Predictive and Prognostic Clinical Variables in Cancer Patients Treated With Adenoviral Oncolytic Immunotherapy

Transcription

1 The Americn Society of Gene & Cell Therpy originl rticle Predictive nd Prognostic Clinicl Vribles in Cncer Ptients Treted With Adenovirl Oncolytic Immunotherpy Kristin Tiple 1, Ilkk Liiknen 1, Anniin Koski 1, Rit Heisknen 2, Ann Knerv 1,3, Otto Hemminki 1, Minn Oksnen 1, Susnn Grönberg-Vähä-Koskel 1, Kri Hemminki 4,5, Timo Joensuu 6 nd Akseli Hemminki 1,7,8 1 Cncer Gene Therpy Group, University of Helsinki, Fculty of Medicine, Helsinki, Finlnd; 2 Trgovx ASA, Helsinki, Finlnd; 3 Deprtment of Obstetrics nd Gynecology, HUCH, Helsinki, Finlnd; 4 Division of Moleculr Genetic Epidemiology, Germn Cncer Reserch Centre (DKFZ), Heidelberg, Germny; 5 Center for Primry Helth Cre Reserch, Lund University, Mlmö, Sweden; 6 Docrtes Cncer Center, Helsinki, Finlnd; 7 Helsinki University Hospitl Comprehensive Cncer Center, Finlnd; 8 TILT Biotherpeutics Ltd., Helsinki, Finlnd The development of oncolytic viruses hs recently mde gret progress towrds being vilble to cncer ptients. With the brekthrough into clinics, it is crucil to nlyze the existing clinicl experience nd use it s bsis for tretment improvements. Here, we report clinicl dt from 29 ptients treted with oncolytic denovirus. Using clinicl vribles nd tretment chrcteristics, we constructed sttisticl models with regrd to tretment response nd overll survivl (OS). Additionlly, we investigted effects of neutrlizing ntibodies, tumor burden, nd peripherl blood leucocyte counts on these outcomes. We found the bsence of liver metstses to correlte with n improved rte of disese control (P =.21). In multivrite evlution, ptients treted with viruses coding for immunostimultory grnulocyte mcrophge colony-stimulting fctor were linked to better prognosis (hzrd rtio (HR).378, P <.1), s well s women with ny cncer type (HR.694, P =.17). In multivrite nlysis for imging response, ptients treted vi intrperitonel injection were more likely to chieve disese control (odds rtio (OR) 3.246, P =.27). Ptients with low neutrophil-to-lymphocyte rtio before tretment hd significntly longer OS (P <.1). These findings could explin some of the vrition seen in tretment outcomes fter virotherpy. Furthermore, the results offer hypotheses for tretment optimiztion nd ptient selection in oncolytic denovirus immunotherpy. Received 12 Jnury 216; ccepted 28 Mrch 216; dvnce online publiction 3 My 216. doi:1.138/mt INTRODUCTION Oncolytic viruses re one modlity of cncer immunotherpy, which hs become n incresingly importnt prt of tretment options for cncer in recent yers. 1,2 After yers of development, first oncolytic viruses re finlly entering clinicl use due to recent recommendtions nd pprovls in Europen Union nd United Sttes. 3,4 In ddition to Tlimogene Lherprepvec (T-VEC; Imlygic ), grnulocyte mcrophge colony-stimulting fctor (GMCSF) coding oncolytic herpes simplex virus, numerous other viruses is being tested in clinicl trils. 5,6 The dt thus fr re promising with good sfety cross the spectrum of viruses used nd with signs of tretment efficcy linked to severl specific products. 6,7 However, our current knowledge on fctors tht influence tretment outcomes of oncolytic viruses is still quite limited. 8 Clinicl trils hve indicted smll number of prmeters tht re ssocited with better or worse tretment responses. In the phse 3 tril of T-VEC for dvnced melnom, disese stge nd previous tretments hd cler effect on durble response rte, which ws the primry objective of the tril. 9 Ptients with metsttic stge IVM1b or IVM1c disese (metstses in lungs or other orgns) received little benefit from T-VEC, s opposed to ptients stged s IVM1, IIIC or IIIB (only lymph node or cutneous metstses t the primry tumor or distnt loctions for stge III nd IV, respectively). Tretment benefits were lso smller in ptients receiving T-VEC s secondline or greter therpy. In phse 2 tril of oncolytic vccini virus JX-594 lso coding for GMCSF for liver cncer, higher tretment dose ws found to correlte with significntly longer overll survivl (OS). 1 Ptients with multiple tumors were found to hve poor survivl compred to ptients with single tumor. On the other hnd, presence or bsence of neutrlizing ntibodies before tretment ws not ssocited with length of survivl. Oncolytic denoviruses hve been used in clinicl trils nd ptient ccess progrms. Previous reports hve indicted tht preexisting systemic immune ctivtion could be linked to inferior responses to oncolytic immunotherpy. 11,12 In cse control estimtion of tretment efficcy, denovirus-treted ptients with good performnce score hd improved survivl when compred to mtched controls. 13 In the sme report, ovrin cncer ptients seemed to receive significnt benefit from tretment, with lmost qudrupled medin survivl versus controls not treted with denovirus. The im of this retrospective clinicl epidemiologicl nlysis is to identify fctors influencing outcomes of denovirus-bsed immunotherpy. We mesured pre nd posttretment neutrlizing ntibody titers from ptients receiving their first oncolytic denovirus tretment. We lso evluted the initil tumor burden of the The uthors I Liiknen nd A Koski contributed eqully to this work. Correspondence: A Hemminki, University of Helsinki, P.O. Box 21, 14 Helsinki, Finlnd. E-mil: kseli.hemminki@helsinki.fi Moleculr Therpy vol. 24 no. 7, jul

2 Predictive nd Prognostic Fctors in Adenovirl Therpy The Americn Society of Gene & Cell Therpy ptients by mesuring primry tumors nd metstses in different orgns. Different clinicl prmeters from ll of the 29 ptients treted in Advnced Therpy Access Progrm were incorported into Cox regression model to estimte prognostic fctors significnt in the context of oncolytic denovirus therpy. In ddition, logistic regression ws used to find independent fctors predictive of disese control. Finlly, peripherl white blood cell counts before nd fter tretment were collected. RESULTS Absence of neutrlizing ntivirl ntibodies t bseline correltes with longer survivl fter tretment with oncolytic denovirus Neutrlizing ntibodies were mesured in 17 ptients, who were then for sttisticl nlysis divided into two ctegories bsed on the presence of neutrlizing ntibodies. 119 ptients did hve neutrlizing ntibodies (ny titer) before tretment, while 51 ptients did not (titer = ). Ptients without neutrlizing ntibodies hd significntly longer OS compred to ptients with preexisting ntibodies, medins 239 nd 122 dys, respectively (P =.22) (Figure 1). This finding ws lso confirmed by multivrite Cox regression model using ptient chrcteristics s confounding fctors (P =.33) (Figure 1). However, when compring imging responses insted of OS, no significnt difference ws seen (Figure 1b). Ptients with bseline neutrlizing ntibodies were further divided into low nd high groups. There ws no difference in medin survivl between these two groups (12.5 nd 126 dys), lthough men survivl ws longer in the ltter group (219 nd 318 dys, not significnt) (see Supplementry Figure S1). After tretment, neutrlizing ntibodies incresed in ptients s expected. 14 Interestingly, increse ws not observed in five ptients (see Supplementry Tble S1). These ptients hd ll different cncers (rectum, hed nd neck, ovrin, Wilms tumor, nd srcom) nd received different virus tretments. Imging responses of these ptients vried (1 prtil response, 1 minor response, 1 stble disese, nd 1 progressive disese), but the ptients tended to hve longer thn verge OS (17 89 dys). Presence of liver metstses is ssocited with worse response rte Tumor lod ws ssessed in 154 ptients. As mesure of initil tumor lod, size of the lrgest tumor lesion ws mesured. Ptients 1 Antibodies present t bseline b 1 8 No ntibodies t bseline 6 4 Percent of ptients , 2,25 2,5 c Age Sex (femle/mle) Tumor type (versus Pnc/chol/HCC) WHO (low/high) Bseline neutrlizing ntibodies (no/yes) CRC/gstric Melnom Lung Gynecologicl Other P vlue <.1.33 Antibodies t bseline No ntibodies t bseline Progressive disese Disese control HR for cncer mortlity (n = 167) HR (95% CI) 1.6 ( ).637 ( ).72 ( ).594 ( ).565 ( ).555 (.32.96).44 ( ).444 ( ).689 (.49.97) Figure 1 Overll survivl (OS) nd disese control rte in ptients with or without bseline neutrlizing ntivirl ntibodies. Ptients with zero neutrlizing ntibody titer in pretretment smples were considered ntibody-negtive. () Ptients with no neutrlizing ntibodies hd longer OS (medin OS 239) compred to ptients with preexisting neutrlizing ntibodies (medin OS 122, P =.22). (b) Overll imging disese control rte in ptients with vilble neutrlizing ntibody smples ws 5.4% (n = 115). No significnt difference between ptients with or without preexisting ntibodies ws observed. (c) Multivrite Cox regression nlysis for prognostic vlue of bseline neutrlizing ntivirl ntibodies. CRC/gstric, colorectl crcinom nd gstric cncer; gynecologicl, brest cncer, ovrin cncer, endometril cncer nd cervicl cncer; HR, hzrd rtio; pnc/ chol/hcc, pncretic cncer, cholngiocrcinom nd heptocellulr crcinom vol. 24 no. 7 jul. 216

3 The Americn Society of Gene & Cell Therpy Predictive nd Prognostic Fctors in Adenovirl Therpy were divided into either hving or not hving lrge bulky ccording to size of the lrgest tumor. In ddition, the presence of metstses ws recorded for lymph nodes, liver, lungs, bones, peritonel cvity, nd other sites. Bsed on these dt, tumor lod score ws ssigned to ech ptient to indicte disese burden. In survivl nlyses, ptients with high tumor lod score hd significntly shorter OS (P =.3) (Figure 2). Bulkiness of the lrgest tumor or presence of metstses did not correlte significntly with OS, lthough some trend for ssocition ws seen between liver metstses nd worse survivl (P =.65) (Figure 2b d). In predictive comprisons, ptients with high nd low tumor lod score hd no difference in response rtes (Figure 3). However, the bsence of liver metstses indicted significntly higher probbility for imging response (P =.21) (Figure 3c). Interestingly, peritonel metstses seemed to be ssocited, lbeit not significntly, with better response rtes (Figure 3d). Tumor type nd virus trnsgene re independent prognostic fctors in ptients receiving oncolytic denovirus Clinicl vribles from 29 ptients (see Supplementry Tble S2) were used to construct Cox proportionl hzrds model. These included ptient ge, gender, tumor type, WHO performnce sttus, 15 virl trnsgene, cpsid nd promoter, dministrtion route, concomitnt tretments, nd prior immunotherpy (Tble 1). OS fter the first virus tretment ws deemed s dependent vrible. The Cox model provided severl prognostic fctors for ptients receiving oncolytic denovirus tretment. Ptients with different tumor types hd differences in OS (P =.6), nd since this is known in oncology, it cn be considered n internl test of vlidity of the model. Ptients with pncretic/biliry trct/ heptocellulr cncer, melnom, nd colorectl/gstric cncer hd worse survivl compred to ptients with lung cncer nd gynecologicl cncers. More interestingly, other significnt fctors in the model included gender, WHO performnce sttus, nd virus switch (Figure 4). Of prticulr interest, lso the use of trnsgene in the tretment virus ws ssocited with significntly (P =.1) better OS (Figure 4d). Administrtion route is n independent predictive fctor for disese control The sme clinicl vribles used in the Cox model were included in predictive logistic regression model (Tble 1) nd 175 ptients with vilble imging response dt were included in the nlysis. The 1 b 1 High tumor lod score Low tumor lod score Bulky tumor Not bulky tumor , 2,25 2,5 5 1, 2,25 2,5 c 1 Liver metstses No liver metstses d 1 Peritonel metstses No peritonel metstses , 2,25 2,5 5 1, 2,25 2,5 Figure 2 Overll survivl (OS) in different tumor lod groups. ( d) OS ws compred between ptients with high or low tumor lod score nd presence or bsence of bulky disese, liver metstses, nd peritonel metstses. Ptients with low tumor lod score hd significntly longer OS (P =.3), while no significnt difference ws found between bulky disese (P =.26), peritonel (P =.272) or liver metstses (P =.65) groups. Moleculr Therpy vol. 24 no. 7 jul

4 Predictive nd Prognostic Fctors in Adenovirl Therpy The Americn Society of Gene & Cell Therpy 1 1 b 8 8 Progressive disese Percent of ptients 6 4 Percent of ptients 6 4 Disese control 2 2 High tumor lod score Low tumor lod score Bulky tumor Not bulky tumor c 1 d 1 8 * 8 Progressive disese Percent of ptients 6 4 Percent of ptients 6 4 Disese control 2 2 Liver metstses No liver metstses Peritonel metstses No peritonel metstses Figure 3 Imging response rtes in different tumor lod groups. Asterisk indictes P <.5. Tumor lod nd imging response were evluble from 148 ptients. (,b) Ptients with high or low combined tumor lod score or presence or bsence of bulky tumor hd no significnt differences in disese control rtes. (c) Absence of liver metstses before tretment ws ssocited with higher response rte (P =.21). (d) Ptients with peritonel metstses hd higher proportion of imging responses, but the difference ws not considered significnt (P =.94). only clinicl vribles reching significnt odds rtio (OR) for disese control were WHO performnce sttus (P =.49) nd dministrtion route of virus tretments (P =.32). Ptients who received most of the tretments vi intrperitonel or intrpleurl injection hd on verge over three times higher probbility for disese control (OR = 3.246, P =.27), compred to ptients treted mostly or exclusively with intrtumorl injections. Ptients treted with intrvenous virus injection lso hd high OR, which filed to rech sttisticl significnce (P =.114) due to smll smple size (n = 8). Differences in white blood cell counts before nd fter tretment ssocite with OS nd tretment response Although oncolytic viruses were initilly developed s trgeted tumor-lysing gents, recent dt from ptients suggest tht n importnt proportion of the benefit ssocited with tretment might be ttributble to lymphocyte ctivtion ginst tumor In contrst, some innte immune cells my dmpen this immunotherpeutic efficcy, while some re lso beneficil. 12 Therefore, we ssessed white blood cell counts in ptients before nd fter therpy. Bseline white blood cell counts were vilble for 112 ptients. OS ws compred between ptients with below or bove medin leucocyte, neutrophil nd lymphocyte counts, nd neutrophil-to-lymphocyte rtio (NLR). Higher bseline leucocyte nd neutrophil counts correlted with shorter OS (P =.8 nd P =.2, respectively) (Figure 5,b). There ws lso trend, lthough not significnt, for ssocition of lymphocyte counts with longer OS (Figure 5c). Ptients with low pretretment NLR hd significntly longer OS (P <.1), compred to ptients with high NLR (Figure 5d). Additionlly, this difference ws confirmed by multivrite Cox model using ptient chrcteristics s confounding fctors (P <.1) (see Supplementry Tble S3). Differences in tretment responses between the groups were not significnt (see Supplementry Figure S2). Cell counts showed cler chnges in posttretment smples (see Supplementry Figure S3). In generl, t erly time points, lymphocytes decresed following tretment while leucocytes incresed. We compred reltive cell count chnges between ptients who showed imging response (disese control or better) fter tretment nd ptients who hd progressive disese. The mgnitude of the chnges ws similr, lthough temporl ptterns vried between the groups (see Supplementry Figure S3). We compred disese control rtes in more detil between ptients who hd increse or decrese in leucocyte nd neutrophil counts on dy 1 posttretment (see Supplementry Figure S4). Ptients with increses in leucocytes nd neutrophils hd higher response rtes, lbeit the differences were not significnt (P =.55 nd P =.116, respectively). DISCUSSION In prllel with other forms of immunotherpy, oncolytic virotherpy hs been ssocited with limited response rte, but mny of the responses tht re seen seem to be durble. 9,2,21 Moreover, vol. 24 no. 7 jul. 216

5 The Americn Society of Gene & Cell Therpy Predictive nd Prognostic Fctors in Adenovirl Therpy Tble 1 Results from Cox proportionl hzrds regression model for overll survivl nd logistic regression for imging disese control HR for cncer mortlity (n = 29) OR for disese control (n = 175) P vlue HR (95% CI) P vlue OR (95% CI) Age ( ) ( ) Sex (femle/mle) ( ) ( ) Tumor type (versus pnc/chol/hcc b ) CRC/gstric c ( ) ( ) Melnom ( ) ( ) Lung ( ) ( ) Gynecologicl d.6.59 ( ) ( ) Other.1.51 ( ) ( ) WHO (low/high) <.1.41 ( ) ( ) Trnsgene.1.68 (versus no trnsgene) CD4L ( ) ( ) GM-CSF < ( ) ( ) Virus cpsid (versus Ad5) Ad ( ) ( ) Ad5/ ( ) ( ) Promoter (tumor specific/no) ( ) ( ) Virus switch (virus switched/not) e < ( ) ( ) Averge dose (high/low) ( ) ( ) Administrtion route f (versus most i.t.) Most i.p ( ) ( ) Only i.v ( ) ( ) Concomitnt tretment g (versus no concomitnt trt) CP ( ) ( ) CP + TMZ ( ) ( ) Concomitnt trt switch (switched/not) e ( ) ( ) Prior immunotherpy (yes/no) ( ) ( ) HR, hzrd rtio; OR, odds rtio. b Pncretic cncer, cholngiocrcinom, nd heptocellulr crcinom. c Colorectl crcinom nd gstric cncer. d Brest cncer, ovrin cncer, endometril cncer, nd cervicl cncer. e Switched lter to different virus nd/or concomitnt tretment thn the one used in first tretment. f i.t., intrtumorl; i.p., intrperitonel; i.v., intrvenous. g CP, cyclophosphmide; TMZ, temozolomide. gin in ccord with other potent immunotherpies, survivl could be ffected more thn tumor size. 9,2,21 Thus, with existing tretment options, it is importnt to develop clinicl criteri to select the right ptients for therpy, 17 s well s to find biomrkers 11,22 to determine which ptients re benefiting from the therpy. Clinicl trils with oncolytic viruses hve reported smll number of potentil cndidtes for predictive nd prognostic fctors. 9,1 Thus, the work towrds estblishing cler principles for evlution of likelihood of tretment benefit from oncolytic virotherpy hs only begun. We hve reported here the ssocitions of severl clinicl vribles nd lbortory tests on tretment outcomes of ptients receiving oncolytic immunotherpy with denovirus. We found the presence of ntivirl neutrlizing ntibodies before tretment s well s high overll tumor lod to correlte significntly with shorter survivl. Additionlly, high pretretment totl leucocyte counts, neutrophil counts, nd, especilly, NLRs were linked to worse survivl. In prognostic regression model for OS, certin tumor types hd significntly smller hzrds rtios, s well s tretments with viruses tht were rmed with GMCSF. A predictive model for imging response indicted higher OR for disese control for ptients who were treted with intrperitonel virus injection. Bseline neutrlizing ntibodies hd significnt effect on OS. This finding is in contrst with previous report from phse 2 tril of oncolytic vccini virus, where neutrlizing ntibodies hd no effect on survivl. 1 The smller number of ptients in the vccini study (n = 3) could ffect conclusions, but if there is discrepncy, it might reflect difference in the biology of the two viruses. Vccini is thought to be ble to evde the immune system to some degree, 23 nd this could lso trnslte to lower sensitivity to ntibody-dependent inctivtion, compred to denovirus which lcks cell-derived envelope nd just hs protein cpsid. However, it is key to keep in mind tht responses nd/or long survivl were seen cross ntibody ctegories. Thus, while ntibodies re n interesting topic for scientific discussion, they re not useful s biomrker for ptient selection. This becomes pprent when considering tht in lmost ll ptients ntibodies increse Moleculr Therpy vol. 24 no. 7 jul

6 Predictive nd Prognostic Fctors in Adenovirl Therpy The Americn Society of Gene & Cell Therpy 1..8 Tumor_type Pnc/HCC/cho CRC/gstric Melnom Lung Gynecologicl Other b 1..8 Sex Mle Femle Cumultive survivl.6.4 Cumultive survivl c , 1,2 WHO_low WHO >1 WHO >2 d , 1,2 Trnsgene_1 None CD4L GMCSF.8.8 Cumultive survivl.6.4 Cumultive survivl , 1, , 1,2 Figure 4 Hzrd functions obtined from the Cox proportionl hzrds model. () Tumor type hd significnt effect on overll hzrd for tumor mortlity (P =.6). Hzrd rtios were smller for gynecologicl cncers, lung cncer nd other cncer types, compred to melnom, pncretic/ heptocellulr/biliry trct cncers nd colorectl/gstric cncers. (b-c) WHO performnce sttus nd gender were significnt fctors in Cox model (P <.1 nd P =.17, respectively). (d) Ptients who received virus coding for immunostimultory trnsgene s first tretment hd smller hzrd rtios compred to other ptients (P =.1). fter tretment, regrdless of tretment benefits, which my lst even yers in the presence of high ntibody titers. Moreover, virus circultion is seen often for weeks or even months despite ntibody titers. 18,19,24 Accordingly, there ws no difference in response rtes in ptients with or without ntibodies t bseline. Further clrity would result from evlution of neutrlizing ntibodies nd survivl in n even lrger ptient popultion. While neutrlizing ntibodies cn block trnsduction, they cn lso be conducive for immune responses through complement ctivtion or ntibody-dependent cellulr cytotoxicity. Thus, in some ptients ntibodies cn be beneficil while being hrmful in others. One specultion is tht the former hve smller number of tumors which cn be injected intrtumorlly while intrvsculr dissemintion of virus is more importnt in the ltter. 25 However, gin the picture is probbly more complicted, since viruses my be ble to escpe neutrliztion by hiding inside cells, or they my simply overwhelm the cpcity of the body to opsonize, given tht huge mounts of virus re being produced by tumors for extended periods. 25 There is one powerful exmple of the bility of oncolytic serotype 5/3 chimeric denovirus to rech distnt tumors despite ntibodies. Ptient N6 ws treted with n intrvenous injection nd the tretment virus could be grown out of brin metstses. 25 His bseline nti-denovirus 5/3 ntibody titer ws 16 (=medium), reported here for the first time. Thus, virus ws ble to rech distnt tumors through the blood strem, replicte, nd new infectious virions were produced despite neutrlizing ntibodies present in blood. It is tntlizing tht in our ptient series, if we focus on ptients with neutrlizing ntibodies present t bseline, those ptients who hd high titers seemed to do better thn those with medium titers (men survivl ws 94 dys longer), which would be comptible with ntibody-dependent direction of immune rections s described bove vol. 24 no. 7 jul. 216

7 The Americn Society of Gene & Cell Therpy Predictive nd Prognostic Fctors in Adenovirl Therpy 1 b 1 8 High bseline leucocytes Low bseline leucocytes 8 High bseline neutrophils Low bseline neutrophils , 2,25 2,5 5 1, 2,25 2,5 c 1 d 1 8 High bseline lymphocytes Low bseline lymphocytes 8 High bseline NLR Low bseline NLR , 2,25 2,5 5 1, 2,25 2,5 Figure 5 Overll survivl (OS) in ptients with different bseline white blood cell counts. Bseline cell counts were mesured 1 dy before or on the sme dy of the first tretment. (,b) Leucocyte nd neutrophil counts tht were below medin before tretment were ssocited with significntly longer survivl (P =.8 nd P =.2, respectively). (c) No significnt difference in OS between different bseline lymphocyte count groups ws observed (P =.119). (d) Ptients with lower thn medin neutrophil-to-lymphocyte rtio (NLR) hd significntly longer OS compred to ptients with high NLR (P <.1). Five ptients in this study did not develop increses in neutrlizing ntibodies fter tretments, lthough ll hd preexisting neutrlizing ntibodies t bseline. It cn be postulted tht one explntion for this might be poor virus repliction, but ll five ptients hd virus present in their blood s mesured by quntittive polymerse chin rection fter tretment. 24,26,27 This suggests tht, for some reson, the ptients were not ble to produce dditionl ntibodies ginst the virus, perhps becuse their B cells were depleted by previous chemotherpies nd rdition. Alterntively, immunosuppression generted by the tumor could impct ntibody responses nd obviously these scenrios re not mutully exclusive. Previously, immune suppression hs been proposed linked to worse responses with oncolytic virotherpy, 8,12,28 but these five ptients hd good performnce scores, tretment resulted in reltively good survivl nd some hd lso mesurble imging responses. Thus, there ws nothing to suggest tht they would hve been prticulrly immunosuppressed on systemic level. Nevertheless, if the immunosuppression would preferentilly ffect ntibody formtion in B cells, it might result in enhnced denovirus repliction nd consequently stronger direct oncolytic effect on the tumor. 29 Nonetheless, this phenomenon is seemingly rre, since it ws seen in only five ptients, nd thus difficult to study more extensively. Overll, we think tht some ptients my benefit from pure oncolysis, while others from pure oncolytic immunotherpy (where the bility of the virus to lyse tumor cells hs little relevnce), lthough the mjority fll in between these extremes, keeping in mind tht oncolysis is n incredibly powerful wy to induce ntitumor immunity. 3,31 All tretments described herein utilized repliction competent denoviruses with oncolytic ctivity. High overll tumor lod ws ssocited with shorter survivl, s expected, becuse it could indicte more dvnced disese where the ptient is closer to deth. However, no significnt correltion ws seen with survivl nd disese bulkiness or presence of liver or peritonel metstses. It ws interesting tht tretment response rtes were not correlted with tumor lod, but there were significntly less responses in ptients with liver metstses. Thus, two conclusions cn be drwn: (i) ptients my benefit regrdless of tumor size, (ii) the orgn where metstses re present my be criticl, probbly becuse of immunologicl resons. The liver nd peritonel cvity represent opposite ends of the spectrum when it Moleculr Therpy vol. 24 no. 7 jul

8 Predictive nd Prognostic Fctors in Adenovirl Therpy The Americn Society of Gene & Cell Therpy comes to microenvironments fvorble to immune responses s will be discussed. Our dt re in line with the phse 3 T-VEC tril, 9 where melnom ptients hving deep viscerl metstses (liver being the most common such orgn) were not likely to benefit from tretment. There re two resons why liver metstses might be poor trgets for oncolytic immunotherpy: (i) liver is probbly one of the most immunosuppressive of ll humn orgns, 32 nd (ii) the liver is n importnt site of virus clernce. 8 With regrd to the ltter, the liver might ct s locl sink for virions being produced by the tumor. Alterntively, the opposite could be hypothesized. Metstses might interfere with the norml processing of virl prticles by heptic Kupffer cells, thus hindering the development of ntivirl immunity. This might then inhibit immunostimultory effects of viruses t the tumor site, in contrst to the lytic effect discussed erlier. For these specultions, it should be noted tht no ptients with cler liver dysfunction were ccepted in the tretment progrm nd thus no ptient hd extensive liver dmge. In the Cox regression model, low WHO performnce score nd femle gender were significntly ssocited with better OS. Effect of low performnce score is evident, s it is linked to less dvnced disese, nd these ptients might hve lived longer thn high WHO ptients, even without ny tretment. However, it could be relevnt tht performnce score is lso linked to immune competence. Mny forms of immunotherpy work better in ptients in better condition, probbly becuse when the tumor ffects the condition of the ptient more, there is lso more immunosuppression. The suggestion tht women benefit more from oncolytic immunotherpy is intriguing but in line with previous specultions. Lower survivl rtes for mles hve been described erlier for severl cncer types, nd vrious fctors, such s differences in led time nd comorbidity, hve been proposed to be underlying cuses for this phenomenon. 33 Thus, if men hd more dvnced disese, it could men tht there ws more immunosuppression. However, this my not be the full explntion since women hve higher prevlence of utoimmune diseses, 34 which is interesting in the context of immunotherpy. This could potentilly indicte tht women hve more ctive immune system, which in turn could men higher responsiveness to immunotherpeutic interventions. Tumor types for mle nd femle ptients were prtly different, but this effect ws tken into ccount s confounding fctor in the multivrite nlysis. Tumor types hd different hzrd rtios, which for some prt reflect the typicl courses of these diseses. However, different immunogenicity 35 nd stroml composition of the tumors 36 could lso directly ffect the efficcy of virotherpy. In contrst to previous findings with oncolytic vccini virus, 1 we found no significnt correltions between tretment dose nd OS, which is logicl since denovirus replictes exponentilly, nd the input dose should, thus, hve limited relevnce. Interestingly, ptients receiving GM-CSF coding virus s first-line oncolytic therpy (168/29 ptients, 58% totl) hd significntly better OS. This supports the widely ccepted principle of rming viruses with immonomodultory cytokines for enhnced efficcy. 17,37 Adenoviruses coding for GM-CSF hve been previously shown to induce ntitumor T-cell responses, 16,18 which is likely to contribute to the improved outcomes seen. In logistic regression nlysis predictive of imging results, low (=better) WHO performnce scores nd the intrperitonel dministrtion route were significntly correlted with higher OR for disese control. High OR in low WHO score ptients could indicte tht ptients with erlier stge disese respond more frequently to denovirus therpy thn ptients with more dvnced cncer. As lredy mentioned, probbly the most importnt mechnism by which tumors could escpe the effects of denovirus is immunosuppression, which is more common in dvnced tumors. 38,39 Higher OR in intrperitonelly treted ptients compred to intrtumorlly treted hs two importnt implictions for oncolytic immunotherpy. First, locoregionl delivery my be n optiml scenrio becuse much of the virus ends up in the vicinity of tumors. When virus replictes in metstses, e.g., in the liver, mny of the dughter virions probbly enter the blood strem nd re lost to nontrget orgns. In the peritoneum, newly replicted virus my be ble to gin disseminte loclly. Importntly, peritonel tumor msses re often nonbulky, presenting s crcinomtosis, resulting in fvorble surfce re to volume rtio which could be useful for both virl trnsduction nd the recruitment nd ctivity of lymphocytes. Second, the peritonel cvity cn be seen s one lrge immune orgn, 4,41 nd thus intrperitonel tretment could result in more effective ctivtion of ntitumor immunity, e.g., by ctivting the infiltrting NK cell nd T-cell subsets. 42,43 Further, in contrst to blood, which is the usul loction for circulting neutrlizing ntibodies, the presence of ntibodies in the peritoneum depends on the ccumultion of scites nd if it ws drined prior to the intrperitonel virus injection. 44 Tumor size ssessments re difficult in oncolytic immunotherpy, becuse of the tremendous mount of inflmmtion cused by virus repliction, nd subsequent swelling of tumors. 45 Nevertheless, in our ptient series, the ssocition of intrperitonel disese nd tretment benefits is supported by both imging dt nd OS dt. Of course, mny ptients with intrperitonel disese nd receiving locoregionl virus injection re in fct ovrin cncer ptients, since peritonel crcinomtosis is their typicl clinicl sitution. Thus, tumor type-specific resons could hve some impct on these dt. Bseline leucocyte counts could offer interesting clues bout the immune system before tretment. Neutrophil counts nd NLRs hve both been previously found to be prognostic in severl cncers. 46,47 We lso sw this in our dt, nd especilly high NLR ws remrkbly ssocited with worse OS. We hve previously reported evidence of link between poor prognosis nd bseline ctivtion of innte immunity. 12 The ssocition of high NLR nd shorter survivl is well in ccord with this dt. To our knowledge, this is the first study demonstrting the prognostic potentil of NLR in the context of oncolytic virotherpy, nd this could be vrible with possible prcticl utility. However, the exct phenotype of the neutrophils 48 nd existence of tumor infiltrting neutrophils, possibly in comprison to tumor infiltrting lymphocytes, should be evluted in future studies to better understnd the role of neutrophils in this setting. In summry, we hve investigted the role of neutrlizing ntibodies, tumor lod, white blood cell counts, nd severl other vol. 24 no. 7 jul. 216

9 The Americn Society of Gene & Cell Therpy Predictive nd Prognostic Fctors in Adenovirl Therpy clinicl fctors in the context of OS nd tretment response in comprehensive cohort of ptients treted with oncolytic denoviruses. In prognostic nlyses, the presence of neutrlizing ntibodies nd high neutrophil or NLR vlues before tretment were indictive of poor survivl. Ptients treted with GMCSF coding viruses were more likely to survive longer compred to viruses not coding for immunostimultory cytokines. Predictive nlyses suggested tht performnce score nd the loction of metstses, especilly liver versus peritoneum, were significntly ssocited with tretment response. These results hve implictions for designing tretments with oncolytic denovirus. Additionlly, the findings offer bsis for further development of tools for clinicl decision-mking in the context of oncolytic virotherpy. However, to be trnslted to prctice on the field of personlized oncology, these concepts require prospective investigtion in clinicl trils. MATERIALS AND METHODS Ptients. All ptients reported here were treted with oncolytic denovirus in the Advnced Therpy Access Progrm, which ws personlized therpy progrm ongoing Before tretment in Advnced Therpy Access Progrm, ptients hd solid tumors tht were refrctory to stndrd tretments nd no mjor orgn dysfunctions were present. Other exclusion criteri were s previously reported. 5 A written informed consent ws received from ll of the ptients prticipting in the progrm. Anlysis of the dt reported here ws pproved by the Helsinki University Centrl Hospitl Opertive Ethics Committee (HUS 62/13/3/2/213). Oncolytic viruses nd tretments. Viruses tht were used in the tretments hve been previously published. 14,18,19,24,26,37,51 53 All of the viruses, such s Ad5-d24-GMCSF, re bsed on the serotype 5 denovirus, excluding Ad3- htert-e1, which is bsed on the serotype 3 denovirus. Ad5-d24-RGD, Ad5-RGD-d24-GMCSF, nd ICOVIR-7 hve RGD modifiction in the fiber region. Some viruses, such s Ad5/3-d24-GMCSF, Ad5/3-Cox2L-d24, nd Ad5/3-E2F-d24-GMCSF hve chimeric 5/3 cpsid, where the Ad5 knob is replced by the serotype 3 knob. Additionlly, severl viruses re rmed with immunostimultory trnsgene GMCSF, wheres Ad5/3- htert-cd4l codes for CD4 lignd nd Ad5/3-d24-hNIS expresses sodium iodide symporter protein. Viruses were mde tumor selective by introducing 24-bp deletion in the retinoblstom binding site of E1A nd/or by inserting tumor-specific promoter, such s E2F, htert or Cox2L. Viruses were dministered intrtumorlly, using ultrsound or CT guidnce when needed, or by intrperitonel/intrpleurl or intrvenous injection. If no contrindictions were present, ptients received lso low-dose cyclophosphmide to reduce the number of regultory T cells. 54 A subset of ptients received lso orl temozolomide to induce utophgy in combintion with the virus tretment. 55 Response evlution. Tretment responses were evluted by chnges of tumor size nd/or metbolic ctivity in pre nd posttretment imging, performed by computer tomogrphy (CT) or positron emission tomogrphy with CT (F18-FDG-PET-CT). Modified RECIST 1.1 criteri 16 were used for evluting CT results, nd custom PET criteri 45 were used to interpret PET-CT imging results. Mgnetic resonnce imging ws used rrely. 56 Responses were grded into following ctegories: progressive disese or progressive metbolic disese (PD/PMD), stble disese or stble metbolic disese (SD/SMD), minor response or minor metbolic response (MR/MMR) nd complete response or complete metbolic response (CR/ CMR). Posttretment imging ws typiclly done 2 3 months fter the tretment 45 (rnge months, medin 2.3 months). The durtion of the responses ws not ssessed. OS ws clculted from the dte of the first virl tretment. Neutrlizing ntibodies. Neutrlizing ntivirl ntibody titer ws obtined by mesuring trnsgene ctivity from cells infected with luciferse-coding denovirus fter incubtion with different dilutions of ptient serum, s described previously. 26,52 Serotype of the virus used in the ssy ws lwys selected to mtch exctly the cpsid of the virus tht ws used in the tretment of given ptient. Tumor lod. Tumor lod score evlution ws bsed on the pretretment CT nd/or PET-CT scn imges. Lrgest tumor dimeter vlues were mesured nd orgns with metstses were identified. Metstses in lymph nodes, liver, lungs, bones, peritonel cvity, nd other sites were grded with score from to 3 (zero indicting no detectble metstsis). If the lrgest xil tumor dimeter exceeded 64 mm, the tumor ws considered bulky nd grded with score of 3 (otherwise zero). Scores from metstses nd tumor bulkiness were combined to form totl score for disese burden ( 21 points). Peripherl blood cell counts. Bseline peripherl blood smples were obtined from ptients on the dy of the tretment or 1 dy before. Posttretment smples were obtined on vrying dtes during dys 1 3 fter tretment. Smples were immeditely nlyzed by the lbortory of the treting hospitl using stndrd techniques. Neutrophil count ws derived by subtrcting the lymphocyte count from totl leucocyte count. Neutrophil to lymphocyte rtio ws clculted s the quotient from the division of neutrophil nd lymphocyte counts. Sttisticl nlyses. Sttisticl nlyses were performed using SPSS Sttistics v23 (Interntionl Business Mchines Corportion, Armonk, NY), Microsoft Excel (Microsoft Corportion, Redmond, WA), nd GrphPd Prism (GrphPd Softwre, L Joll, CA). Log-rnk test ws used to compre OS between different neutrlizing ntibody, tumor lod or blood cell count groups. Differences in tretment responses between groups were nlyzed with Fisher s exct test. Hzrd rtios for clinicl vribles were clculted using Cox proportionl hzrds regression model. ORs for disese control were obtined using logistic regression. P vlues of less thn.5 were considered sttisticlly significnt. SUPPLEMENTARY MATERIAL Figure S1. Overll survivl in different bseline neutrlizing ntibody groups. Figure S2. Response rtes in different bseline cell count groups. Figure S3. White blood cell counts fter tretment. Figure S4. Response rtes bsed on dy 1 cell count chnge. Tble S1. Chrcteristics of ptients without post-tretment neutrlizing ntibodies. Tble S2. Ptient chrcteristics. Tble S3. Multivrite nlysis for prognostic vlue of bseline neutrophil-to-lymphocyte rtio. ACKNOWLEDGMENTS We thnk Tiin Hkonen for expert ssistnce. 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