Oncolytic virotherapy: new weapon for breast cancer treatment

Veronica Martini1,2a , Francesca D’Avanzo1, Paola Maria Maggiora1, Feba Maria Varughese1,2, Antonio Sica3,4b and Alessandra Gennari1,2c

1Division of Oncology, Department of Translational Medicine, University of Eastern Piedmont, Novara 13100, Italy 2Center for Translational Research on Autoimmune & Allergic Diseases – CAAD, Novara 28100, Italy 3Department of Pharmaceutical Sciences, University of Eastern Piedmont, A Avogadro 28100, Italy 4Department of Inflammation and Immunology, Humanitas Clinical and Research Center–IRCCS, Rozzano (MI) 20089, Italy ahttps://orcid.org/0000-0002-0887-4082 bhttps://orcid.org/0000-0002-8342-7442 chttps://orcid.org/0000-0002-0928-2281

Abstract

The recent introduction of viruses as a weapon against cancer can be regarded as one of the most intriguing approaches in the context of precision medicine. The role of immune checkpoint inhibitors has been extensively studied in early and advanced cancer stages, with extraordinary results. Although there is a good tolerability profile, especially when compared with conventional chemotherapy, severe immune-related adverse events have Review emerged as a potential limitation. Moreover, there are still treatment-resistant cases and thus further treatment options need to be implemented. Several in vitro and in vivo stud- ies have been conducted and are ongoing to develop oncolytic viruses (OVs) as a tool to modulate the immune system response. OVs are attenuated viruses that can kill cancer cells after having infected them, producing microenvironment remodelling and antitu- mour immune response. The potential of oncolytic virotherapy is to contrast the absence of T cell infiltrates, converting ‘cold’ tumours into ‘hot’ ones, thus improving the perfor- mance of the immune system. Breast cancer, the second most common cause of cancer- related deaths among women, is considered a ‘cold’ tumour. In this context, oncolytic virotherapy might well be considered as a promising strategy. This review summarises the Correspondence to: Alessandra Gennari current status, clinical applications and future development of OVs, focusing on breast Email: [email protected] cancer treatment. ecancer 2020, 14:1149 https://doi.org/10.3332/ecancer.2020.1149 Keywords: OVs, immunotherapy, BC Published: 03/12/2020 Received: 11/06/2020 Publication costs for this article were supported by ecancer (UK Charity number 1176307). Background Copyright: © the authors; licensee ecancermedicalscience. This is an Open Access The immune system plays an important role in controlling and eradicating cancer. How- article distributed under the terms of the ever, malignant cells can develop multiple mechanisms of immune suppression [1]. In Creative Commons Attribution License (http:// creativecommons.org/licenses/by/3.0), which recent years, a number of immunomodulating agents have been developed as antican- permits unrestricted use, distribution, and cer therapies and introduced into the clinical practice. Among these, immune checkpoint reproduction in any medium, provided the original inhibitors (ICIs) have demonstrated an impressive level of activity as antitumour treatment work is properly cited.

ecancer 2020, 14:1149; www.ecancer.org; DOI: https://doi.org/10.3332/ecancer.2020.1149 1 that show arole for OVs inbreast cancer therapy. of immunosurveillance thatare thebasison OVsthe which are builtanddeveloped. In thesecond part, we discuss preclinical andclinicaldata This review provides anoverviewdevelopment onthe of oncolytic virus immunotherapy. In thefirst part, we briefly describethemechanisms monotherapy or aspartof combination therapy [7]. combination melanoma ICIagainst with OVs, (Canerpaturev—C-REV)as HF10 such andCVA21 (CAVATAK), are now actively beingdeveloped inphaseIIasmonotherapies, or in patients Medicines Agencyapproved (EMA) ofuse the talimogene laherparepvec (T-VEC, Imlygic®) asoncolytic viral therapy for advanced melanoma Several OVs have beensuccessfully tested indifferentpreclinical models[8].In 2015,theFood andDrug Administration (FDA) andEuropean at distant tumour sites [7]. antitumour immunity. Thetumour dying cells release alotof factors that engage antitumour immunity, therapeutic inducing responses also ing to tumour cell lysis. Alongside thisdirectand localantitumour activity, OVs canalsoinduce apotent, systemic andpotentially durable Oncolytic Viruses (OVs) are native or genetically modified viruses that selectively infectand replicate tumourwithin cells, eventually lead- molecular pattern (DAMPs)) andof tumour antigens from virus-infected cells [7]. responsesthrough differentofas induction such mechanisms Immunogenic Cell Death (ICD), release of danger signals(damage-associated activity of theICItreatment [5,6].Manyare studies alsoexploring theability of viruses to killtumour cells by initiating systemic immune chemotherapy hasbeenevaluated asapossiblestrategy to reshape tumour milieuandget immuneinfiltrate compatible anexpectedwith approacheshave been identified to overcomeabsencethe of T cell infiltrates, thusconverting ‘cold’ tumours into ‘hot’ ones. Among these, pancreas andothers, this classofwith agents may becharacterised by suboptimal responses andresistance. In thisperspective, different [4] are considered lessresponsive. In thisscenario, thetreatment of immune‘cold’ tumours,astumours such of thebreast, prostate, ovary, expression of neoantigens) andlow expression of antigen presentation markers, asmajor such histocompatibility complex classI(MHCI) havehigher the chance of response Lymphocytes (TILs), expression of programmeddeath-ligand ontumour-associated1 (PD-L1) immunecells andpossiblegenomic instability response.immune the tioning In fact,has beenshownit that so-called hotthe tumours, characterised by thepresence of Tumour-Infiltrating and complementary strategies needto bedeveloped.In particular, tumour microenvironment (TME)might exertan important role incondi- re-educated to fightcanceragainst cells. Yet,activitythe ofclass of this agents isheterogeneous within thesametumour type,andinnovative involvesaction theremoval of inhibitory pathways thateffectiveblock anti-tumour T cell responses; asaconsequence, theimmunesystem is in differenttumour types ecancer 2020, 14:1149; www.ecancer.org; DOI:https://doi.org/10.3332/ecancer.2020.1149 rectly by cancer cells: inthefirst case,tumour cells canpresent neoantigens by meansof MHC-IandMHC-IImolecules;alternatively, T cell progression. Therefore,role the of TILs hasbeeninvestigated inthe TME. CD8+andCD4+ T lymphocytes canbeactivated directly or indi- can recognise andkillthem. They include T,cells BandNK [12]. The interactions between TILs andcancer cells play acriticalrole indisease TILs were defined as lymphocytes that moved from thebloodinto the tumour bedand, directly and/or opposing surrounding tumour cells, motion andmaintenance of tumour immuneescape( spread TNF-related apoptosis-inducingcharacterisedphase, escape ligands and(3)the byand uncontrolled cell growthperforin, andproliferation(IFNγ), that canleadto metastatic g interferon including macromolecules as well as cells, (NK) Killer Natural and subsets T-cellγδ T-cells, equilibrium/editingphase, where there is acontinuous T-cell-mediated eradication of malignant cells via effector responses includingCD8+ three phases:(1)theelimination phase(i.e.,immunosurveillance), where themalignant cells are detected andcleared by immunecells; (2)the systemimmune attackcan establishanimmunosuppressive microenvironment. This highly dynamicimmunoeditingprocess isdefined by thelial cells, andeventually a variety ofcells immune that canmount aresponse againsttumour cells. Those tumour cells that escapefrom is composedTME of many cellthe originalcancerincluding types, cell with genetic alterations andother cells, asfibroblasts, such endo- Cancer cells andhostimmuneresponses: mechanismof immunosurveillance [11]. TILs, tumour-associated macrophages (TAMs) andmyeloid-derived suppressor cells (MDSCs) play animportant role inthepro- [9]. In glioma,several OVs have clearly demonstrated both safety andapromising efficacy inthephaseIclinicaltrials[7].Various , such as advanced[2], such non-small cell melanoma, lungcancer andbreastcancers [2,3]. The major mechanismof [4]. Conversely, ‘cold’ tumours scarcely expressing characterised PD-L1, by low mutational burden (low [10]. Theoftrials clinical OVs againstpancreatic cancer have not yetdemonstrated efficacy aseither Figure 1). 2

Review ecancer 2020, 14:1149; www.ecancer.org; DOI:https://doi.org/10.3332/ecancer.2020.1149 reactive oxygen speciessuchasnitricoxide [29]. as arginine andcysteine, arewhich required forsurvival the of T cells. are MDSCs alsothoughtto mediate their inhibitory functionsthrough also secreteMDSCs cytokines suchasIL-6 andIL-4 allowing expansion MDSC andsubsequent sequestration of essential aminoacidssuch strated thatinduce MDSCs inhibitionof T cells,cells NK andDCs, while stimulating immuneregulators as such Th2 T cells, T-reg, and TAMs. intoMDSCs immune-activating cells, including macrophages or retinoicgene-I acid–inducible (RIG-I)–like receptors, inhibit theimmunosuppressive functions of and/orMDSCs induce differentiation of CpGincluding ecules, oligodeoxynucleotide andpolyinosinic-polycytidylic acid(polyI:C), which are ligands of toll-like receptors (TLRs) and/ fluorouracilincluding and gemcitabine, reduced numbers the of intumour-bearing MDSCs hosts[30].In addition,immunostimulatory- mol demonstrated thata decrease inimmunosuppressiveleads to MDSCs theactivation ofimmunity, host andthat several chemotherapy drugs, cells towards atumour diversionfunctional of myeloid cells in response to microenvironmental signals. This multistep process drives the reprogramming of myeloid myeloid precursors from recruitment theBM; ofand MDSCs TAMs precursors into both secondary lymphoidorgans and/or tumour tissues; tion to TAMsis andtheresultMDSCs of a process driven by cancer-related inflammation, involving: altered myelopoiesis; mobilisation of mouse and CD11b+CD14−CD15+ or CD11b+CD14−CD66+ cells in human) CD11b+Ly6G+Ly6Cloin as cells identified (PMN-MDSCs, MDSCs granulocytic and human) in cells CD11b+CD14+HLA−DRlow/−CD15− reactionsimmune [29]. These cells comprise at least two subsets: monocytic MDSCs (identified as CD11b+Ly6G−Ly6Chi cells in mouse and in cancertissues and neoplastic patients,exerting a suppressive effect onantitumour immunity, by inhibitingboth innate andadaptive are a heterogenous group of immune cells from themyeloid lineage, particularly enriched in the peripheral blood, bone marrow (BM), Along with TAMs,are MDSCs characterised by thecapacity to suppress T cell tumour andsupport functions progression [28].MDSCs tion of TAM-associated molecules proposed,suppressionas the such of TAMrecruitment, depletionthe of their number, thepromotion of M1-polarised TAMs andtheinhibi- cell line decreases proliferation and up-regulates tumour suppressor genes Moreover, it was demonstrated thattreatment anti-humanwith ofCD47 cancer stem cells isolated from the Triple-Negative MDA-MB-231 data, obtained by Immunohistochemistry (IHC),showed amajor expression of both CD47andSIRP1ain TNBC with respect to Luminal BC. CD47 werein studied various preclinicalmodels andare currently beingtested inongoing clinicaltrials[24].Our preliminary unpublished by innate surveillance. immune Since high CD47mRNA expression is associated with poor survival of cancer patients on TAMsexpressed inhibitory effect on phagocytosis, called ‘don’t eat me’ signal. This is mediated by CD47 binding to signal-regulatory protein (SIRPα), which is however, is impaired by the ‘don’t eat’ me signal CD47, expressed by many cancer cells anti-tumour ing specific immunity [21].In thisscenario, phagocytosis of cancer cells by TAMs provides additionalantitumour activity, which, TAM and tumour-associated dendritic cells (TADCs). ICD activates TADC and TAM sustaining enhanced antigen presentation - and support models cytotoxicsome agents are ableto favourantitumour the of activities TAMs, at leastinleukaemiaand/or immunogenic (transplant) tumour ronment and in response to severalsuch as inflammation,stimuli, undergo M1 (classical) or M2 (alternative) activation. It is well known that activity,promoting thus tumour growthand progression [19]. TAMs derive from peripheral bloodmonocytes recruited into themicroenvi- The reductionof effectorof functions TILs,promotion the of T-reg andthesecretion of inhibitory cytokines are theconsequence of TAMs with apoor prognosis, whereas high levels of CD8+ TILs seemto beassociated with animproved prognosis [18]. in Triple-Negative BreastCancer patients (TNBC) to several factors,a low including tumour mutational load[17].Indeed, specific TIL subtypes have beenassociated with different outcomes breast tumours isassociated anadversewith relapse-free andoverall survival [16].Breast cancer isnot considered to beimmunogenic, due gic andautoimmune disorders [15].In thisregard, ithasbeendemonstrated that enhanced presence of Treg cells inbiopsy samplesof the [14]. In normal conditions, Treg cells are involved in maintaining immune homeostasis by protecting hosts against the development of aller a negative effect onseveral cancers, dueto theenhancement of tumour aggressiven [12]. TILs exert agood prognosticeffect in patients treated with ICI(i.e.,anti-PD-1/PD-L1pathway) not only becausethey activate [20]. Inregard, this chemotherapyinduce can ICD is triggeredwhich by innate immunecells or infiltrating immunecells, including [13]. Activated cytotoxicCD8 T cellscells andNK have beenassociated with agood prognosis; incontrast,the presence of Treg has [22]. CD47 is overexpressed in manyof types cancerhuman cells and protects them from being recognised and cleared -promoting phenotypeand remotely controls thecomposition of thetumour-microenvironment. Previous studies [26, 27]. [17]: inparticular, highlevels of PD-1+ TILs or FOXP3+ TILs have beenshown to correlate [31]. MDSC’s hasbeen function studied in breast cancer. These studies demon- [25]. In this scenario, several TAM-centred strategies have been [28]. Accumulation of myeloid progenitors andtheir differentia- [20]. CD47 is a transmembrane glycoprotein with an [23], antibodies anti- 3 -

Review ecancer 2020, 14:1149; www.ecancer.org; DOI:https://doi.org/10.3332/ecancer.2020.1149 Piao of exosomesin immunity,and host TME andsuggestexosomes as potential therapeutic multi-functional agents for [33].Moreover,TNBC EVsPD-L1+ only inthetumour,and endothelial lymphoid compartment of breast cancer patients. Preclinical therole studiesalsosupport blood concentration of EVs resulted significantly higher incancer patients inhealthy than volunteers. Moreover, asexpected, we detected has been recently hypothesised in BC [34]. Our pilot preliminary data confirmed previously reported data to-cellmediators possible of oncogenicinformation, through horizontal transfer of various bioactive asproteins molecules,such andmRNAs, frommembraneplasma the andapoptoticbodies, represent EVs circulating component [33]. The role of exosomes aslocalandsystemic cell- nano-sized membrane vesiclesproduced cellendosomal in the compartment and,together with microvesicles (MVs) generated by budding has been recently hypothesiSed as a possible determinant of response to ICI, in patients with lung cancer and melanoma undertypes havestimuli, specific alsobeenproposed aspotential biomarkers incancer. In particular, therole of exosomal-PD-L1 fraction Recently,involvement the of extracellular vesicles(EVs) in cancer pathophysiology hasbeendescribedand,given their release from allcell Complex ClassI. The figure was modified from Servier Medical Art,http://smart.servier.com/ balance from elimination. escape and equilibriumto Treg: T regulatory; MDSCs: Myeloid Derived Suppressor Cells; MCHI: Major Histocompatibility expression, immunosuppressive cytokines, MDSCs,andexpression of negative regulatory receptors. Various forms of immunotherapy aim to shiftthe In theequilibriumphase(middle), thetumour andtheadaptive immune system coexist. Tumour escape (right) occurs whenthere ispoor antigenic often occurs inearly tumour development, highly antigenic tumour clones are recognised andeliminated by both innate andadaptive immune systems. Figure 1.Immunoediting. The three phases of thecancer immunoediting process: elimination, equilibriumandescape. Intumour elimination (left), which [40] (Figure 1). activated CD8+, may elicit a direct cytotoxic response to eradicate cancer cells, through the generation of IFNg, perforin-1, and granzyme B antigens, andtheantigen presentation to T cells [39]. As aconsequence, theincrease inthenumber of TILs, highratiowith CD8+/Treg, and orsecondaryupon HMGB1 necrosis, stimulatewhich recruitment the of DCsinto thetumour bed,theuptake andprocessing of tumour calreticulin(CRT) on the cell surface prior to death and release DAMP asadenosinetriphosphate such molecules, (ATP) duringapoptosis a strategy to induce ICD, ofa type tumour cell death, an which primes anticancer immune response. In response to ICD, tumour cells expose The reactivationof immunosurveillancefor iscritical better prognosis and improved patient survival; to ithasbeendeveloped thispurpose, tumourigenic microenvironment to suppress thetumour growth metastasis. and their molecular cargo [37,38] . In terms of therapeutics,exosomes cargoes canbeartificially modulated to provide pro-inflammatory, anti- fate of macrophage phenotypes,either M1,classically activated macrophages, or M2,alternatively activated macrophages onthebasisof node metastasis,by tumour modifying microenvironment.Recently, ithasbeenshown thatexosomes have thepotential to change the et al [36], using an orthotopic model, foundTNBC that TNBC-derived exosomes were able to polarize macrophages, favouring lymph . [35], demonstrating that the overall [32]. Exosomes are 4

Review ecancer 2020, 14:1149; www.ecancer.org; DOI:https://doi.org/10.3332/ecancer.2020.1149 MedicalServierArt, CTLA-4: Cytotoxic T-lymphocyte-associated protein 4;IFNI:Interferon typeI;CCL3/4: Chemokine (C-C motif) ligand3or 4. The figure wasmodified from respective targets oneither cancer or immune cells (right panel). NK: Natural killer;PD-1: Programmed death-1; PD-L1: Programmed death ligand-1; attract immune cells expressing PD-1or CTLA-4. These events make tumour ‘hot’, and whenICIs are administered subsequently, they canbind to their by theproduction of cytokines, suchasIFNtypeIthat stimulates PD-L1 expression onthesurface of cancer cells,andchemokines,like CCL3and CCL4, and for thisreason, theresponse to ICItherapy isinefficient (left panel). Oncolytic virotherapy promotes strong antiviral immune response accompanied Figure 2.Oncolytic virusesmake tumours ‘hot’. ‘Cold’ tumours poorly infiltrated by immune cellsand have low expression of PD-L1oncancer cellssurface, immune response. Most available oncolytic viruses are genetically modifiedto reduce virulence for non-neoplastichostcells. OVs areor RNA- DNA-attenuated able toviruses killtumour cells after having infected them,inducing remodellingTME andantitumour Oncolytic viruses (OVs) responseated with T cells,protein PDL1 andIFNg gene expression inresponders. Baseline CD8+ T cell infiltration or abaselineIFNg signature was not associ- attenuated herpes simplex 1 (HVS-1),virus type and an anti-PD-1 therapy resulted in a 33% complete response rate, with increased CD8+ expression,and PD-L1 creating a favourable forTME subsequent ICItherapy oncolyticOrthoreovirus has been shown to increase T cell infiltration inbrain tumours andto up-regulate IFN-regulated gene expression (Figure 2). Yet,several aretrials clinical evaluatingcombinationthe of oncolytic virotherapy andICI[41].In theclinicalsetting, intravenous perspective,this natural or engineered able toviruses, transforma ‘cold’ into a‘hot’ TME, increasedwith immunecell andcytokine infiltration Theof aim oncolytic virotherapy isto stimulate anti-cancer response, immune targeting andkillingtumour cells while sparingnormalcells. In Oncolytic virotherapy cancer cells. as astrategy to stimulateproduction, ICD-hallmarks particularly IFN, which takes advantage of competent replicating viruses to destroy A lot of chemotherapeuticdrugs have been used to induce ICD [39], but in the recent years, the interest has focused on oncolytic virotherapy http://smart.servier.com/ [43]. Oncolytic viral therapy istherefore apotential future option to skew the towardsTME anICIresponsive phenotype. . [42]. In melanoma patients, the sequential administration of an 5

Review European countries [52]. derived Oncorine for headandneckcancers treatment inChina[51]andnative Echovirus 7(Rigvir) for thetreatment of melanomainseveral Laherparepvec (T-VEC), oncolytica modified HSV-1 for clinicalusein US, Europe and Australia [50],along with the clinical use of adenovirus- earlytrials, clinical morewith 570 ongoing than testingtrials clinical OVs [10]. This activity hasnow granted theapproval of Talimogene To date, ninedifferentfamilies of bothincluding viruses, DNA andRNA viruses, have successfully transitioned from preclinical studiesto glioma xenograft was achieved [49]. (dlsptk), depletedwith thymidinekinase or Infected cell protein (ICP)34.5, were selectively replicating only inproliferating cells of human With thefirst experiences recombinantwith DNA technology, in1991,thefirst demonstration that herpessimplex virus (HSV) mutants associated with measlesinfection. oncolyticimmunity. In thenext years, several viruses were in studied various leukaemiamodels,Hodgkins’ diseaseandBurkitt’s lymphoma enhanced humoral immunity againsttumour cell antigens, via thesecretion of immunoglobulin andcytotoxic antibodies, was dueto post- Far East Virus to inhibitthegrowthof five transplantedsarcoma mouse 180[47].In 1965,LindenmannandKlein[48]demonstrated that led tomelanoma aremarkableagainst virus remissionpartial tumours, ratherthe effects thanstudying of these on tumourviruses growth. The first clinicaltrial was reported in1940, when anattenuated Since 1904many researchers havethe relationship beenstudying between viruses andtumour cells [44,45],focused on virus growth in ecancer 2020, 14:1149; www.ecancer.org; DOI:https://doi.org/10.3332/ecancer.2020.1149 uninfected tumour cells may bekilledby thecytotoxic perforins andgranzymes released by theinfected tumour cells [61]. clones may beableto circulate andkillantigen-expressing cancer cells, includingcancer cells that were not infected by the virus. The nearby ICI [59,60]. with The presence of theseneo-antigens isimportant since itmay trigger animmuneresponse andthenewly generated T cell releasethe of novel cancer antigens (neo-antigens) thatmay have beenpreviously hiddenfrom theimmunesystem, aneffectalready known oftype the cells immune the within potentiatingTME systemic immunity againstthe cancer [58] . Moreover, oncolytic cell death caninduce TAMsOVs andMDSCs. can modify thissuppressive microenvironment through a variety of mechanismsthat alter thecytokine milieuand Cancer cells canovercome theimmune-mediated attackby modifying TME, facilitating therecruitment of immune-suppressive cells, as such and CD11b). Different DCsubsets induce different T helper (Th) cell polarization [57]. and, finally, prime T cells activation and differentiation. There are different DC subsets distinguished from CDexpression (CD8, CD4, CD103 antigen andexhibitit (i.e. CCR7) andproducingcytokines inducewhich their migration from peripheral bloodto secondary lymphoidorgans, where they process ture’pathogenupon DCs recognitionor stimulationbecome ‘mature’, expressing co-stimulatory molecules(CD40 andB7),homingreceptors Yet,they activateinnate the cells immune (suchtoas NK) respond to pathogens through non-specificmechanisms[55].In particular, ‘imma- fromneutralising antibodies between innate andadaptive immunity and,for thisreason, may serve asacarrier for someOVs (such asreovirus andmeasles) protecting and interleukin ‑12), promotewhich maturation the of antigen-presenting cells (APCs) asDCs(Figure such ). DCsare 3 considered a‘bridge’ IFNγ (TNFα), factor-αnecrosis tumour IFNs, I type (e.g., cytokines CRT,and HMGB1, acid) proteins,ATP uric shock and heat example, for ing normalcells [53]. As aconsequence of oncolyticcell death,cancer cells alsorelease viral PAMPS (DAMPs; andadditionalICD-hallmarks cancer cell responseto stress, cellprovide signallingandhomeostasis aselective advantage for viral replication andincancer cells, exclud- activity may beabnormal;thus, viral clearance is prevented, allowing increased viral replication. Moreover, theinherent abnormalitiesinthe introduced to thecells by a viral infection.It inducecan cell deathand viral clearance [53].In cancer cells,pathway IFN signallingandPKR activates Proteinis aproteinPKR KinaseR(PKR). kinase,encoded by theEIF2AK2gene, activated by double-stranded RNA (dsRNA) and active intracellular TLRs pathway, coordinate the antiviralwith machinery ininfected cells; thisreinforcesrelease, localIFN which, inturn, In normalcells, pathogen-associated molecular patterns (PAMPs), aselements such of viral RNA capsids,DNA, and viral protein products, autophagy) [7]. and induced viral tropism, and the susceptibility of the cancer cell to the different forms of cell death (apoptosis, necrosis, pyroptosis and ing, viral replicationcelland host antiviral response. Thepotential lytic of oncolytic viruses also dependsonthetypeof virus, dose,natural OVs mediate toxiceffect on tumour cells by director indirectmechanisms. This activity isinfluenced by theefficiency of cell receptor target- Mechanisms of oncolytic virusimmunotherapy MHC class I-II, promoteI-II, class via MHC expression the of corresponding costimulatory moleculeson T cells (CD40L,CD28) [56] . DCs have[54]. DCs animportantrole inthe initiation of immune responses, to control andeliminate viral infections. [46], andin1949,thefirst preclinical study describedtheability of theRussian 6

Review ecancer 2020, 14:1149; www.ecancer.org; DOI:https://doi.org/10.3332/ecancer.2020.1149 nota confirmation of live viraland on-going particles replicationand more informative test are beenrequired. In recent years, the sensitivity tested for thepresence of virus usingIHC andconfirmedby assay. PCR However, PCRisahighly sensitive assay for viral sequences, have but reason,this is important it to assessthe viral presence notonly inthetumouralso intheblood.Normally, lesionbut tumour specimensare the intravenousthus, administration of these viruses maythe effects limit inpatients who have hadprevious treatments or vaccination. For proteins, especially afterinfusions. multiple Of note, naturallyviruses cause viremia and are more susceptible to antibodies neutralisation; systemicin the dilution circulation likelihood andthe of premature neutralisation through anti-viral serum immunoglobulins or other serum and reduced delivery totumour the site followingintravenous delivery [63]. The issues with IV delivery of any OVs includesconsiderable In thecaseof SVV, itcanbedelivered IVof because natural resistance to hemagglutination, aprocess resulting inpremature viral clearance to allow viralat distribution any site, precluding theneedfor additionaltraining andinterventional procedures associated with IT delivery. SenecaValley (SVV) Virus previous couldsection, tobe asolution overcome limitation,this as evidenced inthe T be challengingfor OVs repeated administrationsover time. The ability of OVs to induce systemic anti-tumour response, asdiscussed inthe are not accessible bypalpation; clinical in thiscase,localisation via interventional imagingor surgical exposure might berequired, which may systemicneutralisation andpremature clearance. The maindisadvantage isrepresented by physical inaccessibility for thosetumours that the mainstrategy usedinthemajority ofstudies clinical OVswith since itallows maximaldelivery of high viral titres to tumours, bypassing blood–brain barrier, tumour size andheterogeneity, denseextracellular matrix andpoorly vascularisation [62].Intratumoural (IT)injectionsis spreadthe of OVs,presenceas the such of necrosis, calcification, hypoxia, acidosis,increased proteolytic activity, highinterstitial pressure, One of themajortoOVs issues the use intherapyroute isthe of administration andneedsto take into account physical barriers that canreduce Oncolytic virus transport andbiodistribution suppressor cells. The figure was modified from Servier Medical Art,http://smart.servier.com/ dendritic cell; MCHI:major histocompatibility complex ClassI; Treg: T regulatory; TAMs: tumour-associated microenvironment; MDSCs: myeloid-derived calreticulin; DAMPs: damage-associated molecular pathways; CD:cluster differentiation; TLR4: Toll-like receptor 4;P2RX7: Purin 2 Receptor X7;DC: microenvironment altering thecytokine milieu(red lines) OVs: oncolytic viruses; ATP: adenosinetriphosphate; HMGB1:highmobility Group Box 1;CRT: helper T lymphocytes of theadaptive immune system. The effects of OVs reflect onMDSCs, TAMs and Treg cellsinhibitionandmodify thesuppressive killing of tumour cells isprimarily mediated by natural killer cells of theinnate immunesystem andtumour antigen-specific CD8+cytotoxic and CD4+ antiviral responses andinflux of immune cellsthat mediate thedestruction of residual infected anduninfected tumour cells. The direct recognition and stimulate thehostantitumour immune responses. Cellular detection of viral infection andthe products of oncolysis trigger the rapid activation ofhost of infectious viral progeny that infect nearby cancer cells. Tumour-associated antigens and cellular DAMPs, suchas CRT, HMGB1and cellular ATP, Figure 3:Mechanism of oncolytic virusimmunotherapy. Oncolytic viruses infect cancer cellsandinduce the immunogenic celldeath and release [63] oradenovirus,chimeric the enadenotucirev [64],delivery by intravenously administration (IV) hasbeenshown . ‑ VECphase IIIclinicaltrial[9.]In OPTIM thecaseof 7

Review one cell to another [33].Tang Recenthighlightstudies importancethe of MVs incancer development. These specialisedMVs are ableto transfer bioactive moleculesfrom cells which are p53deficient [70] . somes harbouringtheplasmids were resistant to antibodies neutralising theparent strain, while theplasmidscould only transfect tumour were alsousedinpre-clinical tostudies encapsulate oncolyticalphavirus strain M1(M-LPO) [69]andONYX-015-based plasmid[70].Lipo are ongoing to understand if OVs could beconsidered in combination or as a neoadjuvant therapy with cyclophosphamide [8]. Liposomes antibodies. The concomitant administration of immunomodulatory ascyclophosphamide, drugs,such seemsto rescue OVs [68] . Clinicaltrials [54], mesenchymalstem cells [66]or carcinomacells [67],could beacarrier of viruses to tumour sites, also in thepresence of neutralising Different approaches, suchastheuseof cell carriers, immunomodulators and liposomes have also been proposed. Yet, dendritic and T cells significantly lower when compared to that of real timePCR and accuracy of real-timeassaysPCR have beengreatly improved by thedroplet digitalPCRtechnology (ddPCR), whose detection limitis activity to hepatitis B virus replication hepatocytes [75]. ing to escape host immune recognition. Exosomes deriving from liver nonparenchymal cells can transmit interferon (IFN)-α induced antiviral in establishing a productive infection [74]. Exosomes canalso confer membranes to non-enveloped viruses, as such hepatitis A virus, allow- HIV-1invasion [73].HepatitisC exosomes utilises (HCV) virus to transfer itsgenomic RNA,antisense RNA,andproteins, which aidthe virus tory factors, transactivationresponse elements, viral microRNAs(vmiR-88, vmiR-99 and vmiR-TAR), CCR5, andCXCR4—all of which facilitate oftype and cells.virus For example, exosomes isolated from immunodeficiencyhuman infectedvirus (HIV) cells contained negative regula- needed for invivo treatmentefficacy [72]. Exosomes are MVs (100–150nm) that caninfluence viral infection indifferent ways, inrelation to carrieras aunique be utilised system to deliver oncolyticadenoviruses to tumours, human leadingto highly efficient cytolysis of tumour cells signals, canserve assafe andefficient carriers to deliver chemotherapeutic drugsto tumour cells. Moreover, they reported that T-Mvs can ecancer 2020, 14:1149; www.ecancer.org; DOI:https://doi.org/10.3332/ecancer.2020.1149 miRNA carryingcells, resulting ininhibitionof theIFNpathway andpromotion of viral infection [83]. cancer cellsalso beuptakecan it but bycells. these Ithas beenalsohypothesised thatNDV might useexosomes to entry into neighbouring glioma cells, that frequently downregulate miR ‑7 glioma, where itshowed limited viral replication inneurons that constitutively express high levels of miR‑7, while allowing proliferation in ing (miRTA) canbeblocked from replicating innormalcells, where specificmiRNAs are expressed. This of type construct was designed in Sinceand cancer normal cells exhibitdifferential expressions of cognate miRNA elements, theengineered OVs with miRNA Target Sequenc PSA ishighly active, E1A isselectively expressed only inthesecells, resulting inproliferation of adenovirus and virus mediated cell lysis [81]. ral protein that inhibitscell cycle, was placed under promoter of prostate-specific antigen (PSA) [81].In prostate cancer cells thepromoter of cancer cells. Thisofcase isthe forI trial aphase patients prostatewith cancer, treated with adenovirus (CV706) in which theE1A,anadenovi - pathway targetscellB (BCL) lymphoma acterised by aberrant signallingpathways that promote resistance to cell death andproliferation. For example, Newcastle disease virus (NDV) A longintracellular incubationis neededto time allow OV replication andspread, explaining thepreference for tumour cells infection, char [78]. receptors, as in the case of adenovirus Ad5/3‑Δ24, which was modified to bind to integrins that are highly expressed on ovarian cancer cells and CD54,respectively)HVEM- andcanenter inthecancer cell [76,77];other OVs canbeengineered to directly target uniquecell surface into target cells. SomeOVs, like HSV-1 andCoxsackievirus, naturally recognise theoverexpressed molecules(herpesvirus entry mediator – cancer cell surface (CD46, CD155, CD54, CD55,alfa2beta1, laminin receptor, etc.) and that can be used by OVs to recognise andentry allowed theinvestigation of several strategies to enhance theeffectiveness of OVs. There are somemoleculesthat are overexpressed on selectively. Natural tropismfor surfacealtered molecule, cancer cell pathways together with theflexibility of recombinant engineeringhave The rulesof a well-designedOV approachare mainly two: 1)to attenuate the viral pathogenesis and2)to targetand killtumour cells, Development of oncolytic virusesasdrugs [80]. Furthermore, thepossibility to insert viral genes under active promoters incancer cells, allows limiting viral replication only in et al [79] or Reovirusand Vaccinia virus show natural selectivity for cells with overexpression of theRASsignalling [71] have demonstrated thattumour cell-derived MVs (T-MV), containing both tumour antigens andinnate [82]. Recent evidence hasdemonstrated that exosomal miRNA cannot only besecreted by [65]. 8 - - -

Review distinct adenovirusesdistinct infections cancause inhumans. Adenovirus expresses coxsackie-adenovirus receptor (CAR) andadenoviral early genes Adenovirusesare medium-sized (70–90 nm), non-envelopedicosahedral viruses with double-stranded Many DNA. typesof immunologically Oncolytic adenovirus ecancer 2020, 14:1149; www.ecancer.org; DOI:https://doi.org/10.3332/ecancer.2020.1149 Talimogene laherparepvec (T are permissive to HSV-1 andgenetic manipulations allow virus selectivity, while sparingnormalcells [86]. is not ableto induce insertionalmutagenesis, makingHSV HSV Oncolytic Herpes Simplex Virus(HSV) and clinicaltrials. TNBC up-regulationofinflammatoryinduce to JAK-STATshown metastatic in Nivolumabresponsivenessincrease to to and signalling and TNF-α as anti-PD1/PD-L1,such is reprogramming:TME yet,administrationthe of chemotherapy agents, asdoxorubicin such andcisplatin, hasbeen research is ongoing to characterise the prognostic role of TILs [84]. In this perspective, a powerful strategy to enhance the response to ICI, leading to FDA andEMA approval of atezolizumabin thefirst line treatment ofoverexpressing PD-L1 MBC [5].Moreover,TN extensive In recent years, therole of ICIhasbeenextensivelyin breast studied cancer patients, with particular emphasisontriplenegative disease Oncolytic virustherapy inbreast cancer in vitro and replication to cancer cells and expressing murine Interleukin 12 (IL-12) (instead of GM-CSF). The G47Δ-mIL12 efficacy has been tested both genetically engineered oncolyticHSV (oHSV) that hassimilar genetic modifications to T-VECcontains but ICP6inactivation, limitingHSV a is (14) cancer.G47Δ-mIL12 breastadvanced of model tumour 4T1 a in observed was Recently,G47Δ-mIL12 ofactivity[17]. antitumour safety and efficacy of T-VEC (as a monotherapy or combination therapy paclitaxel)with in patientsTNBC is under evaluation in a clinical trial with T-VEC followed by pembrolizumab, showed an interesting level of activity of the anti-PD-1 agent, related to TME modulation [43]. The poorly responsive to ICIasasingleagent,as breast such cancer. A phase1bclinicaltrial,including21advanced melanomapatients treated T-VEC may representa valuabletool incombination ICIs,with particularly inthosetumours with alow baselinelymphocyte infiltrate that are chills, nausea,fatigue andlocalinjectionsitereactions. patients breastwith cancertrial [87]. inaphaseIclinical Treatment was generally well tolerated, with mostadverse events related to fever, cancer cells [10]. T In preclinical studies, T cell apoptosis [86]. HSV leading tocontainment the of theinfectionin healthy and immune-mediated tissues killof cancer cells that selectively propagate oncolytic colony-stimulating factor (GM-CSF)to improveof the induction antitumour immunity. ICP47 deletion results into viral antigen presentation, tivity andprevention of neural infection. The two ICP34.5 genes have beensubstituted with thegene encoding granulocyte-macrophage and ICP47.ICP34.5isaneurovirulence gene,for critical blockingthehostantiviralresponse; PKR–IFN itsdeletion confers cancer selec tration of CD8+andCD4+ T-cells around tumour islets, of theinduction supporting animmuneresponse [91]. metastaticand subcutaneous cutaneous with breast cancer. A histological examination revealed fibrosis andtumour cell death aninfilwith - apoptotic cells, therefore producing a synergistic antitumour effect in combination bevacizumab,with since bevacizumabenhances viral increases distribution, tumour hypoxia andexpands thepopulation of and provided resistance to malignant cell re-implantsis aspontaneously [89].HF10 mutated HSV-1 andisexpected to beapromising agent Administration ofin immunocompetent HF10 models of mouse colon and breast cancer stimulated specificantitumour immune responses effectively inhibited 4T1tumour growth inaCD8+ T cell-dependent fashion, andprevented metastatic spread [88]. ‑1 isadouble-stranded DNA virus a largewith genome (152kb), member of thealphaherpesvirus family; itreplicates and inthenucleus ‑1. The deletionof also induces ICP47 early the activationof the US11promoter that blocksPKRphosphorylation preventing cancer [85]. Innovative treatment options,as OVs, such are as amonotherapy studied or incombination with ICI or CT, both inpre-clinical in vivo experiments. In vitro, it was able to ‑ VEC hasbeenevaluatedin patientstrials inclinical with melanoma,pancreatic cancer, headandnecktumours, andin ‑ VEC demonstrated potenteffects lytic against several tumour cell lines,most notably melanomaandpancreatic ‑ VEC)commercially isthemost advanced HSV infect and eliminate both murine and human cells.TNBC ‑1 anattractive candidate for therapeutic development. Human breast cancer cells [90]. An investigator-initiated phase Itrial of enrolled HF10 six patients based OV.‑1 T ‑ VEC contains deletions of two genes: ICP34.5 In vivo , G47Δ-mIL12 treatment 9 -

Review of oncolytic adenoviruses engineered interfering lncRNA [94]or targeting TGF-β incancer cells [95]. memory T cells, givingto theoncolyticadenovirus animmunomodulatory effect [93].Other pre-clinical highlighted studies theimportance macrophages and neutrophils, itmay induce the productionand activation of effector T cells and regulate thesurvival and proliferation of may beaneffective target for treatment. Also, by Interleukin-15human carrying (IL-15) gene, cytokine acrucial enhancingtheactivity of proliferationand carcinogenesis, is higher andit expressed inbreastcancer tissuescompared with normal tissues,suggesting that E2F-1 has an inhibitive effect on breast cancer proliferation [93]. The transcriptional factor E2F-1 plays a significant role inthe control of cell cycle, patients differentwith of types cancer [92].Recent findingsdemonstrated that a novel recombinant adenovirus, targeting E2F-1 andIL-15, etanercept (a recombinant dimer of thehuman so far,TNF); this study enrolled two patients with metastatic breast cancer, as well as other ONYX Oncolyticadenovirus provides anovel therapeuticmodality for breast cancer itsappropriatewith targeting property. Adenoviruses in clinicalpractice hasregistered very few adverse events. cell cycle proteins. Some of thestrategies for modification of adenovirus are described,asattenuation, andtheuseof oncolytic adenovirus Normalcells are protected byand pRb p53 activities, whereas incancer cells thesegenes are compromised, andadenovirus caninterfere with (encoded by E1Athat andE1B) allow cell the infectiontargeting suppressors oncogene p53andretinoblastoma-associated protein (pRb). IFN, RANTES, IL RANTES, IFN, ecancer 2020, 14:1149; www.ecancer.org; DOI:https://doi.org/10.3332/ecancer.2020.1149 pression of BCL-X membraneor fusion throughdirect endocytosis of the Cancervirus. cell specificity isconferred by itssensitivity to typeIIFNs andoverex NDV isasingle-stranded RNA envelopedavian paramyxovirusthat ranges insize from 100to 500nm.NDV infects cells through plasma Newcastle disease virus tumours (first-in-human phase1). The study report that it was well-tolerated, showing aselective antitumour activity [101]. selectivity through two targeted gene deletions (vvDD). vvDD were administered viaIT doseescalation in16patients with advanced solid Moreover, the Western Reserve strain of vaccinia most the (VV), virus virulent strain of VV inanimalmodels,hasbeenengineered for tumour provides atumour-specific oncolytic immunotherapy for cancer patients, especially thoseresistant to blockadetherapy PD-1/PD-L1 [100]. on tumourPD-L1 inhibits cellscells andimmune activatecan but tumour neoantigen-specific T cell responses, via GM-CFS stimulation. This that co-expressesinhibitor aPD-L1 andGMCSF hasbeendeveloped. This OV cansecrete inhibitor, thePD-L1 that systemically bindsand antigens, T cell co‑stimulatory molecules and inflammatory cytokines response and the ability of the vaccinia viral genome to accept large transgenes (25 kb), vaccinia virus has been engineered to express tumour proteinI IFN-binding have (B18R) to beenmodified increase cancer cell selectivity andlysis [99].Based ontheimportance of theimmune host genome,the the vaccinia is consideredvirus agood OV. Specifically, viral thymidine kinase, vaccinia growth factor and vaccinia type and replicating inthecytosol. For itslarge capacity of transgenes, easy manipulation, agood safety profile, andtheinability to integrate into Vaccinia virus isadsDNA virus andisamember of thepoxvirus family; they caninfect a wide range of cells entering by endocyticmechanism Vaccinia virus cells when combined with anti-PD-1 therapy [98]. [97]. Recently, inapre-clinicalstudy, has beendemonstrated it thatreovirus was ableto induce animmuneresponse againstbreast cancer combination paclitaxelwith inpatients metastaticwith breast cancer hasshown that thiscombination ismore effective thanpaclitaxel alone an oncolytic virus exploitspathwayRAS the thatis linked toactivation the of PKR uitous, non-enveloped, anicosahedral shaped virus a genomewith of 10segments of double-stranded RNA. The application of reovirus as strain. REOvirus(Respiratory Enteric Orphan), belongingtogenus the Orthoreovirus, family Reoviridae, isa70nm,naturally occurring, ubiq- Reolysin (Oncolytics Biotech Inc., Calgary, Alberta, Canada) liveis a purified replication-competent form of thereovirus serotype 3 Dearing Pelareorep (Reolysin®) ‑015, hasadeletionwhich ofportion inthe that E1B inactivates p53,are currently investigated inaphaseItrialcombination with CSF) and improved‑12, andGMCSF) antigen presentation. The NDV protein haemagglutinin-neuraminidase canactasapotent L . NDV induces apoptosis anddirectly activates theinnate immunesystem through increased cytokine production (type I [7]. Recently, an engineered oncolytic vaccinia virus ((VV)-iPDL1/GM) [96]. A phaseII,randomised, clinicaltrialof pelareorep in 10 -

Review ecancer 2020, 14:1149; www.ecancer.org; DOI:https://doi.org/10.3332/ecancer.2020.1149 also becarefully evaluated in terms of immune system performance. CT or ICIs, to improve performance andresults.Furthermore, intermediate endpoints, aspredictive such biomarkers. Patient selectionmust macodynamicassays, delivery systemsof and type clinical trialdesigns need to be carefully assessed, especially in combination regimens with activity,be optimised someissuesmust in thedevelopment of OVs asanew classof drugs.In fact, appropriate dosages andschedules,phar However, if, ontheone hand,thisform of immunomodulation ischaracterised by moderate toxicity, increased specificity, andhopefully tumour immunity already present,ofcase as inthe ‘hot’ tumours, andthat of systemic immunecells attracted in TME, in‘cold’ tumours. The two mainadvantages inthe use of oncolytic virotherapy are: 1) its specificity for tumour cells and 2)its active role in triggering anti- Viruses usedasa weapon againstcancer isonetherevolutionary discoveries of recent years. Conclusion and NCT02879760). rhabdovirus hasbeen tested ina variety oftumour mouse models[113]andisnow beingevaluated inphase1/2studies (NCT02285816 up-regulationtion, of antigen presentationby tumour cells andproduction of proinflammatory cytokines andchemokines[113].Maraba Oncolyticrhabdoviruses like Maraba and VSV have beenshownto enhance cell–mediated NK killingof tumour cells, dendriticcell matura- is to evaluate thetoxicity of thisapproach inMBC. deliveryvirus to breastcancer cells [112]. To date there isonephaseIclinicaltrialthat iscurrently open(NCT01846091), whose primary aim cellsusing dendritic asacarrier for oMV, itispossibleto overcome theproblem of antibody neutralisation with promising results interms of oncolysis oMV infectivity invitro andinxenograftmodels [110].It hasalsobeenobserved that the Aurora A kinaseinhibitor alisertibenhanced oMV Cell Adhesion Molecule 4 (Nectin 4).Nectin 4 levels canbe downregulated by miRNAspecific (i.e.,miR-31 and miR-128) with the impact on sion ofactivationsignalling lymphocytic as CD46, such molecules specific molecule,thepoliovirus receptor-related 4protein andNectin Oncolyticmeasles viruses (oMV) derived from theattenuated Edmonston-B vaccine strain canrecognise target cells through overexpres- 109]. In particular, coxsackievirusstrains andpoliovirusreported promising results, both invivo andinvitro, agent: coxsackievirus, poliovirus, vesicular stomatitis virus (VSV), measles virus andMaraba virus. There are other underviruses investigationfor their oncolyticactivity both asasingleagent as well asincombination with other anticancer Other trial. PV701 was IV injected twice at lower dosages followed by doseescalation. The treatment regimen was well tolerated [106]. limited number trialsinprogress atpresent Althoughnumerous preclinicalhavestudies suggested that NDV hasantitumour activity againsta wide variety of cancers, there are only a infection; thisresults inIFNpathway inhibitionandpromotion of viral infection of the ‘recipient cell’ [104]. to entry into neighbouringcells,‘recipient theso-called cell’, miRNAs carrying produced by theoriginalcell, calledthe‘parentcell’, after virus intercellular communication,through miRNA. In arecentstudy performed inHeLa cell line,ithasbeenspeculated that NDV usedexosomes been proposed asaninnovative approach for virus delivery. Several studieshave shown that exosomes are involved in viral infections andin ity to applyof theuse OVs andinparticular NDV indifferent strategies to treatbreast cancer. The encapsulation of NDV inexosomes has 2-deoxyglucosea enhances (2-DG), anti-tumour the effectand mouse-breast in human- cancer cells [103]. These results openthepossibil- growthsignificantly (52%)intreated mice [102].Recently, ithasbeendemonstrated that combining NDV virotherapy glucosewith analogue strategy for breast cancer therapy. A newly developed recombinant NDV that expresses IL12(rAF-IL12) hasbeenobserved to inhibittumour are relatively permissive to NDV infection.In vitro andin vivo data have shown NDV infection usually has minimal clinicalsymptoms, makingthis virus optimal for cancer treatment. This is due to the fact that cancer cells suppressive tumour microenvironment into apro-inflammatory environ antigen thataugments cytolytic T cell responsesinfectedagainst cells. NDV-induced apoptosis results intheconversion of animmune- OVs in vitro andimproved outcome in vivo againstbreastcancer xenografts [111].Intriguingly, agroupof researchers demonstrated that, [105]. The effectof NDV (PV701) hasbeenevaluated intwo BCpatients enrolled into aphaseI ment that antitumour supports immuneresponses. ­

the effectiveness of different strains of oncolytic NDV asnovel with favourable toxicity profile [107– 11 -

Review tolerated regimens, andpossibly improving quality of life. and dosages. In conclusion, oncolytic virotherapy seemsto show good promise for breast cancer therapy, with potentially effective and well- approach. On theother hand,anumber of preclinicaltrials are alternative studying combination approaches concerning delivery systems At present,there are severalOVsusing trials I andIIclinical phase to treat breast cancer patients (Table 1);mostof theseuseacombination promising candidate, dueto itstransfection capacity, easy manipulation, good safety profile andinability to integrate into thehostgenome. particular, inbreast cancer, novel reovirus, vaccinia virus andHSV are beendeveloping. Among these, vaccinia virus isconsidered to be very ity perturbatingwithout normalcells, improvingsystem immune activity againstcancer, reducing adverse events andensuringsafety. In The development of new oncolytic virus for thetreatment of breast cancer includesnew viral vectors targeting tumour cells with highaffin- ies, andpreferentially incombination with other anticancer therapies. proteins. To overcome this,itis recommended to use rapid infusion schedules, repeatedwith high doses to avoid neutralisingserum antibod - administrationis associated excessivewith and premature dilution neutralisation through anti-viral serum immunoglobulins or other serum approach,this injection: interventionalthe infact,simplifies procedures andimproves systemic delivery to distant metastasis. However, IV trationhas not yetbeen defined, to reduce adverse eventsand enhance OVs efficacy. In clinicaltrials,IV delivery may bepreferable to IT spontaneously, avoiding viremia. Today, theOVs are administrated viaIT andIV delivery, althoughthemostappropriate route of adminis- adverse eventsbycaused oncolytic virotherapy have rarely beenreported, while moderate adverse events canbecontrolled or disappear increase theknowledgebetweenmechanisms on specific OVs, system immune human andtumour, mainly to prevent adverse events. Severe systemimmune human and ManyTME. oncolytictrials including clinical virotherapy are phaseII/IIItrials(Table 1),dueto theneedto Oncolytic virotherapy islocatedcontext inthe of personalised medicine,since itsbestapplication isassociated to theinterplay between ecancer 2020, 14:1149; www.ecancer.org; DOI:https://doi.org/10.3332/ecancer.2020.1149 Small Cell Lung Cancer. Liver Carcinoma; CRC: Colorectal Cancer; PanA: Pancreatic Cancer; PC:Prostate Cancer; ER:Estrogen Receptor; PR:Progesterone Receptor; NSCLC: Non roidal Sodium Iodide Symporter; TNBC: Triple Negative Breast Cancer; MBC:Metastatic Breast Cancer; HNSCC: Head neckSquamous Cell Carcinoma; LC: Abbreviations: Table 1:Clinical Trials for breast cancer treatment usingoncolytic virotherapy. NCT03564782 NCT03004183 NCT02977156 NCT02779855 NCT02630368 NCT02285816 NCT01846091 NCT01656538 NCT01017185 NCT00636558 NCT00574977 ID Ad: Adenovirus; HSV: Herpes Simplex virus; MV: Measles Virus; VV: Vaccinia Virus; MV-NIS Oncolytic (MV): Measles Virus Encoding Thy - recruiting recruiting recruiting recruiting active, not recruiting recruiting recruiting active, not completed completed completed completed Ststus Phase I/II I/II I/II II II I I I I I I Actual enrolment 118 57 66 50 56 12 81 28 26 6 8 Stage II-IVStage TNBC TNBC, NSCLC CRC TNBC, Mesothelioma, HNSCC, Ductal BC,Ductal Carcinoma SarcomaMBC, AdvanceTissue Gastric Cancer MBC, NSCLC, Esophageal and Stage IV BC, TNBC HER2/Neu+, MBC,PR+,PR-, ER-, ER+,HNSCC, HER2/Neu-, MBC melanoma BC, refractory HNSCC, Skin, BC, Melanoma, PC PanA BC, Melanoma, HNSCC, LC, CRC, Type of disease PVSRIPO (oncolytic poliovirus) ADV/HSV-tk (oAd) Pexa-Vec (oVV) (oHSV) Talimogene Laherparepvec Jx-594 (oVV) vaccine)(AD and AdMA3 MG1MA3 (oncolytic Maraba) MV-NIS (MV) Reolysin (Reovirus) HF10 (HSV) CVA21 (Coxsackievirus) vvDD-CDSR (VV) Virus none stereotactic XRT Pembrolizumab, lpilimumab juvant) Paclitaxel (neoad - phosphamide metronomic cyclo- none none Paclitaxel none none none Additional Theraphy 12

Review 14. 13. 12. 11. 10.

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