkmn, J (6). Antingiogenesis in cncer therpy endosttin nd its mechnisms of ction. Exp Cell Res : He, GA, Xue, G, Xio, L, Wu, JX, Xu, BL, Hung, JL et l. (5). Dynmic distribution nd expression in vivo of humn endosttin gene delivered by denovirl vector. Life Sci 77: 4.. Li, L, Hung, JL, Liu, QC, Wu, PH, Liu, RY, Zeng, YX et l. (4). Endosttin gene therpy for liver cncer by recombinnt denovirus delivery. World J Gstroenterol : Ling, ZH, Wu, PH, Li, L, Xue, G, Zeng, YX nd Hung, WL (4). Inhibition of tumor growth in xenogrfted nude mice with denovirus-medited endosttin gene comprison with recombinnt endosttin protein. Chin Med J 7: vol. no. 6 jun. 4

9 The Americn Society of Gene & Cell Therpy Efficcy nd Sfety of Recombinnt Endosttin 5. Li, L, Liu, RY, Hung, JL, Liu, QC, Li, Y, Wu, PH et l. (6). Adenovirus-medited intr-tumorl delivery of the humn endosttin gene inhibits tumor growth in nsophryngel crcinom. Int J Cncer 8: Li, LX, Zhng, YL, Zhou, L, Ke, ML, Chen, JM, Fu, X et l. (). Antitumor efficcy of recombinnt denovirus encoding endosttin combined with n EB55KD-deficient denovirus in gstric cncer cells. J Trnsl Med : Adhim, Z, Lin, X, Hung, W, Morishit, N, Nkmur, T, Ysui, H et l. (). EA, n denovirus-crrying endosttin gene, drmticlly incresed the tumor drug concentrtion of metronomic chemotherpy with low-dose cispltin in xenogrft mouse model for hed nd neck squmous-cell crcinom. Cncer Gene Ther 9: Bi, RZ, Wu, Y, Liu, Q, Xie, K, Wei, YQ, Wng, YS et l. (9). Suppression of lung cncer in murine model: treted by combintion of recombinnt humn endoststin denovirus with low-dose cispltin. J Exp Clin Cncer Res 8:. 9. Zho, P, Luo, R, Wu, J, Xie, F, Li, H, Xio, X et l. (). EA, n denovirus crrying humn endosttin gene, in combintion with docetxel tretment inhibits prostte cncer growth nd metstses. J Cell Mol Med 4: Hung, BJ, Liu, RY, Hung, JL, Ling, ZH, Go, GF, Wu, JX et l. (7). Long-Term toxicity studies in Cnine of EA, n denovirl vector for humn endosttin gene. Hum Gene Ther 8: 7.. Lin, X, Hung, H, Li, S, Li, H, Li, Y, Co, Y et l. (7). A phse I clinicl tril of n denovirus-medited endosttin gene (EA) in ptients with solid tumors. Cncer Biol Ther 6: Li, HL, Li, S, Sho, JY, Lin, XB, Co, Y, Jing, WQ et l. (8). Phrmcokinetic nd phrmcodynmic study of intrtumorl injection of n denovirus encoding endosttin in ptients with dvnced tumors. Gene Ther 5: Vokes, EE, Weichselbum, RR, Lippmn, SM nd Hong, WK (99). Hed nd neck cncer. N Engl J Med 8: Brizel, DM, Albers, ME, Fisher, SR, Scher, RL, Richtsmeier, WJ, Hrs, V et l. (998). Hyperfrctionted irrdition with or without concurrent chemotherpy for loclly dvnced hed nd neck cncer. N Engl J Med 8: Pignon, JP, Bourhis, J, Domenge, C nd Designé, L (). Chemotherpy dded to locoregionl tretment for hed nd neck squmous-cell crcinom: three metnlyses of updted individul dt. MACH-NC Collbortive Group. Met-Anlysis of Chemotherpy on Hed nd Neck Cncer. Lncet 55: Hddd, RI nd Shin, DM (8). Recent dvnces in hed nd neck cncer. N Engl J Med 59: Tupchong, L, Scott, CB, Blitzer, PH, Mrcil, VA, Lowry, LD, Jcobs, JR et l. (99). Rndomized study of preopertive versus postopertive rdition therpy in dvnced hed nd neck crcinom: long-term follow-up of RTOG study 7-. Int J Rdit Oncol Biol Phys : Gibson, MK, Li, Y, Murphy, B, Hussin, MH, DeConti, RC, Ensley, J et l.; Estern Coopertive Oncology Group. (5). Rndomized phse III evlution of cispltin plus fluorourcil versus cispltin plus pclitxel in dvnced hed nd neck cncer (E95): n intergroup tril of the Estern Coopertive Oncology Group. J Clin Oncol : McCrthy, JS, Tnnock, IF, Degendorfer, P, Pnzrell, T, Furln, M nd Siu, LL (). A Phse II tril of docetxel nd cispltin in ptients with recurrent or metsttic nsophryngel crcinom. Orl Oncol 8: Eder, JP Jr, Supko, JG, Clrk, JW, Puchlski, TA, Grci-Crbonero, R, Ryn, DP et l. (). Phse I clinicl tril of recombinnt humn endosttin dministered s short intrvenous infusion repeted dily. J Clin Oncol : Hnsm, AH, Broxtermn, HJ, vn der Horst, I, Yun, Y, Boven, E, Giccone, G et l. (5). Recombinnt humn endosttin dministered s 8-dy continuous intrvenous infusion, followed by dily subcutneous injections: phse I nd phrmcokinetic study in ptients with dvnced cncer. Ann Oncol 6: Kulke, MH, Bergslnd, EK, Ryn, DP, Enzinger, PC, Lynch, TJ, Zhu, AX et l. (6). Phse II study of recombinnt humn endosttin in ptients with dvnced neuroendocrine tumors. J Clin Oncol 4: Thoms, JP, Arzoomnin, RZ, Alberti, D, Mrnoch, R, Lee, F, Friedl, A et l. (). Phse I phrmcokinetic nd phrmcodynmic study of recombinnt humn endosttin in ptients with dvnced solid tumors. J Clin Oncol :. 4. Wng, J, Sun, Y, Liu, Y, Yu, Q, Zhng, Y, Li, K et l. (5). [Results of rndomized, multicenter, double-blind phse III tril of rh-endosttin (YH-6) in tretment of dvnced non-smll cell lung cncer ptients]. Zhongguo Fei Ai Z Zhi 8: Ling, Z, Wu, J, Hung, J, Tn, W, Ke, M, Liu, R et l. (7). Bioctivity nd stbility nlysis of endosttin purified from fermenttion superntnt of 9 cells trnsfected with Ad/rhEndo. Protein Expr Purif 56: Gering, AJ nd Newmn, W (99). Circulting dhesion molecules in disese. Immunol Tody 4: Knzw, H (7). VEGF, ngiopoietin- nd - in bronchil sthm: new moleculr trgets in irwy ngiogenesis nd microvsculr remodeling. Recent Pt Inflmm Allergy Drug Discov : Kzerounin, S, Yee, KO nd Lwler, J (8). Thrombospondins in cncer. Cell Mol Life Sci 65: Li, Y, Li, LJ, Zhng, ST, Wng, LJ, Zhng, Z, Go, N et l. (9). In vitro nd clinicl studies of gene therpy with recombinnt humn denovirus-p5 injection for orl leukoplki. Clin Cncer Res 5: Pn, JJ, Zhng, SW, Chen, CB, Xio, SW, Sun, Y, Liu, CQ et l. (9). Effect of recombinnt denovirus-p5 combined with rdiotherpy on long-term prognosis of dvnced nsophryngel crcinom. J Clin Oncol 7: Dummer, R, Eichmüller, S, Gellrich, S, Assf, C, Dreno, B, Schiller, M et l. (). Phse II clinicl tril of intrtumorl ppliction of TG4 (denovirus-interferongmm) in ptients with dvnced cutneous T-cell lymphoms nd multilesionl cutneous B-cell lymphoms. Mol Ther 8: Peng, Z (5). Current sttus of gendicine in Chin: recombinnt humn Ad-p5 gent for tretment of cncers. Hum Gene Ther 6: 6 7. Moleculr Therpy vol. no. 6 jun. 4 9