Talimogene laherparepvec and atezolizumab in HER2-negative breast cancer following neoadjuvant chemotherapy: a window-of-opportunity phase II trial (SOLTI-1503 PROMETEO)

Domain: chemistry
Published: 2026-02-16
Editorial outcome: minor_revision

Abstract
Thissingle-arm,phaseII,preoperativewindow-of-opportunitytrial(Clinical-Trials.govIdentifier:NCT03802604)investigatedtheefficacyandsafetyoftalimogenelaherparepvec(T-VEC),anoncolyticvirus,withatezolizumab,ananti–PD-L1antibody,inpatientswithbreastcancerandradiologicallyandpathologicallyconfirmedresidualdiseasepriortosurgery.Eligiblepatientshadtriple-negativebreastcancer(TNBC)orhormonereceptor-positive(HR+)/HER2-negativediseasewithahighproliferationindex(Ki67≥20%)priortoneoadjuvantchemotherapy.TreatmentconsistedofoneintratumoralinjectionofT-VEC(106plaque-formingunits[PFU]/mL),followedbyfourbiweeklyT-VECdoses(108PFU/mL)plusatezolizumab(840mg,intravenously).Amongthe28patientsenrolled,20patients(71.4%)hadHR+/HER2-negativeand8patients(28.6%)hadTNBC.Atsurgery,7patients(26.9%)achievedResidualCancerBurden(RCB)−0/I(primaryendpoint),12(46.2%)RCB-IIand7(26.9%)RCB-III.Safetyprofilewasfavorable,withmostlylow-gradeadverseeventsandnoseriousevents(secondaryendpoint).Therapyinducedimmunemodulation,includingincreasedtumor-infiltratinglymphocytes,elevatedPD-L1expression,andenhancedimmune-relatedgenesignatures(exploratoryendpoints).Thetrialmetitspre-specifiedefficacyandsafetyendpoints.ThesefindingssupportthefeasibilityofT-VECplusatezolizumabasapreoperativeimmunotherapyapproachformanagingHER2-negativeresidualdiseasepost-neoadjuvantchemotherapyandwarrantfurtherexplorationinlargertrials.

Introduction
Despiteadvancesindetectionandtreatment,breastcancerremainsoneoftheleadingcausesofcancer-relatedmortalityinfemalepatientsworldwide. The advent of neoadjuvant chemotherapy (NAC) has improved surgical outcomes and provided prognostic information throughtheassessmentoftumorresponse1.However,thepresenceofresidualcancerafterNACislinkedtoapoorerprognosisandsignifiesanunmetclinicalneed2–4.Pathological complete response (pCR) hasemerged as a surrogatemarkerforlong-termoutcomesinbreastcancer,particularlyinaggressive subtypes such as triple-negative and HER2-positive cancers4.However,asignificantproportionofpatientsfailtoachievepCRandfaceahighriskofrecurrence(ROR)2–4.Thishaspromptedtheexplorationofnewtherapeutic strategies tomoreeffectivelytargetresidualdisease(RD).

Recent studies highlighting the role of the immune system in cancercontrol andthe therapeutic potential ofimmunomodulationhave garnered significant attention. Elevated levels of tumor-infiltratinglymphocytes(TILs)andspecificimmunegenesignaturesareconsistentlyassociated with improved survivaloutcomes across various breast cancer subtypes. This has led to the exploration of immunotherapies,includingimmunecheckpointinhibitors(ICIs)andoncolyticviruses,aspotentialtherapeuticapproaches.

Talimogenelaherparepvec(T-VEC)5–7,anoncolyticherpessimplexvirustype-1,andatezolizumab8,9,ananti-programmeddeath-ligand1(PD-L1) antibody, represent two such investigational therapeutic modalities.T-VECreplicatesexclusivelyintumorcellsandhasbeen genetically engineered to express granulocyte-macrophage colony-stimulating factor (GM-CSF), enhancing the immune response by activating dendritic cells and stimulating T cells5,6,10,11. Preliminary resultshaveshownthatthecombinationofT-VECwithICIscanboostanti-tumorefficacywithoutsignificantlyincreasingsafetyconcerns11–13.

In this work, we present the PROMETEO trial, a phase II study designedtoevaluatethesafety,feasibility,andbiologicalactivityofcombining T-VEC and atezolizumab in patients with HER2-negative breastcancerwithresidualdiseasefollowingNAC.

Results
Patientdemographicsandbaselinecharacteristics
Atotalof28patientswereenrolledinthisstudy(Fig.1).ThefirstpatientwasenrolledonJanuary8,2019,andthelastpatientonFebruary1,2022.Out of 28 patients, 20 (71.4%) had HR+/HER2- breast cancer and 8 (28.6%)hadtriple-negativebreastcancer(TNBC)(Table1).Themedianage was 47 years (range 31 to 70), with 74% of them being pre-menopausal.Atbaseline,priortoreceivingNAC(pre-NAC),60.7%ofthecohort had clinical stage II disease, and 60.7% were clinically node-positive. The median Ki-67 proliferation index was 37.5% (range 20–95%).High levelsofTILs(≥10%)wereobservedin36%ofbaseline samples.Only14%ofpatientshadPD-L1-positivetumors(SP142,immunecells≥1%)atbaseline.Tumormutationalburden(TMB)wasevaluablein 22of28pre-NACsamples,and5ofthese(22.7%)wereclassifiedasTMB-high(≥10mutations/Mb).ThemedianTMBwas3mutations/Mb(range 0–19),withnotumorsidentifiedasmicrosatelliteinstability-high.

At screening, after receiving NAC (post-NAC), the mean tumor size by magnetic resonance imaging (MRI) was 28.3mm (range 10–93mm).TheCONSORTdiagramandthebreakdownofavailable samplesforeachbiomarkerateachtimepointareshowninExtended DataFig.1.

Efficacyofthetreatment
Theprimaryendpointwasresidualcancerburdenclassof0/I(RCB-0/I) rate at surgery. Secondary outcomes included pCR rate and radiologicalresponse.Outofthe28patientsenrolled,26wereevaluable fortheprimaryendpoint(Fig.2a).Twopatientswerenotevaluable: one withdrew consent after the first dose of T-VEC, and another experienceddiseaseprogressionaftercycle2andbegancapecitabine priortosurgery.Threeofthe28patients(10.7%)didnotcompletethe fullstudytreatmentandproceededdirectlytosurgery.Onepatient discontinuedafterCycle3duetotheonsetoftheCOVID-19pandemic, whiletwopatientsstoppedafterCycle4duetodiseaseprogression (ExtendedDataFig.1).

ARCB-0/Iwasobservedin7patients(25.0%,95%CI:10.7–44.9%), allofthemachievingpCR(RCB-0),showingastatisticallysignificant improvementoverthenullhypothesis(p-value<0.001)(Fig.2a).RCB-IIwasachievedin12patients(42.9%,95%CI:24.5–62.8%),andRCB-IIIin 7 patients (25.0% 95% CI: 10.7–44.9%). Among those with pCR, 6 (86.0%)hadHR+/HER2-disease.Overall,thepCRratesforHR+/HER2- and TNBC were 30.0% (95% IC: 11.9–54.3%) and 12.5% (95% IC: 0.3–52.7%),respectively(Fig.2a).Additionally,onepatientintheRCB-IIcategoryexhibitedpCRinthebreastbutnotintheaxilla.

RegardingtheradiologicalresponsebyMRI,15patients(58%)had stabledisease(SD),2(8%)showedapartialresponse(PR),1(4%)hada completeresponse(CR),and8(31%)experiencedprogressivedisease (PD).Importantly,theradiologicalresponsedidnotalwaysalignwith thepathologicalresponse;PRandCRwereobservedinsomecases classified as RCB-II/III but not in cases with a pCR, while PD was observedinsomepatientswhoachievedapCR(Fig.2b).

Figure2cillustratestheclinicalcharacteristicsandresponsesoftwo patients. Figure 2d summarizes the baseline characteristics of the patients who achieved pCR after treatment in the trial. Notably, the patientwithTNBCwhoachievedpCRhadapre-NACPAM50Basal-like subtype,TILsof35%,PD-L1IHCof3%,andaTMBof19.Despitethe differencesinthe6patientswithHR+/HER2-tumors,patientsexhibited significantTILsandanotablepresenceoftertiarylymphoidstructures (TLS)andPD-L1expression,whichmaycorrelatewiththeirfavorable responses,leadingtopCR.AllHR+/HER2−patientswhoachievedpCR hadhighlevelsofestrogenreceptorexpression,rangingfrom80%to 100%byIHC.Additionally,theclinicalandmolecularcharacteristicsofthethreepatientswhoexperienceddiseaseprogressionduringtreatmentanddidnotcompletetheplannedfivecyclesaresummarizedina supplementarytable(SupplementaryTable1).

Safetyandtolerability
Safetywasassessedasasecondaryoutcome.Amongthe28patientswho received the study treatment, 92.9% experienced treatment-emergentadverseevents(AEs)ofanygrade(Table2).Themostcommon grade 1 or 2 AEs were fever (11 patients), increased alanine aminotransferase and aspartate aminotransferase (AST) levels (8 patients each), arthralgia (6 patients), and anemia (3 patients). One patient experiencedgrade3reversibleneutropenia.Nograde4or5AEswere reported,underscoringthetreatment’soverallmanageabilityandsafety. Importantly, no serious AEs were reported, and no deaths occurred duringthestudy.

Impactontumorbiology
Exploratoryanalyzeswereperformedtoevaluatebiologicalchangesin thetumoranditsmicroenvironment.Asexpected,allPAM50subtypes wereidentifiedandshowedsignificantdifferencesaccordingtohormone receptor status (p<0.001). In HR+ disease, the majority of tumors were classified as Luminal B (n=9), followed by Luminal A (n=6), Basal-like (n=1), Normal-like (n=1), and HER2-enriched (HER2E) (n=1). In TNBC, tumors were primarily identified as Basal-like(n=6)andNormal-like(n=2).Asanticipated,NACenrichedthe populationforLuminalAandNormal-likePAM50subtypesattimeof screening.TreatmentwithT-VECandatezolizumabfurtherincreased theproportionofNormal-liketumors.Notably,atthetimeofsurgery, theonlyTNBCtumorthatachievedpCRwasclassifiedasNormal-like, while the remaining TNBC tumors were Basal-like. Meanwhile, the majority of HR+/HER2- tumors were classified as Normal-like, even whenRDwaspresent(ExtendedDataFig.2).

Consistentwiththesubtypingresults,LuminalAandNormal-like signatureswereenrichedatscreening,whileotherPAM50signatures, including the proliferationsignature,weredownregulated following NAC. This pattern was particularly evident in the HR+ population, whereasnosignificantchangeswereobservedinTNBCtumorsafter NAC treatment. During treatment with T-VEC and atezolizumab, no notable changes were detected in the PAM50 signatures (Extended DataFig.3).

Impactontumormicroenvironment:TILs,TLS,andPD-L1 expressionacrosstimepoints
ThecombinationofatezolizumabandT-VECsignificantlyaffectedtumor microenvironment(Fig.4a).TILsshowedamarkedincrease(P=0.0009) throughoutthetherapy.TheproportionsofTILs≥10%atbaseline(pre-NAC),screening(post-NAC),cycle2day1(C2D1),cycle3day1(C3D1),andsurgerywere37.0%,14.8%,34.6%,53.8%,and65.4%,respectively (P=0.0056).SimilartrendswereobservedforTLS,thedistributionof tumor-immunephenotypes,andPD-L1IHCexpression(Fig.4b–d).

The percentages of TILs and PD-L1 IHC in baseline samples showedasignificantdifferencebetweenpatientswhoachievedapCR andthosewithRD.ThemedianbaselineTILswere15%forpatientswith pCR versus 2% for patients with RD (odds ratio [OR]: 1.08; 95% CI: 1.004–1.16,P=0.008),andthemedianbaselinePD-L1IHCwas1%for pCRpatientsversus0%forRDpatients(OR:3.04;95%CI:0.78–11.8; P=0.017)(ExtendedDataFig.4).Additionally,thepercentageofPD-L1 cellpositivity(usinga≥1%cutoff)inbaselinesampleswashigherin patientswhoachievedpCRcomparedtothosewithRD(75%vs.18.18%; Chi-squaredP=0.021).

At screening, the TIL percentages continued to differ between pCRandRDgroups,thoughthisdifferencewasnotstatisticallysignificant(OR:1.13;95%CI:0.93–1.37;P=0.19)(ExtendedDataFig.5). Notably,theonlysamplewithpositivePD-L1expressionfollowingNAC wasfromapatientwhoachievedpCR.

Importantly,patientswithpCRandRDshowedanincreaseinTILs andPD-L1expressionfollowingtreatmentwithT-VECandatezolizumab, suggesting that the therapy induces changes in the tumor microenvironmentregardlessofthefinalpathologicalresponse(Fig.5a,b).

Impactontumormicroenvironment:immunegeneexpression signaturesacrosstimepoints
Consistent with the observed increases in histological data, gene expression analysis revealed significant changes in immune gene expression throughout the study treatment. A total of 201 immune gene expressionsignatures(iGESs)werederivedfromRNAsequencing (RNAseq) data, with a comprehensive list of signatures provided in Supplementary Data 1 and 2. Supervised hierarchical clustering was performed on study samples, and the resulting heatmapof median signatureexpressionisshowninFig.4e.Thisheatmapshowedapro-minentclusterofsignatureswithincreasedexpressionatCycle2and Cycle3,whichremainedelevatedinsamplescollectedatthetimeof surgery.Conversely,asmallerclusterofsignaturesshoweddecreased expressionfrombaselinetosurgery,primarilyassociatedwithinterferon,exhaustedTcells,andTh2andTh17Tcellsignatures(Fig.4e) (SupplementaryData3).

Pairedtwo-classsignificanceanalysisofmicroarrays(SAM)analysiscomparingsurgicalsamplestoscreeningsamplesindicatedthat 178signatures(88.6%)hadincreasedexpression,while23signatures (11.4%)showeddecreasedexpression(Fig.4f,g).Notably,91.5%ofthe signaturesexhibitedastatisticallysignificantchange(falsediscovery rate[FDR] < 1%)(SupplementaryData4).ThemostsignificantchangeswereobservedfollowingthefirstdoseofT-VEC(C2D1)andthefirst dose of the combined T-VEC and atezolizumab treatment (C3D1) (Fig.4h).Nosignificantdifferenceswereobservedwhentheanalysis wasrestrictedtoTNBCorHR+/HER2-breastcancersubtypes(Fig.4i).

When examining the iGESs in baseline samples, 22 out of the 201signatures(10.9%)weresignificantlycorrelatedwithpCR.Notable signatures included those more associated with adaptive immune responses, including interferon signaling, TLS, activated dendritic cells, and MHC-Iexpression(Extended Data Fig. 5). When the same analysiswasconductedonscreeningsamples,33outofthe201signatures(16.4%)werecorrelatedwithpCRfollowingthetrialtreatment (Extended Data Figure 6). These show a tendency toward innate immuneactivation,characterizedbythepresenceofM2macrophages, granulocytes,andnaturalkillercells.Similartotheobservedincrease inTILs,therewasalsoanincreaseiniGESsthroughoutthetreatmentin bothpatientswhoachievedpCRandthosewhodidnot(Fig.5c).

Patientlong-termoutcomes
A post-hoc exploratory analysis of disease-free survival (DFS) was conductedtoassesslong-termoutcomes.Followingsurgery,among patients with TNBC, one patient with pCR and four patients with residual disease received adjuvant capecitabine. Additionally, one TNBCpatientreceivedadjuvantdocetaxelincombinationwithbevacizumab. Among patients with HR+/HER2− disease, a total of 19 receivedadjuvantendocrinetherapy,includingonewhowastreated withabemaciclibfor2years.Allpatientsunderwentadjuvantradiotherapyasclinicallyindicated.

Atamedianfollow-upof44.6months,atotaloffourDFSevents (9.4%)wereobserved,allcorrespondingtodistantmetastaticrecurrences,twoinpatientswithTNBCandtwoinpatientswithHR+/HER2- disease.Twooftheseeventsresultedinbreastcancer-relateddeath. Notably,noneoftheDFSeventsoccurredinpatientswhoachieved pCR.The4-yearDFSratewas100%amongpatientswithpCRand77.4% amongthosewithRCBII/IIIdisease(Fig.3).

Methods
Studydesignandparticipants
PROMETEO was a single-arm preoperative window-of-opportunity phaseIItrialcarriedoutin4sitesacrossSpain.Thefirstpatientwas enrolledonJanuary8,2019,andthelastpatientwasonFebruary1, 2022.Eligiblefemaleparticipantswereaged≥18yearswithanECOG performance status of 0 or 1 and an operable early breast cancer, specificallytriple-negativeorLuminalB-like/HER2-subtypes,withRD evidentaftercompletionofNACpriortosurgery.Thestudyrecruited only female participants due to the lack of experience with T-VEC injectionsinmenwithbreastcancer.Eligibleparticipantsmusthave hadRDwithatleastonelesionmeasurablebyMRIcompletingNAC andpriortosurgery,withthelongestdiameterbeing≥10mm,confirmedbyacoreneedlebiopsyduringthescreeningprocess,andwere candidates for primary surgery. Tumor markers such as estrogen receptor (ER), progesterone receptor (PR), and HER2 status were determinedinpre-NACtumorbiopsiesaccordingtoAmericanSociety of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines, with Luminal B-like disease defined as ER≥10% and/or PgR≥10%,andKi-67levelsabove20%.

Patients were also required to have completed ≥80% of an anthracycline/taxane-based neoadjuvant regimen, as recommended bytheNationalComprehensiveCancerNetwork(NCCN)guidelines, with an option to include carboplatin. Eligibility also depended on adequate hematologic and organ function, and no significant psychological,familial,sociological,orgeographicconstraintsthatcould affecttheadherencetotheprotocol.Inaddition,patientsshouldhave resolved any acute toxic effects of prior NAC to a National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 4.03 grade ≤1, excluding alopecia and other non-safety related toxicities. Lastly, prior therapy with an anti-programmed death (PD)-1, anti-PD-L1, anti-PD-L2, anti-CD137 antibody, or anti-cytotoxicT-lymphocyteassociatedprotein-4(CTLA-4)antibodycompound,T-VECoranyotheroncolyticimmunotherapywasnotallowed.

Thestudydesignandconductwereexecutedinfullcompliance withallrelevantregulationsconcerningtheuseofhumanstudyparticipantsandadheredtotheprinciplesoftheDeclarationofHelsinki and Good Clinical Practice guidelines and local regulatory requirements.Writteninformedconsenttoparticipateinthetrialandconsent topublishany identifiable images ofthe researchparticipantswere obtainedfromallpatients.

The PROMETEO trial was registered at ClinicalTrials.gov (NCT03802604).ThefullstudyprotocolandStatisticalAnalysisPlan (SAP)areavailableintheSupplementaryInformationunderthesection“SupplementaryNote.”

Intervention
TheinterventionconsistedofintratumoralinjectionsofT-VECfollowed byintravenousadministrationsofatezolizumabpriortosurgery.The initialdosewassetat106PFU/mL,servingasaprimingdosetoinitiate theoncolyticprocessandpreparetheimmunesystemforamorerobust response. Three weeks after the initial dose, the treatment regimen continuedwithT-VECataconcentrationof108PFU/mL.Thishigherdose wasadministeredbiweeklyforatotalof4doses.Thedoseescalation aimedtomaximizeoncolyticactivitywithinthetumormicroenvironmentandfacilitateastrongerlocalandsystemicimmuneresponse.

T-VEC was administered by a breast radiology specialist under ultrasoundguidancetoensureaccuratedeliveryintothetumormass. AllpatientshadaclipplacedinthetumorpriortothestartofNACas partofstandard-of-careprocedures,whichfacilitatedlocalizationof residualdisease.

Concurrent with the second dose of T-VEC, atezolizumab was introduced into the treatment regimen. Atezolizumab was administered intravenously at a doseof 840mg, consistent with the T-VEC treatment schedule. It was administered every 2 weeks, concurrent withT-VECadministration,foratotalof4cycles.

AtC2D1andC3D1,acore-needlebiopsywasmandatoryjustprior totheT-VECinjectionprocedure.MRIwasperformedaftercompletion ofNACandpriortostudyentrytoconfirmthepresenceofmeasurable residualdisease(longestdiameter≥10mm),aspartoftheeligibility assessment.AsecondMRIwasperformedbetweenthelastdoseofT-VEC/atezolizumabandsurgery,toallowforradiologicalassessmentof responseaccordingtoResponseEvaluationCriteriainSolidTumors (RECIST)v1.1criteria.

Surgery was performed between 1 and 3 weeks after the last treatmentdose.Thelastsafetyfollow-upvisitwasdone30daysafter surgery.Standardadjuvanttherapywasadministeredatthediscretion ofthetreatingphysician.

Objectivesandoutcomemeasures
Theprimaryobjectivewastoevaluatetheefficacyofthecombination treatment in achieving RCB-0/I at surgery. Secondary objectives include evaluation of the safety and tolerability of the combination treatment,overallresponserates,pCRrates,DFS,andchangesinthe expressionofimmune-relatedgenesignatures.Surgicalresectionwas performed1to3weeksafterthelasttreatmentdose.pCRwasdefined astheabsenceofresidualinvasivecancerinbreastandaxillafollowing clinicaltrialtreatmentwithT-VECandatezolizumab(ypT0/isypN0). TheRCBindexwascalculatedaccordingtoSymmansetal.31.

TheradiologicalresponsewasaccordingtoRECISTassessedby MRIandcorebiopsyatmultipletimepointsthroughoutthetreatment regimen.

Pathologyreview
Thestudyincludedarobusttranslationalresearchcomponentaimed atidentifyingbiomarkersoftreatmentresponse.Atotaloffivebiopsiesperpatientweresystematicallyobtainedatdifferenttimepoints: baseline (pre-NAC), screening (post-NAC), C2D1, C3D1, and surgical specimen. All biopsies were formalin-fixed and paraffin-embedded (FFPE)forsubsequenthistopathologicalandbiomarkeranalyzes.

Hematoxylinandeosin(H&E)stainedsectionsfromeachsample were subjected to an independent central pathology review. At screening,oneH&Esectionwasexaminedtoconfirmthepresenceof invasive tumor cells and to determine the minimum tumor surface area.ForsamplesobtainedatC2D1,C3D1andsurgery,thosewithout invasive tumor cells were also profiled. Baseline samples were collectedretrospectivelyfrompatientsenrolledinthestudy.

An immunohistochemical (IHC) study was performed for PD-L1 (VENTANA®PD-L1[SP142]assay,RocheDiagnostics),andpositivitywas evaluated in immune cells32. PD-L1 IHC was conducted on tumor samplesobtainedatbaseline,post-NACscreening,andsurgery.

TILsat alltime points wereevaluated centrally on H&E-stained whole-mounttumortissue,blindedtoclinicopathologicandoutcome data.PercentagesofTILsatbaseline,screening,C2D1andC3D1were scoredinslidesofcorebiopsies.TILswerequantifiedaccordingtothe GuidelinesdevelopedbytheInternationalTILsWorkingGroup33,34.The reproducibility of this method has been described previously. TLS weredefinedasspatiallyorganizedclustersofimmunecellaggregates withinthetumor,withorwithoutgerminalcentersonH&Eslides35.The spatialdistributionofTILswasanalyzed,classifyingtumorsintothree distinctcategoriesbasedonTILslocationrelativetotumorcells:(1) immune-desert, (2) immune-excluded, and (3) immune-inflamed, basedontheminimalorabsenceofTILs,thepresenceofTILspredominantlyattheperipheryofthetumormargin,andtheinfiltration ofTILsamongtumorcells36.

IntrinsicPAM50subtyping
RNAwasextractedfrombaselinepre-treatmentFFPEtumorsamples usingtheHighPureFFPETRNAisolationkit(Roche,Indianapolis,IN, USA).Onetofive10-μmFFPEslidesdependingontumorcellularity were used for each tumor sample, and macrodissection was performed, when needed, to avoid normal tissue contamination. A minimum of 100ng of total RNA was analyzed on the nCounter platform37(NanostringTechnologies,Seattle,USA)usinga192-gene custompanel38,whichincludesthe50genesofthePAM50testand5 housekeepinggenes(ACTB,MRPL19,PSMC4,RPLP0,andSF3A1).Data were log base 2 transformed and normalized. All tumors were assigned to an intrinsic molecular subtype (Luminal A, Luminal B, HER2E,Basal-like)ortheNormal-likegroupusingtheresearch-based PAM50 subtype predictor39. The PAM50 ROR was calculated using weightedcoefficientsforthefoursubtypesandaproliferationscore usingapreviouslyreportedandvalidatedformula40.PAM50analysis wasperformedonallavailabletumorsamplescollectedatdifferent timepointsthroughoutthestudy(baseline,post-NACscreening,C2D1, C3D1, and surgery). Extended Fig. 1B shows the molecular analyzes conductedpersampleforeachpatient.

TotalRNAsequencingandimmunegeneexpressionsignatures
TotalRNAseqlibrarieswerepreparedbytheLinebergerComprehensive CancerCenter(LCCC)TranslationalGenomicsLab(TGL)from100to 1000ngoftotalRNAusingaHamiltonNGSSTARliquidhandlerandthe TruSeqStrandedTotalRNALibraryPrepGoldkit(Illumina,#20020599) following the manufacturer’s protocol (Illumina, #1000000040499). RNAseq library quality and quantity were measured using the 4200 TapeStationsystem(Agilent,#G2991AA)andQubitTMFlexFluorometer (Thermo Fisher Scientific, #Q33327), respectively, pooled at equal molar ratios, and denatured following the manufacturer’s protocol. SequencingwasperformedatTGLusingtheNovaSeq6000S4system (Illumina)witha2×50bppaired-endconfigurationtoadepthof~110 million clusters per library on average. Raw sequencing data were processed and analyzed by the LCCC Bioinformatics Core. Quality-control-passed reads were aligned to the human reference GRCh38/ hg38genomeusingtheSplicedTranscriptsAlignmenttoaReference (STAR)aligner41.Transcriptabundanceestimatesforeachsamplewere performed using Salmon41, an expectation-maximization algorithm usingtheUniversityofCaliforniaSantaCruz(UCSC)genedefinitions. Raw read countsfor all RNAseq sampleswere normalized toafixed upper quartile41. The raw read files are available in the European Genome-phenome Archive (EGA) (accession EGAS50000001499). RNAseq analysis was performed on all available tumor samples collectedatdifferenttimepointsthroughoutthestudy(baseline,post-NAC screening, C2D1, C3D1, and surgery). Extended Fig. 1B shows the molecularanalyzesconductedpersampleforeachpatient.

Wequantifiedtheexpressionof202iGESsderivedfrom43studies, detailedinSupplementaryData1and2.TheseiGESsweresubsequently organizedinto22distinctimmunecategoriesbasedongeneontology analyzes,usingCIBERSORTmethodforreferencecategorization42,43.

TargetedDNAsequencing
Inaddition,weperformedDNAsequencingofbaselinetumorsamples usingtheFoundationOne®CDx(F1CDx,Foundation Medicine) panel, whichincludesmutationalstatusof335cancergenesandinformation regardingTMBandMicrosatelliteInstability(MSI).

Samplesizeandstatisticalmethods
Forthesamplesizecalculation,weinitiallyassume≥20%RCB0/Iin pathologic tumor response at surgery according to MD Anderson CancerCenterrecommendations31bylocalassessmentwiththestudy treatment. The null hypothesis was to have 5% RCB 0/I, while the alternative hypothesis was to increase this percentage to 20% with T-VEC with atezolizumab. Considering a type I error of 0.05 (one-sided)and80%statisticalpower,therequirednumberofpatientsfor thefinalanalysiswas26evaluablepatientsinasingle-stagedesign.The hypothesistestwasconductedusingaone-proportionexactbinomial testintheintent-to-treat(ITT)population.Assuminga12%attrition rate, 30 patients will be included. The statistical framework of the studyisdesignedtoprovidepreliminaryefficacydataforthedesignof largerconfirmatorytrials.

An interim analysis was planned in the safety phase after the fourthpatientcompletedthetreatmentperiodordiscontinuedstudy treatment due to PD or toxicity. An Independent Safety Data Monitoring Committee (ISDMC) reviewed all study data. Following this initial analysis, the ISDMC supported continuation of the study to enrollanadditional6patients.Uponcompletionofthesecondanalysis,theISDMCdeemedthestudytobesafeandupto20additional patientswereenrolled.

Secondary endpoints including pCR rate, ORR, and safety outcomeswereanalyzedusingdescriptivestatistics.Continuousvariables, suchaschangesingeneexpressionortumorsize,weresummarized using the mean, median, standard deviation, and interquartile range. Categoricalvariables,suchasincidenceofadverseevents,weresummarizedusingfrequenciesandpercentages.Associationofeachvariable withpCRwasdeterminedbyunivariatelogisticregressionanalysis.Odds ratiowitha95%confidenceinterval(CI)wasestimated.

SurvivalendpointswereestimatedusingtheKaplan–Meiermethod. StratifiedunivariableCoxproportionalmodelswereusedtoestimate hazard ratios. The median follow-up time was calculated using the reverseKaplan–Meiermethod.Nodataimputationwascarriedoutand allanalyzeswereperformedusingRstatisticalsoftwareversion4.3.2.

To identify genes and signatures whose expression was significantlydifferentbetweenpairedorunpairedsamples,weusedSAM,a permutation-basedmethodthatcontrolstheFDR,withanFDRthresholdsetat<1%44.Allstatisticaltestsweretwo-sided,andthesignificance level was set at <0.05. No data imputation was performed, consequently,onlyavailableinformationwasusedforthebiomarkeranalysis. AllanalyzeswerecarriedoutusingRstatisticalsoftwareversion4.3.2.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript submitted by Pascual et al. describes a single-arm, preoperative window phase II trial evaluating the combination of talimogene laherparepvec (T-VEC) and atezolizumab in patients with HER2-negative residual breast cancer after neoadjuvant chemotherapy and before surgery. The authors are to be commended for their innovative and well-executed study, which provides valuable insights into the immunomodulatory effects of this novel combination therapy. The manuscript can be further improved by addressing the following comments:\nTitle and Abstract:\n• The trial design is not clear from the title nor the abstract. This is a preoperative window study. The term “residual disease” traditionally refers to residual disease after surgery, not before surgery. It is strongly suggested to edit title, abstract, and other aspects in the paper to more clearly define and describe the window trial design.\nMethods:\n• In the “Study Design and Participants” section of the methods, there are two sentences that are redundant and should be streamlined. Specifically, the sentences: (1) “The study recruited only female participants due to the lack of experience with T-VEC injections in men with breast cancer.” (Lines 296-297); (2) “The inclusion of male patients was not considered due to the lack of previous studies of intratumoral administration in this population.” (Lines 304-305). These sentences convey the same information. To improve clarity and conciseness, please consider omitting one of these sentences.\n• How as the T-VEC administered? Who administered the agent (radiology? Surgery?)? Was radiology used to target the residual disease? Was a clip present in the tumor, as with a number showing pCR, could they have radiologically visualized?\n• For RECIST analysis of response, when was the MRI performed? Should this be added to the Figure 1 Schema?\n• Serial biopsies: can it be specified which biopsies are fresh biopsies and which are FFPE only?\n• PAM50 subtyping: Which samples were used for this analysis? All samples? Which timepoints?\n• RNA sequencing and immune gene signature: which samples were used for this analysis? Please consider if information should be added to Figure 1 to indicate which correlative analysis were performed at each timepoint.\n• Statistics: first sentence: is there a reference for predicted rates of RCB MDACC recommendations? Please either add a reference or describe why there is no reference.\nResults:\n• In the results section, the authors report that “patients with pCR and RD showed an increase in TILs and PD-L1 expression following treatment with T-VEC and atezolizumab, suggesting that the therapy induces changes in the tumor microenvironment regardless of the final pathological response (Fig 4a, 4b).” Subsequently, in the discussion, the authors mention that “the increase in TILs observed in the trial supports the hypothesis that the combination therapy not only initiates an innate immune response but also sustains an adaptive immune response capable of recognizing and eliminating tumor cells.” While the results suggest that changes in the tumor microenvironment occur regardless of the pathological response, the discussion implies a direct link between these changes and the elimination of tumor cells. These interpretations appear to go in opposite directions. Please justify how their suggestion in the discussion is supported by the results section and elaborate on whether the observed immune changes are sufficient to infer tumor cell elimination or merely reflect immune activation without definitive clinical impact.\n• For the 6 patients who had pCR, what was their ER percentage?\n• Of the 28 patients enrolled, three experienced disease progression during treatment. Please provide more information about these patients. What subtype of disease? What other characteristics were notable?\n• Impact on Tumor Biology section – there appears to be a repetitive sentence about enrichment of luminal A and Normal PAM50 at screening. Please adjust and remove repetitive text.\n• Tumor biology, tumor microenvironment, etc. The organization of these sections is difficult to interpret in terms of assay and timepoints, as the text jumps back and forth in each section. It is recommended to reorganize this section for improved clarity. For example, one section about TILs, timepoints, trends. One section on iGES – what timepoints was this assessed, what were the iGES identify, and how did they change over time? One section on PD-L1 testing, and consider adding ER percentage if that is known.\n• Fig. 1 | Study design. Please specify the clinical stages of breast cancer included in the study rather than using the general term “early breast cancer.”\n• Table 1 under “Ethnicity” appears to mix race and ethnicity, as it includes classifications such as “White,” “Arab,” and “Hispanic,” which encompass both racial and ethnic categories. Please clarify the criteria used for this classification. To improve clarity and consistency, consider aligning with standardized approaches for reporting race and ethnicity.\n• Table 1: Can ER percentage be added? Especially given activity of IO in ER low disease observed in KN756 and Checkmate 7FL.\nDiscussion:\n• The authors correctly acknowledge in the discussion that the prognostic value of RCB and pCR in this context, where residual disease post-NAC achieves a pCR after two months of innovative therapy, remains unclear. This is a valid point, as RCB and pCR have been increasingly challenged as a surrogate for long-term outcomes in immunotherapy trials (e.g., GeparNuevo). This is particularly relevant in the PROMETEO trial, where 20 of the 28 included patients had ER+ tumors, a population for which pCR is a questionable surrogate for survival outcomes. It is also worth noting that EFS or DFS is not captured as a secondary endpoint in the trial, which further limits the understanding of the long-term impact of the intervention. The authors should include this limitation, explain the rationale for choosing pCR as the primary endpoint in this study, and detail plans to report the effect of the intervention on EFS or other long-term outcomes.\n• Immunotherapy has now been incorporated into the standard neoadjuvant treatment of patients with early-stage TNBC, with immune checkpoint inhibitors (ICIs) being used in combination with chemotherapy. In the PROMETEO trial, residual disease (RD) was defined after neoadjuvant chemotherapy alone, without the inclusion of ICIs, which no longer reflects the current standard of care. This distinction is critical, as the evolving treatment landscape could significantly affect the efficacy and safety outcomes observed in this study. Furthermore, the lack of a comparator arm with chemotherapy and systemic immunotherapy alone, without the addition of T-VEC, limits the ability to isolate and interpret the specific contribution of each treatment component in the observed outcomes. The authors are encouraged to contextualize their results within this framework, highlighting how these factors might influence the generalizability and relevance of their findings in the current therapeutic landscape.\n• In the early-stage TNBC setting, the IMpassion030 and IMpassion031 trials evaluated the addition of atezolizumab to standard chemotherapy in patients with early-stage TNBC. However, these studies did not demonstrate a significant improvement in outcomes. Please discuss how the choice of atezolizumab as the systemic immunotherapy backbone in this study might have influenced their results. It would be important to discuss whether the observed efficacy in their trial is specific to the combination with T-VEC or whether it may be limited by the inherent performance of atezolizumab in this setting.\n• This type of clinical trial design, offering a window of treatment before surgery for clinical RD, is a novel design and, like I-SPY, could be an interesting platform for future trial design. Please consider adding some text to the discussion about the trial design and future applicability/challenges.\n• This is a very small pilot study which shows feasibility; it is too small to draw firm conclusions, but suggests further study is of interest. Therefore the text of the Discussion should be more restrained. For example, at Line 234, these results do not suggest adding T-VEC to NAC could improve RCB rate in TNBC as that was not at all studied in this trial. However one could say that future analysis of this combination would be of interest for further study. Line 236: would not call rate of pCR “high” (only 7 patients) but instead would refer to it as a signal of activity. Line 261, would remove the phrase “groundbreaking approach.” Line 283: would remove the word “revolutionize” and replace with a toned down word, for example “advance.” Line 284: would remove the word “high” pCR rates, as this is a signal of activity, not a high rate in a well powered study.", "reviewer_2": "Pascual et al. report on an interesting clinical trial of T-VEC with atezolizumab in patients with residual breast cancer after neoadjuvant chemotherapy in the SOLTI-1503 PROMETEO phase II trial. This trial is the first to report on combination T-VEC with immune checkpoint blockade in localized breast cancer in the neoadjuvant setting. The investigators used an innovative clinical space for this concept by treating in the window before surgery in patients that had residual disease after standard of care neoadjuvant chemotherapy. In addition to reporting on clinical outcomes, which were especially intriguing in the pCR rate in the ER+ population, they demonstrating longitudinal increase in immune inflammation with treatment through carefully collected and analyzed patient samples. This manuscript would be further improved by addressing the following points:\n1) For patient baseline characteristics section of results, it would be interesting to also include the % of tumors with high TMB.\n2) One main limitation in the concept and using pCR/RCB as endpoint is that T-VEC is a local therapy. Therefore there is a high chance that improved outcomes by these measures translate to a local effect and it is unclear what significance is from systemic standpoint. Discussion of this should be expanded.\n3) Along the lines of the local effects, are there systemic samples that can be analyzed to understand effect on systemic immunity?\n4) Samples without presence of tumor were included in C2D1 and later timepoints. This could theoretically impact results if tumor bed for example was not adequately sampled and enrich for microenvironment from non-tumor tissue. Acknowledging this is an inherent challenge of translational research, it would be helpful to clarify as needed or address this further in results and control for potential confounding of results.\n5) As the authors acknowledged it is difficult to distinguish clinical effects of T-VEC vs atezo in this tough some correlative data support T-VEC has some immune role. Especially in the TNBC cohort where the pCR rate is relatively low and comparable to the increase achieved with addition of neoadjuvant ICB to standard chemotherapy such as in KEYNOTE-522, is this interpreted to really be an increase from that achieved from ICB itself?\n6) For the gene expression profiling, lack of single cell resolution or spatial context is a limitation. It would be helpful even if for select cases to perhaps look at spatial expression to better support findings.", "reviewer_3": "This is a single-arm phase 2 trial with primary objective is to evaluate the efficacy of the combination treatment (T-VEC + atezolizumab) in achieving RCB 0/I at surgery. The study reported primary and secondary outcomes as well as the biomarkers of treatment response of the total 28 eligible patients. The presentation is clear and the data analysis method is reasonable and the results are presented according to the SAP. The work is considered original, and the results support the claims.\nOne minor suggestion about the results: add the 95% CI for the primary endpoint.\nHere are some concerns and limitations that must be considered:\nNeed more details of the SAM analysis, at least add the reference to the method.\nLine 417-425: the sample size was determined based on the hypothesis test of RCB 0/1 rate of 20% vs 5%: “The null hypothesis (p0) was RCB-0/I observed in 5% of cases (p0 = 0.05), while the alternative hypothesis was p1 = 0.20. Considering a type I error of 0.05 and a type II error of 0.20 in a two-sided test, the required number of patients for the final analysis was 26 evaluable patients”. However, there is no statistical test reported in the SAP or the manuscript. Also, there is no justification of selecting p0=5% in the protocol. Please clarify.\nIncomplete Adjustment for Confounding Factors\nLine 437. The study uses univariate logistic regression to assess the relationship between tumor biomarkers (e.g., TILs, PD-L1) and response to treatment, but does not adjust for confounding variables like tumor stage, previous treatment response, or molecular subtype. A multivariate analysis would help clarify which factors truly influence response rates.\nHandling of Missing Data is Unclear\nLine 424. Sample size includes 12 % attrition rate which mean that some patients did not complete treatment (e.g., one withdrew consent, two had disease progression before surgery), but it is not clear how their data were handled in the analysis. Similarly, the study does not explicitly state whether missing values in gene expression data were imputed or simply excluded. Could you please clarify how the missing data were addressed, and how missing values in gene expression data were handled?", "reviewer_4": "This paper describes a Phase II study giving an oncolytic herpes simplex virus, talimogene laherperepvec (T-Vec) with an anti-PDL1 antibody, atezolizumab, to patients with residual disease after neoadjuvant chemotherapy (NACT) for breast cancer. These patients had triple negative breast cancer (TNBC, 8 patients), or HR+/HER2- breast cancer (20 patients), with a high proliferation index prior to NACT. It is important to note (and should be discussed in the manuscript) that, whilst immunotherapy (IO) with immune checkpoint blockade (ICB) is now standard of care as part of neoadjuvant/adjuvant treatment in TNBC, ICB remains unproven in HR+/HER2- disease. Hence this cohort represents a mixture of patients in terms of their likely response to IO based on previous evidence from the more advanced disease setting, and in the context of current treatment regimes. Nevertheless, in this early trial setting, inclusion of both TNBC and HR+/HER2- groups is reasonable, and the findings of activity in HR+ disease may in particular be of wider significance, suggesting that IO can benefit more patients with breast cancer than currently thought.\nThe treatment had an acceptable safety profile, and led to promising pathological responses by the time of surgery. In particular, and as already alluded to, the 30% pCR rate in the HR+/HER2- group of patients is encouraging,\nWhilst the clinical and translational readouts suggest an encouraging response to T-Vec/anti-PDL1 in this setting, the small number of patients and mixture of TNBC and HR+/HER2- precludes any too firm conclusions being drawn. This is an understandable and inevitable limitations of such studies, but a key question I would like to see addressed is what the investigators would propose as the next study. Might this focus on HR+ disease, and/or should T-Vec be incorporated as an additional treatment to ICB, in neoadjuvant TNBC?\nThis study adds important data to the application of oncolytic virotherapy as part of combination strategies, in breast and other cancers. However, there should be some discussion of negative findings, in particular the Phase III study of anti-PD1 +/- T-Vec in advanced melanoma (https://ascopubs.org/doi/full/10.1200/JCO.22.00343). In relation to this, the scheduling of the T-Vec and anti-PDL1 warrants further discussion. There was one lower dose ‘seroconversion’ dose of T-Vec; thereafter cyles of T-Vec and anti-PDL1 were given concurrently. This issue is important because one reason for the failure of T-Vec/anti-PD1 in the randomised Phase III melanoma trial has been suggested to be the switch from starting T-Vec before anti-PD1 in the Phase Ib part of the study, to starting concomitantly in the Phase III. The paradigm of the virus ‘sculpting’ the TIME for subsequent benefit from checkpoint blockade is an important one to highlight and, indeed, is supported by the translational readout comparisons presented between C2D1 and C3D1 in the current dataset.\nWhilst there is stratification by response in Fig 4, the authors could usefully do the same with regard to the immune effects by outcomes, by stratifying some of the analyses in Fig 3. This would help acknowledge the distinction between immune heat against virus versus heat against tumour, and that not all heat may be beneficial. This distinction, balance and development of the anti-viral and anti-tumour immune response is critical, very poorly understood and worthy of discussion.\nThe effects in the HR+ part of the cohort are interesting, with the caveat of small sample size. It is also worth noting the study with a different oncolytic virus, reovirus, which did not demonstrate any additional benefit from the addition of an anti-PDL1 (avelumab) (NCT04215146). The reovirus trial was in the different context of metastatic HR+ breast cancer patients, but wider discussion of the different settings, viruses etc, would be worthwhile in terms of how to move forward in evaluating the constituents and scheduling of these increasingly complex chemo/surgery/oncolytic virus/ICB combinations in future studies.\nIn terms of translational data, accepting the small number and heterogeneity of trial patients, there is convincing evidence that NAC increased the immunological ‘heat’ of the tumour, which rose further on treatment with T-Vec and then with anti-PDL1. The translational data could, however, have been more extensive. Specifically, additional readouts could have included TCR repertoire analysis, ideally in blood as well as tumour during treatment. Given that there is longitudinal RNAseq data, the TCR (and BCR) could be extrapolated using analysis such as MIXCR. This would be relatively straightforward, make more of the available data, and usefully add to the immune readouts. In addition, more extensive, quantitative pathology using multiplex immunofluorescence might also have been informative.\nOtherwise, the data as shown is well presented, although it should be clarified whether any of the differences shown in Fig 4 are statistically significant.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_6": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We sincerely thank the reviewer for the thoughtful review of our manuscript and truly appreciate the positive comments.\nTitle and Abstract:\n• The trial design is not clear from the title nor the abstract. This is a preoperative window study. The term “residual disease” traditionally refers to residual disease after surgery, not before surgery. It is strongly suggested to edit title, abstract, and other aspects in the paper to more clearly define and describe the window trial design.\nWe thank the reviewer for this comment. We agree that clarifying the preoperative window-of-opportunity design is important to avoid confusion. We have revised the title to:\n“A Preoperative Window-of-Opportunity Phase II Trial of Talimogene Laherparepvec and Atezolizumab in HER2-Negative Breast Cancer Following Neoadjuvant Chemotherapy: Results from SOLTI-1503 PROMETEO”.\nWe have also updated the abstract and relevant sections in the manuscript to more clearly define and describe the window trial design.\nMethods:\n• In the “Study Design and Participants” section of the methods, there are two sentences that are redundant and should be streamlined. Specifically, the sentences: (1) “The study recruited only female participants due to the lack of experience with T-VEC injections in men with breast cancer.” (Lines 296-297); (2) “The inclusion of male patients was not considered due to the lack of previous studies of intratumoral administration in this population.” (Lines 304-305). These sentences convey the same information. To improve clarity and conciseness, please consider omitting one of these sentences.\nWe thank the reviewer for pointing out this redundancy. As suggested, we have removed the second sentence and retained the more concise version: “The study recruited only female participants due to the lack of experience with T-VEC injections in men with breast cancer.”\n• How as the T-VEC administered? Who administered the agent (radiology? Surgery?)? Was radiology used to target the residual disease? Was a clip present in the tumor, as with a number showing pCR, could they have radiologically visualized?\nWe thank the reviewer for these pertinent questions. We have clarified the procedures in the Methods section. T-VEC was administered by a breast radiology specialist under ultrasound guidance to ensure accurate intratumoral delivery. In addition, all patients had a clip placed in the tumor bed prior to the start of NAC, as per standard-of-care procedures.\n• For RECIST analysis of response, when was the MRI performed? Should this be added to the Figure 1 Schema?\nWe thank the reviewer for this important observation. As clarified in the revised Methods section, MRI was performed twice: first, after completion of NAC and prior to enrollment, to confirm measurable residual disease; and second, after the final dose of T-VEC and atezolizumab but before surgery, to assess radiological response per RECIST criteria. We have also updated Figure 1 to reflect the timing of MRI assessments.\n• Serial biopsies: can it be specified which biopsies are fresh biopsies and which are FFPE only?\nWe thank the reviewer for this question. All tumor biopsies collected during the study, including those obtained at screening and on-treatment (C2D1 and C3D1), were formalin-fixed and paraffin-embedded (FFPE) for histological and immunohistochemical analysis. No fresh-frozen tissue was collected. This information has been added to the Pathology review section of the manuscript for clarification.\n• PAM50 subtyping: Which samples were used for this analysis? All samples? Which timepoints?\nWe thank the reviewer for this question. PAM50 subtyping was performed on all available tumor samples from each patient, including those collected at different timepoints (baseline, post-NAC/screening, C2D1, C3D1, and surgery). This information has been added to the Intrinsic PAM50 subtyping section of the manuscript. As also illustrated in Extended Figure 1B, each patient is represented with all available samples and the corresponding molecular analyses performed at each timepoint.\n• RNA sequencing and immune gene signature: which samples were used for this analysis? Please consider if information should be added to Figure 1 to indicate which correlative analysis were performed at each timepoint.\nWe thank the reviewer for this helpful comment. RNA sequencing were performed on all available tumor samples collected throughout the study (baseline, post-NAC screening, C2D1, C3D1, and surgery). This information has been added to the “Total RNA sequencing and immune gene expression signatures” section of the manuscript. Extended Figure 1B provides a detailed overview of the molecular analyses performed per sample and per patient.\n• Statistics: first sentence: is there a reference for predicted rates of RCB MDACC recommendations? Please either add a reference or describe why there is no reference.\nWe thank the reviewer for this observation. The corresponding reference for the predicted rates of RCB as per MD Anderson Cancer Center recommendations has now been added in the Statistics section of the manuscript.\nResults:\n• In the results section, the authors report that “patients with pCR and RD showed an increase in TILs and PD-L1 expression following treatment with T-VEC and atezolizumab, suggesting that the therapy induces changes in the tumor microenvironment regardless of the final pathological response (Fig 4a, 4b).” Subsequently, in the discussion, the authors mention that “the increase in TILs observed in the trial supports the hypothesis that the combination therapy not only initiates an innate immune response but also sustains an adaptive immune response capable of recognizing and eliminating tumor cells.” While the results suggest that changes in the tumor microenvironment occur regardless of the pathological response, the discussion implies a direct link between these changes and the elimination of tumor cells. These interpretations appear to go in opposite directions. Please justify how their suggestion in the discussion is supported by the results section and elaborate on whether the observed immune changes are sufficient to infer tumor cell elimination or merely reflect immune activation without definitive clinical impact.\nWe thank the reviewer for this observation. We agree that it is essential to distinguish between evidence of immune activation and definitive proof of immune-mediated tumor eradication.\nIn response to this comment, we have revised the Discussion section to clarify our interpretation. Specifically, we now emphasize that the observed increases in TILs and PD-L1 expression following treatment,seen in both pCR and RD cases,support the occurrence of local immune activation. However, we explicitly state that it remains uncertain whether this activation is sufficient to achieve tumor cell elimination.\nOur revised text reflects a more cautious interpretation of the data, suggesting that while the combination of T-VEC and atezolizumab appears to promote a more immune-permissive tumor microenvironment, additional factors likely determine whether this translates into complete pathological response. We appreciate the reviewer’s input, which helped us improve the nuance and clarity of our conclusions.\n• For the 6 patients who had pCR, what was their ER percentage?\nWe thank the reviewer for this question. The ER expression levels by IHC for the six patients who achieved pCR are shown in Figure 2D. As now stated in the Results section, all HR+/HER2− patients who achieved pCR had high ER expression, ranging from 80% to 100%.\n• Of the 28 patients enrolled, three experienced disease progression during treatment. Please provide more information about these patients. What subtype of disease? What other characteristics were notable?\nThank you for your comment. We have now included a supplementary table in the extended material of the manuscript detailing the clinicopathological characteristics of the three patients who experienced disease progression during treatment. This table includes information on tumor subtype, baseline clinical features, and other relevant data that may help contextualize their early progression.\nPD-L1\nPatient Histologic Histologic Ki67 ER IHC PR IHC sTILs TMB\nAge cT cN IHC subtype RCB ORR PAM50 IHC TLS TIP\nID type grade IHC (%) (%) (%) (%) (Muts/Mb)\n(%)\n1 50 NOS G2 cT2 cN0 45 0 3 TNBC III PD Basal 0 15 Absence D 9\n6 48 NOS G2 cT2 cN1 30 100 80 HR+/HER2- II PD LumA 0 0.9 Absence D NE\n22 63 NOS G3 cT2 cN0 30 100 6 HR+/HER2- NE NE LumB NE 10 Absence D NE\n• Impact on Tumor Biology section – there appears to be a repetitive sentence about enrichment of luminal A and Normal PAM50 at screening. Please adjust and remove repetitive text.\nWe thank the reviewer for this observation. We have removed the repeated sentence to improve clarity. We would like to clarify that the first paragraph in this section focuses on changes in PAM50 intrinsic subtypes, while the second paragraph discusses changes in PAM50 gene expression signatures, including the proliferation signature. The revised text now avoids redundancy while maintaining this conceptual distinction.\n• Tumor biology, tumor microenvironment, etc. The organization of these sections is difficult to interpret in terms of assay and timepoints, as the text jumps back and forth in each section. It is recommended to reorganize this section for improved clarity. For example, one section about TILs, timepoints, trends. One section on iGES – what timepoints was this assessed, what were the iGES identify, and how did they change over time? One section on PD-L1 testing, and consider adding ER percentage if that is known.\nThank you for this thoughtful suggestion. We agree that clarity in the presentation of assays and timepoints is essential. Based on your comment, we have revised the structure within the “Impact on tumor microenvironment” section to better distinguish the key components:\n A clearer paragraph structure has been applied to separate TILs, TLS, and PD-L1 dynamics across timepoints.\n A distinct paragraph is now dedicated to immune gene expression signatures (iGES), with explicit mention of the timepoints assessed and the main trends observed.\n We have also clarified that ER expression percentages are provided earlier in the results section (see paragraph describing HR+/HER2− patients who achieved pCR).\nWhile we considered reorganizing the full Results subsections as suggested (e.g., creating fully separate subsections for each assay), most reviewers did not raise concerns regarding the structure. Therefore, to maintain consistency across the manuscript and facilitate the reading flow, we opted not to implement large-scale structural changes at the subsection level. Nonetheless, we trust that the revisions introduced in paragraph organization and timepoint clarity now address your concern and improve the readability of this part of the manuscript.\nChanges are marked in the revised version of the manuscript.\n• Fig. 1 | Study design. Please specify the clinical stages of breast cancer included in the study rather than using the general term “early breast cancer.”\nWe thank the reviewer for this suggestion. We have updated the figure to specify the clinical stages of breast cancer included in the study. The enrolled population consisted of patients with stage I–III breast cancer.\n• Table 1 under “Ethnicity” appears to mix race and ethnicity, as it includes classifications such as “White,” “Arab,” and “Hispanic,” which encompass both racial and ethnic categories. Please clarify the criteria used for this classification. To improve clarity and consistency, consider aligning with standardized approaches for reporting race and ethnicity.\nWe thank the reviewer for this important observation. We acknowledge the inconsistency in terminology. The variable in Table 1 reflects self-reported patient background collected as part of standard demographic information at enrollment, without a formal distinction between race and ethnicity. We have updated the table heading to “Race/Ethnicity” and added a footnote to clarify this point.\n• Table 1: Can ER percentage be added? Especially given activity of IO in ER low disease observed in KN756 and Checkmate 7FL.\nWe thank the reviewer for this insightful comment. We have added baseline ER expression data (local assessment) to Table 1, including the mean, standard deviation, median and range.\nDiscussion:\n• The authors correctly acknowledge in the discussion that the prognostic value of RCB and pCR in this context, where residual disease post-NAC achieves a pCR after two months of innovative therapy, remains unclear. This is a valid point, as RCB and pCR have been increasingly challenged as a surrogate for long-term outcomes in immunotherapy trials (e.g., GeparNuevo). This is particularly relevant in the PROMETEO trial, where 20 of the 28 included patients had ER+ tumors, a population for which pCR is a questionable surrogate for survival outcomes. It is also worth noting that EFS or DFS is not captured as a secondary endpoint in the trial, which further limits the understanding of the long-term impact of the intervention. The authors should include this limitation, explain the rationale for choosing pCR as the primary endpoint in this study, and detail plans to report the effect of the intervention on EFS or other long-term outcomes.\nWe thank the reviewer for this thoughtful and important comment. We fully agree that the prognostic value of pCR and RCB in the context of residual disease post-NAC—particularly in ER-positive tumors—is uncertain, and that relying solely on these endpoints has limitations.\nIn the revised manuscript, we have now included a new section reporting disease-free survival outcomes, which was a predefined secondary endpoint of the study. Importantly, median follow-up of 44.6 months, allowing us to provide meaningful data on the potential long-term impact of the intervention.\n“Patient long-term outcomes\nFollowing surgery, among patients with triple-negative breast cancer (TNBC), one patient with pCR and four patients with residual disease received adjuvant capecitabine. Additionally, one TNBC patient received adjuvant docetaxel in combination with bevacizumab. Among patients with HR+/HER2− disease, a total of 19 received adjuvant endocrine therapy, including one who was treated with abemaciclib for two years. All patients underwent adjuvant radiotherapy as clinically indicated.\nAt a median follow-up of 44.6 months, a total of four disease-free survival (DFS) events (9.4%) were observed, all corresponding to distant metastatic recurrences, two in patients with TNBC and two in patients with HR+/HER2- disease. Two of these events resulted in breast cancer–related death. Notably, none of the DFS events occurred in patients who achieved pCR. The 4-year DFS rate was 100% among patients with pCR and 77.4% among those with RCB II/III disease (Fig. 3).”\nWe have also expanded the discussion to explain the rationale for selecting pCR as the primary endpoint. While we acknowledge its limitations—especially in ER+ disease—pCR remains a commonly used and pragmatic endpoint in early-phase neoadjuvant immunotherapy trials. Crucially, all patients enrolled in PROMETEO had biopsy-confirmed residual disease after completing standard NAC, and the achievement of pCR in some of these cases likely reflects true biological activity of the T-VEC and atezolizumab combination. Nevertheless, we agree that long-term outcomes are essential, and we emphasize that DFS data provide important complementary information.\n• Immunotherapy has now been incorporated into the standard neoadjuvant treatment of patients with early-stage TNBC, with immune checkpoint inhibitors (ICIs) being used in combination with chemotherapy. In the PROMETEO trial, residual disease (RD) was defined after neoadjuvant chemotherapy alone, without the inclusion of ICIs, which no longer reflects the current standard of care. This distinction is critical, as the evolving treatment landscape could significantly affect the efficacy and safety outcomes observed in this study. Furthermore, the lack of a comparator arm with chemotherapy and systemic immunotherapy alone, without the addition of T-VEC, limits the ability to isolate and interpret the specific contribution of each treatment component in the observed outcomes. The authors are encouraged to contextualize their results within this framework, highlighting how these factors might influence the generalizability and relevance of their findings in the current therapeutic landscape.\nWe thank the reviewer for this thoughtful and highly relevant comment. We fully agree that the standard of care for early-stage TNBC has evolved significantly since the initiation of the PROMETEO trial, particularly with the integration of immune checkpoint inhibitors (ICIs) into neoadjuvant chemotherapy regimens. As the reviewer correctly notes, all patients in PROMETEO had residual disease after standard chemotherapy alone, without prior exposure to immunotherapy—an important distinction that reflects the standard of care at the time of study design and recruitment.\nWe now explicitly highlight this point in the revised Discussion section, noting that the efficacy and immunomodulatory effects observed may not be directly extrapolable to patients previously treated with neoadjuvant chemo-immunotherapy. Nonetheless, we believe that PROMETEO provides valuable biological and clinical insights, particularly regarding the potential of intratumoral oncolytic immunotherapy in the post-NAC window, an area with limited therapeutic options and where residual disease still confers high risk of relapse.\nWe also agree that the lack of a comparator arm—including systemic ICIs or chemotherapy—limits the ability to isolate the contribution of T-VEC. This is a recognized limitation of the study design, and we have emphasized it in the revised Discussion. Future studies in this setting should consider randomized comparisons and incorporate current standards of care.\n• In the early-stage TNBC setting, the IMpassion030 and IMpassion031 trials evaluated the addition of atezolizumab to standard chemotherapy in patients with early-stage TNBC. However, these studies did not demonstrate a significant improvement in outcomes. Please discuss how the choice of atezolizumab as the systemic immunotherapy backbone in this study might have influenced their results. It would be important to discuss whether the observed efficacy in their trial is specific to the combination with T-VEC or whether it may be limited by the inherent performance of atezolizumab in this setting.\nWe appreciate this insightful comment. We agree that the choice of atezolizumab as the systemic immunotherapy backbone may have influenced the observed outcomes in PROMETEO. As correctly noted, the IMpassion030 and IMpassion031 trials did not demonstrate a significant benefit from the addition of atezolizumab to standard neoadjuvant chemotherapy in early-stage TNBC, raising important questions about its overall efficacy in this setting. In PROMETEO, the observed clinical and immunologic activity may therefore be specific to the combination of atezolizumab with T-VEC, rather than attributable to atezolizumab alone. We now discuss this point explicitly in the revised Limitations section, acknowledging that the limited efficacy of atezolizumab in prior studies complicates the interpretation of its role in our trial and underscores the need for randomized studies to better define the contribution of each component of the combination.\n• This type of clinical trial design, offering a window of treatment before surgery for clinical RD, is a novel design and, like I-SPY, could be an interesting platform for future trial design. Please consider adding some text to the discussion about the trial design and future applicability/challenges.\nWe thank the reviewer for this insightful comment. We fully agree that the preoperative window-of-opportunity design used in PROMETEO offers a valuable framework to evaluate the activity of novel therapies in a high-risk population with residual disease following NAC.\nAs suggested, we have expanded the Discussion: “The PROMETEO trial also represents a new approach in the treatment of chemoresistant residual tumors in breast cancer, particularly by utilizing a preoperative treatment window to evaluate the biological and clinical activity of innovative therapies in patients with pathologically confirmed residual disease after NAC. This strategic timing addresses the challenge of treating tumors that have demonstrated resistance to conventional NAC and provides a unique opportunity to assess drug efficacy in a controlled, pre-surgical setting. Similar to the I-SPY model (27), this type of design could serve as a platform for accelerated evaluation of promising agents, enabling early insights into biological effects, mechanisms of action, and potential efficacy before proceeding to large-scale adjuvant trials in patients with RD after surgery. Importantly, such window-of-opportunity studies also allow for the integration of robust translational endpoints and serial sampling, which can deepen our understanding of treatment-induced changes. However, challenges remain in terms of patient selection, timing of surgery, and the interpretation of short-term endpoints such as pCR in the absence of long-term outcomes. Despite these challenges, the PROMETEO approach could significantly enhance our understanding of therapeutic responses in chemoresistant tumors, offering hope for more targeted and effective interventions for this difficult-to-treat population.”\nWe believe this addition strengthens the contextual relevance of the trial and highlights its potential impact on future study design in early breast cancer.\n• This is a very small pilot study which shows feasibility; it is too small to draw firm conclusions, but suggests further study is of interest. Therefore the text of the Discussion should be more restrained. For example, at Line 234, these results do not suggest adding T-VEC to NAC could improve RCB rate in TNBC as that was not at all studied in this trial. However one could say that future analysis of this combination would be of interest for further study. Line 236: would not call rate of pCR “high” (only 7 patients) but instead would refer to it as a signal of activity. Line 261, would remove the phrase “groundbreaking approach.” Line 283: would remove the word “revolutionize” and replace with a toned down word, for example “advance.” Line 284: would remove the word “high” pCR rates, as this is a signal of activity, not a high rate in a well powered study.\nWe thank the reviewer for this thoughtful and constructive feedback. We agree that, as a small single-arm pilot study, PROMETEO is best interpreted as a demonstration of feasibility and biological activity, rather than definitive clinical efficacy. In response to the reviewer’s suggestions, we have revised the Discussion to adopt a more cautious and balanced tone. Specifically:\n- At Line285, we have clarified that PROMETEO did not evaluate the addition of T-VEC to NAC, and now frame the reference to earlier studies as a rationale for further exploration of that approach.\n- The phrase “groundbreaking approach” (Line 314) has been changed to “new approach.”\n- At Line 356, the word “revolutionize” has been replaced with “advance” to more accurately reflect the exploratory nature of the findings.\n- At Lines 279 & 357, we have replaced “the high rate of pCRs” with “the rate of pCRs” to avoid overstatement.\nReviewer #2 (Remarks to the Author):\nPascual et al. report on an interesting clinical trial of T-VEC with atezolizumab in patients with residual breast cancer after neoadjuvant chemotherapy in the SOLTI-1503 PROMETEO phase II trial. This trial is the first to report on combination T-VEC with immune checkpoint blockade in localized breast cancer in the neoadjuvant setting. The investigators used an innovative clinical space for this concept by treating in the window before surgery in patients that had residual disease after standard of care neoadjuvant chemotherapy. In addition to reporting on clinical outcomes, which were especially intriguing in the pCR rate in the ER+ population, they demonstrating longitudinal increase in immune inflammation with treatment through carefully collected and analyzed patient samples. This manuscript would be further improved by addressing the following points:\n1) For patient baseline characteristics section of results, it would be interesting to also include the % of tumors with high TMB.\nWe thank the reviewer for this insightful comment. We have now included the percentage of tumors classified as TMB-high among those with available TMB data in the Results section. Specifically, TMB was evaluable in 22 of 28 baseline samples, and 5 of these (22.7%) were identified as TMB-high (≥10 mutations/Mb). This information has been added to the baseline characteristics paragraph for clarity.\n2) One main limitation in the concept and using pCR/RCB as endpoint is that T-VEC is a local therapy. Therefore there is a high chance that improved outcomes by these measures translate to a local effect and it is unclear what significance is from systemic standpoint. Discussion of this should be expanded.\nWe appreciate the reviewer’s thoughtful observation regarding the potential limitations of using pCR/RCB as endpoints in a study evaluating a locoregional therapy such as T-VEC. We agree that these measures primarily reflect local response and may not fully capture systemic antitumor activity.\nTo address this point, we have expanded the Discussion section to explicitly acknowledge this limitation. In addition, EFS was included as a pre-specified secondary endpoint of the trial, and we have now incorporated EFS data into the Results section and Figure 3, as the minimum follow-up of three years required by the study protocol has been reached. These data help to contextualize the pathological responses from a systemic perspective.\nFig. 3 DFS curve for the study population.\nWe believe this additional information strengthens the interpretation of the study outcomes and addresses the reviewer’s concern.\n3) Along the lines of the local effects, are there systemic samples that can be analyzed to understand effect on systemic immunity?\nWe thank the reviewer for this relevant and forward-looking question. As part of the PROMETEO trial, plasma samples were collected longitudinally during treatment and throughout the 3-year follow-up period for analysis of circulating tumor DNA (ctDNA). In addition, peripheral blood immunophenotyping was performed in a subset of patients to explore systemic immune modulation.\nHowever, the minimum follow-up period has only recently been completed, and these translational analyses have not yet been conducted. We plan to analyze and report these data in future communications, which we hope will provide further insight into the systemic immunologic effects of the treatment combination.\n4) Samples without presence of tumor were included in C2D1 and later timepoints. This could theoretically impact results if tumor bed for example was not adequately sampled and enrich for microenvironment from non-tumor tissue. Acknowledging this is an inherent challenge of translational research, it would be helpful to clarify as needed or address this further in results and control for potential confounding of results.\nThank you for raising this important point. We acknowledge that tumor cellularity can vary across on-treatment biopsies, particularly at later timepoints. However, based on histopathological review, we observed that TILs increased not only within tumor nests but also in the surrounding stroma. Therefore, we believe the immune activation we report is not merely an artifact of sampling non-tumor tissue.\nTo further assess the potential impact of tumor content, we evaluated the correlation between CD8A gene expression and histologically assessed TILs across all timepoints. The strength and direction of the correlation remained relatively consistent, with Spearman coefficients ranging from 0.23 to 0.58, despite variations in mean cellularity (ranging from ~19% to 56%) at each timepoint. For example, the correlation at surgery was 0.58 (with 33.9% mean cellularity), while at C3D1, despite lower cellularity (~19%), the correlation remained positive (r = 0.23). These findings suggest that immune gene expression levels, including CD8A, reflect genuine biological changes rather than being driven by tumor content alone.\nSPEARMAN Mean tumor cellularity\nSurgery 0.582214536 33.88\nC3D1 0.233934693 18.95\nC2D1 0.380402419 24.07\nScreening 0.307728283 35.74\nBaseline 0.37498605 56.11\nMoreover, we observed the emergence and progressive increase of tertiary lymphoid structures (TLS) throughout treatment, as well as an increase in PD-L1 expression by immunohistochemistry, even in some samples with limited tumor content. These findings further support that the immune modulation induced by therapy is evident both histologically and at the gene expression level, and not an artifact of sample composition.\nAlthough we chose not to include these exploratory analyses in the main manuscript, we believe they provide strong reassurance regarding the robustness of our findings and help mitigate concerns about potential bias related to tumor cellularity.\n5) As the authors acknowledged it is difficult to distinguish clinical effects of T-VEC vs atezo in this tough some correlative data supports that T-VEC has immune effects. Especially in the TNBC cohort where the pCR rate is relatively low and comparable to the increase achieved with addition of neoadjuvant ICB to standard chemotherapy such as in KEYNOTE-522, is this interpreted to really be an increase from that achieved from ICB itself?\nWe thank the reviewer for this insightful and important question. Indeed, in KEYNOTE-522, the addition of pembrolizumab to standard neoadjuvant chemotherapy in early-stage TNBC resulted in a 13.6% absolute increase in pCR rates compared to chemotherapy alone. It is important to highlight that pembrolizumab in KEYNOTE-522 was administered concurrently with systemic chemotherapy, and in a treatment-naïve population.\nIn contrast, the PROMETEO trial enrolled patients with radiologically residual TNBC of at least 1 cm following multi-agent neoadjuvant chemotherapy—representing a high-risk population with poor prognosis and low expected pCR rates in the absence of further systemic therapy. In this context, achieving any pCR, particularly in patients previously considered non-responders, suggests potential synergistic immunologic activity beyond that expected from immune checkpoint blockade alone.\nImportantly, atezolizumab was not administered until the second dose of T-VEC, allowing for the evaluation of early immunologic effects of T-VEC alone. Translational analyses support this design, as an increase in immune activation signatures was already observed after the first dose of T-VEC, prior to the introduction of atezolizumab. This temporal separation further suggests that T-VEC may have contributed to the reactivation of an antitumor immune response in a previously immunologically “cold” and treatment-exposed setting.\nWhile the single-arm design limits definitive attribution, these findings collectively support the hypothesis that the observed pathological responses may be driven, at least in part, by T-VEC-induced immune effect in combination with atezolizumab.\n6) For the gene expression profiling, lack of single cell resolution or spatial context is a limitation. It would be helpful even if for select cases to perhaps look at spatial expression to better support findings.\nWe thank the reviewer for this valuable suggestion. We fully agree that single-cell or spatial transcriptomic analyses would provide additional insights, particularly regarding intratumoral heterogeneity and immune microenvironment context. However, these approaches were not included in the original study protocol, and unfortunately, tissue availability after completion of the planned analyses was insufficient to allow for additional spatial profiling.\nReviewer #3 (Remarks to the Author):\nThis is a single-arm phase 2 trial with primary objective is to evaluate the efficacy of the combination treatment (T-VEC + atezolizumab) in achieving RCB 0/I at surgery. The study reported primary and secondary outcomes as well as the biomarkers of treatment response of the total 28 eligible patients. The presentation is clear and the data analysis method is reasonable and the results are presented according to the SAP. The work is considered original, and the results support the claims.\nWe thank the reviewer for the thorough and positive evaluation of our study.\nOne minor suggestion about the results: add the 95% CI for the primary endpoint.\nThank you for your helpful suggestion. We have now included the 95% confidence intervals according to Clopper-Pearson method for the primary endpoint and related pathological response rates in the Results section. Specifically, we have added the following:\n“A residual cancer burden (RCB) class of 0/I (RCB-0/I) was observed in 7 patients (25.0%, 95% CI: 10.7 - 44.9%), all of them achieving pCR (RCB-0), showing a statistically significant improvement over the null hypnosis (p-value<0.001). RCB-II was achieved in 12 patients (42.9%, 95% CI: 24.5 – 62.8%), and RCB-III in 7 patients (25.0% 95% CI: 10.7 - 44.9%). Among those with pCR, 6 (86.0%) had HR+/HER2- disease. Overall, the pCR rates for HR+/HER2- and TNBC were 30.0% (95%IC: 11.9 – 54.3%) and 12.5% (95%IC: 0.3 - 52.7%), respectively”\nHere are some concerns and limitations that must be considered:\nNeed more details of the SAM analysis, at least add the reference to the method.\nWe appreciate the reviewer’s suggestion. We have now expanded the description of the SAM (Significance Analysis of Microarrays) method in the Statistical Analysis section to provide more clarity. In addition, we have included the original reference describing the method:\n“Significance Analysis of Microarrays (SAM), a permutation-based method that controls the false discovery rate (FDR), was used to identify differentially expressed genes and signatures, with an FDR threshold set at <1% [Tusher VG, Tibshirani R, Chu G. Proc Natl Acad Sci U S A. 2001;98(9):5116–5121].”\nLine 417-425: the sample size was determined based on the hypothesis test of RCB 0/1 rate of 20% vs 5%: “The null hypothesis (p0) was RCB-0/I observed in 5% of cases (p0 = 0.05), while the alternative hypothesis was p1 = 0.20. Considering a type I error of 0.05 and a type II error of 0.20 in a two-sided test, the required number of patients for the final analysis was 26 evaluable patients”. However, there is no statistical test reported in the SAP or the manuscript. Also, there is no justification of selecting p0=5% in the protocol. Please clarify.\nIn response to the reviewer’s comment, we have now included a one-proportion exact binomial test in the manuscript, conducted using the binom.test function in R. The corresponding details have been incorporated into both the Methods and Results sections.\n“A residual cancer burden (RCB) class of 0/I (RCB-0/I) was observed in 7 patients (25.0%, 95% CI: 10.7 - 44.9%), all of them achieving pCR (RCB-0), showing a statistically significant improvement over the null hypnosis (p-value<0.001)”.\nWith respect to the choice of a 5% null hypothesis, this study involves a population with radiologically and pathologically confirmed residual disease prior to surgery. Therefore, in the absence of additional treatment, the expected response rate would be close to zero. However, since a 0% response rate cannot be used for sample size calculation—and acknowledging the residual possibility of observing a response—we specified a 5% null hypothesis.\nIncomplete Adjustment for Confounding Factors\nLine 437. The study uses univariate logistic regression to assess the relationship between tumor biomarkers (e.g., TILs, PD-L1) and response to treatment, but does not adjust for confounding variables like tumor stage, previous treatment response, or molecular subtype. A multivariate analysis would help clarify which factors truly influence response rates.\nWe thank the reviewer for their comment. We agree that the only way to determine whether the relationship between tumor biomarkers (e.g., TILs, PD-L1) and response to treatment is not influenced by other variables is through a multivariable model. However, with only 28 patients, this study is clearly underpowered to support multivariable analyses. Considering this limitation, the exploratory nature of these analyses, and the fact that they were not pre-specified in the SAP of the study, we prefer not to include them in the report, in order to focus on the pre-defined analyses.\nHandling of Missing Data is Unclear\nLine 424. Sample size includes 12 % attrition rate which mean that some patients did not complete treatment (e.g., one withdrew consent, two had disease progression before surgery), but it is not clear how their data were handled in the analysis. Similarly, the study does not explicitly state whether missing values in gene expression data were imputed or simply excluded. Could you please clarify how the missing data were addressed, and how missing values in gene expression data were handled?\nThe reviewer raises a relevant point. Overall, no data imputation was conducted in this study. The trial design accounted for the possibility that some patients might not complete treatment. Ultimately, this was the case for 2 out of 28 patients—one who withdrew consent and one who experienced disease progression prior to surgery. For the primary analysis, the intention-to-treat (ITT) population was used, and these patients were classified as non-responders. The RCB0/1 rate was therefore calculated as 25% (7/28), using the ITT population as the denominator. Likewise, the Table 1 of the study show the characteristics of the 28 patients. No data imputation was performed in the gene expression analysis; the analysis was conducted based on the available data. This clarification has been included in the revised manuscript.\nAlso, the study was designed as early phase I (phase 0) per the protocol and the NCT registration. But based on the manuscript, this is a single-arm phase 2 trial with primary objective is to evaluate the efficacy of the combination treatment (T-VEC + atezolizumab) in achieving RCB 0/I at surgery.\nThe study was originally designed and registered as an early phase I (phase 0) trial, primarily focusing on feasibility, safety, and biological activity of the combination of T-VEC and atezolizumab in early-stage HER2-negative breast cancer. However, as the protocol evolved and after the first two patients were enrolled, the primary objective was refined to assess efficacy in terms of pathological response (RCB 0/I), aligning more closely with a single-arm phase II design.\nWhile the manuscript emphasizes the response rate to reflect the clinical interest and potential of the combination strategy, the trial remains exploratory in nature and was not powered for formal hypothesis testing. If necessary, we would be happy to clarify in the manuscript that this is an early-phase trial, to ensure consistency with the original registration and protocol.\nReviewer #4 (Remarks to the Author):\nThis paper describes a Phase II study giving an oncolytic herpes simplex virus, talimogene laherperepvec (T-Vec) with an anti-PDL1 antibody, atezolizumab, to patients with residual disease after neoadjuvant chemotherapy (NACT) for breast cancer. These patients had triple negative breast cancer (TNBC, 8 patients), or HR+/HER2- breast cancer (20 patients), with a high proliferation index prior to NACT. It is important to note (and should be discussed in the manuscript) that, whilst immunotherapy (IO) with immune checkpoint blockade (ICB) is now standard of care as part of neoadjuvant/adjuvant treatment in TNBC, ICB remains unproven in HR+/HER2- disease. Hence this cohort represents a mixture of patients in terms of their likely response to IO based on previous evidence from the more advanced disease setting, and in the context of current treatment regimes. Nevertheless, in this early trial setting, inclusion of both TNBC and HR+/HER2- groups is reasonable, and the findings of activity in HR+ disease may in particular be of wider significance, suggesting that IO can benefit more patients with breast cancer than currently thought.\nWe appreciate the reviewer’s thoughtful comment. We fully agree that immune checkpoint blockade is now part of the standard of care in early-stage TNBC, whereas its role in HR+/HER2− breast cancer remains investigational, with limited clinical benefit observed to date in the advanced disease setting. This distinction highlights the biological heterogeneity of the enrolled population and the need to interpret the findings within the context of tumor subtype.\nAs suggested, we have now included a sentence in the Discussion to address this point explicitly and to emphasize that the preliminary signals of activity observed in HR+/HER2− tumors in our study could have broader implications for the future development of immunotherapy strategies in this subtype.\n“However, this no longer reflects current clinical practice in early-stage TNBC (28), where neoadjuvant multi agent chemotherapy plus pembrolizumab has become standard or care. This distinction is critical when considering the applicability of our findings to today’s therapeutic landscape, particularly in TNBC. Fourthly, the encouraging pCR rate observed in HR+/HER2- patients, an outcome that is uncommon in this subtype, warrants further investigation. Future studies should focus on elucidating the biological mechanisms involved, including detailed analysis of the tumor microenvironment and immune response markers before and after treatment.”\nThe treatment had an acceptable safety profile, and led to promising pathological responses by the time of surgery. In particular, and as already alluded to, the 30% pCR rate in the HR+/HER2- group of patients is encouraging,\nWhilst the clinical and translational readouts suggest an encouraging response to T-Vec/anti-PDL1 in this setting, the small number of patients and mixture of TNBC and HR+/HER2- precludes any too firm conclusions being drawn. This is an understandable and inevitable limitations of such studies, but a key question I would like to see addressed is what the investigators would propose as the next study. Might this focus on HR+ disease, and/or should T-Vec be incorporated as an additional treatment to ICB, in neoadjuvant TNBC?\nWe thank the reviewer for this insightful and important comment. As noted, the small sample size and inclusion of both TNBC and HR+/HER2− subtypes preclude definitive clinical conclusions, although the translational and pathological signals observed are encouraging. We agree that the next logical step is to further explore the role of this combination in specific clinical contexts.\nBased on the observed immune activation and pCRs in HR+/HER2− tumors—a population typically less responsive to neoadjuvant chemotherapy—we believe that high-risk patients with radiological or pathological residual disease following standard neoadjuvant therapy represent a particularly relevant subgroup for future research. In these patients, novel perioperative strategies such as the administration of an oncolytic virus plus a checkpoint inhibitor prior to surgery could potentially improve long-term outcomes. We have now incorporated this perspective into the final paragraph of the Discussion section of the manuscript, as follows:\n\"In conclusion, the findings from the PROMETEO trial, supported by previous studies on T-VEC in breast cancer, suggest the potential of oncolytic viruses combined with immune checkpoint inhibitors to advance the treatment landscape for HER2− early breast cancer. The pCR rates observed in HR+/HER2− are particularly promising, suggesting a new avenue for research and therapeutic intervention in a subset of patients traditionally associated with lower responsiveness to NAC. In this context, future studies could focus on high-risk HR+/HER2− patients with radiological residual disease after neoadjuvant chemotherapy, where novel perioperative immunotherapy strategies, such as oncolytic virus plus checkpoint inhibitor prior to surgery, may help improve long-term outcomes. Further randomized controlled trials with larger patient cohorts and longer follow-up are essential to validate these results and explore the full potential of this combination therapy in different breast cancer subtypes.\"\nWe hope this addresses the reviewer’s question and highlights the rationale for future investigation in this space.\nThis study adds important data to the application of oncolytic virotherapy as part of combination strategies, in breast and other cancers. However, there should be some discussion of negative findings, in particular the Phase III study of anti-PD1 +/- T-Vec in advanced melanoma (https://ascopubs.org/doi/full/10.1200/JCO.22.00343). In relation to this, the scheduling of the T-Vec and anti-PDL1 warrants further discussion. There was one lower dose ‘seroconversion’ dose of T-Vec; thereafter cyles of T-Vec and anti-PDL1 were given concurrently. This issue is important because one reason for the failure of T-Vec/anti-PD1 in the randomised Phase III melanoma trial has been suggested to be the switch from starting T-Vec before anti-PD1 in the Phase Ib part of the study, to starting concomitantly in the Phase III. The paradigm of the virus ‘sculpting’ the TIME for subsequent benefit from checkpoint blockade is an important one to highlight and, indeed, is supported by the translational readout comparisons presented between C2D1 and C3D1 in the current dataset.\nWe thank the reviewer for this insightful comment and fully agree on the importance of discussing the sequencing of immunotherapy combinations, particularly in light of prior studies in melanoma. We have now expanded the Discussion to address this explicitly.\n“Importantly, this study introduced atezolizumab three weeks after the second dose of T-VEC, allowing for an initial priming phase with oncolytic virotherapy prior to checkpoint blockade. This sequencing was designed based on the hypothesis that T-VEC could reshape the tumor immune microenvironment by promoting antigen release, inflammation, and immune cell recruitment, thereby enhancing the efficacy of subsequent PD-L1 inhibition. The immunological changes observed betweenC2D1 (after T-VEC monotherapy) and C3D1 (after the first dose of combination therapy) support this hypothesis, showing increased TILs, TLS, and iGES. This paradigm aligns with the findings from the phase Ib trial of MASTERKEY-265(15) in which sequential administration of T-VEC followed by pembrolizumab in 21 patients with advanced melanoma yielded promising clinical outcomes. In contrast, the phase III trial(16), which used concurrent administration of both agents, failed to demonstrate additional clinical benefit. This discrepancy highlights the potential importance of treatment sequencing in optimizing the synergy between oncolytic viruses and immune checkpoint inhibitors, and should be carefully considered in the design of future combination immunotherapy trials.”\nWhilst there is stratification by response in Fig 4, the authors could usefully do the same with regard to the immune effects by outcomes, by stratifying some of the analyses in Fig 3. This would help acknowledge the distinction between immune heat against virus versus heat against tumour, and that not all heat may be beneficial. This distinction, balance and development of the anti-viral and anti-tumour immune response is critical, very poorly understood and worthy of discussion.\nWe thank the reviewer for this thoughtful and important comment. We agree that distinguishing between antiviral and antitumor immune responses is a critical and emerging area of research. In the PROMETEO study, our immune analyses were structured to address this complexity across complementary figures: Fig. 3 (now appears as Figure 4) presents the longitudinal evolution of immune-related markers across all patients, while Fig. 4 (now Figure 5) stratifies these immune parameters by pathological response (pCR vs RD) to highlight associations with clinical outcome.\nGiven the overlap in the variables shown (e.g., TILs, PD-L1, immune gene expression signatures), and to avoid redundancy, we opted to distribute the information across both figures rather than duplicate analyses. We believe this approach preserves clarity while allowing the reader to interpret both the general immune dynamics (Fig. 3) and the response-specific differences (Fig. 4). We have clarified this in the figure legends to better guide the reader through the complementary nature of the two figures.\nThe effects in the HR+ part of the cohort are interesting, with the caveat of small sample size. It is also worth noting the study with a different oncolytic virus, reovirus, which did not demonstrate any additional benefit from the addition of an anti-PDL1 (avelumab) (NCT04215146). The reovirus trial was in the different context of metastatic HR+ breast cancer patients, but wider discussion of the different settings, viruses etc, would be worthwhile in terms of how to move forward in evaluating the constituents and scheduling of these increasingly complex chemo/surgery/oncolytic virus/ICB combinations in future studies.\nWe thank the reviewer for this valuable comment. We agree that the negative findings from the trial combining reovirus and avelumab in metastatic HR+/HER2- breast cancer are important to consider when interpreting the immune and clinical signals observed in PROMETEO. As noted, the two studies differ markedly in patient population, disease stage, and therapeutic context. PROMETEO focused on high-risk, early-stage HER2-negative patients with residual disease post-NAC, a setting in which immune remodeling and subsequent checkpoint blockade may have distinct effects compared to the metastatic setting.\nWe have now included a sentence in the Discussion acknowledging this prior trial and emphasizing the need for a more systematic comparison of different oncolytic platforms, tumor settings (early vs metastatic), and scheduling strategies. These variables will be critical in guiding the rational development of future combination regimens.\n“Of note, a recent trial evaluating the combination of a different oncolytic reovirus with the anti–PD-L1 antibody avelumab in patients with metastatic HR+/HER2- breast cancer failed to demonstrate clinical benefit (REF). This highlights the importance of carefully considering the type of virus, disease setting, and timing of immunotherapy delivery when designing future trials that integrate oncolytic virotherapy into multimodal treatment regimens.”\nIn terms of translational data, accepting the small number and heterogeneity of trial patients, there is convincing evidence that NAC increased the immunological ‘heat’ of the tumour, which rose further on treatment with T-Vec and then with anti-PDL1. The translational data could, however, have been more extensive. Specifically, additional readouts could have included TCR repertoire analysis, ideally in blood as well as tumour during treatment. Given that there is longitudinal RNAseq data, the TCR (and BCR) could be extrapolated using analysis such as MIXCR. This would be relatively straightforward, make more of the available data, and usefully add to the immune readouts. In addition, more extensive, quantitative pathology using multiplex immunofluorescence might also have been informative.\nWe thank the reviewer for these valuable suggestions. We fully agree that a deeper characterization of the T and B cell receptor repertoire, as well as functional immune phenotyping, would enhance the translational insight of the trial. In the current study, the available translational data were limited by the use of FFPE tissue samples, which constrained certain types of analyses, including robust TCR/BCR repertoire reconstruction from RNAseq.\nHowever, as part of an ongoing translational extension of the PROMETEO study, we have collected fresh-frozen tumor biopsies at multiple timepoints (screening, C2D1, C3D1, and surgery) from a subset of 14 patients treated at Hospital Clínic. These high-quality samples are now being used to perform TCR and BCR sequencing, as well as other exploratory analyses including ELISPOT assays for IFN-γ secretion, CD40L induction, and activation marker profiling following therapy with T-VEC and atezolizumab. In addition, we are conducting deep immunophenotyping in peripheral blood, which will provide complementary insight into systemic immune dynamics.\nThese expanded analyses are currently ongoing and will be the subject of future communications. We appreciate the reviewer’s suggestions and believe they will strengthen the translational depth of our research program.\nOtherwise, the data as shown is well presented, although it should be clarified whether any of the differences shown in Fig 4 are statistically significant.\nWe thank the reviewer once again for their thorough review of the manuscript. Following the latest suggestion, we have modified Figure 4 to indicate with an asterisk (*) the comparisons that are statistically significant. The figure legend has also been updated to clarify that the highlighted results correspond to comparisons with an unadjusted p-value < 0.05, using the Wilcoxon test for unpaired samples\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe thank the reviewer and their co-reviewer for their time, thoughtful assessment, and valuable contributions to the evaluation of our manuscript. We fully support initiatives that promote training and recognition for Early Career Researchers in the peer review process.\nReviewer #6 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe sincerely thank the reviewer and their co-reviewer for their time, thoughtful evaluation, and constructive feedback on our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have responded to the requests and queries appropriately.", "reviewer_2": "Pascual et al. report on an interesting clinical trial of T-VEC with atezolizumab in patients with residual breast cancer after neoadjuvant chemotherapy in the SOLTI-1503 PROMETEO phase II trial. This trial is the first to report on combination T-VEC with immune checkpoint blockade in localized breast cancer in the neoadjuvant setting. The investigators used an innovative clinical space for this concept by treating in the window before surgery in patients that had residual disease after standard of care neoadjuvant chemotherapy. In addition to reporting on clinical outcomes, which were especially intriguing in the pCR rate in the ER+ population, they demonstrating longitudinal increase in immune inflammation with treatment through carefully collected and analyzed patient samples. This manuscript would be further improved by addressing the following points:\n1) For patient baseline characteristics section of results, it would be interesting to also include the % of tumors with high TMB. *This has been addressed.\n2) One main limitation in the concept and using pCR/RCB as endpoint is that T-VEC is a local therapy. Therefore there is a high chance that improved outcomes by these measures translate to a local effect and it is unclear what significance is from systemic standpoint. Discussion of this should be expanded. *The addition of the DFS curve for the study population is helpful to addressing this concern.\n3) Along the lines of the local effects, are there systemic samples that can be analyzed to understand effect on systemic immunity? *While these samples are available, this analysis has not yet been completed at this time so the authors were unable to address and plan to in a future publication.\n4) Samples without presence of tumor were included in C2D1 and later timepoints. This could theoretically impact results if tumor bed for example was not adequately sampled and enrich for microenvironment from non-tumor tissue. Acknowledging this is an inherent challenge of translational research, it would be helpful to clarify as needed or address this further in results and control for potential confounding of results. *The authors found increased TIL in tumor and stroma and showed correlation between CD8 on gene expression and TIL across timepoints. The potential limitation should be acknowledged in discussion.\n5) As the authors acknowledged it is difficult to distinguish clinical effects of T-VEC vs atezo in this trial, correlative data supports that T-VEC has immune effects. Especially in the TNBC cohort where the pCR rate is relatively low and comparable to the increase achieved with addition of neoadjuvant ICB to standard chemotherapy such as in KEYNOTE-522, is this interpreted to really be an increase from that achieved from ICB itself? *The discussion acknowledges the limitations of not have a PD-L1 only control arm. Their response partially addresses this in highlighting the high risk nature of this population and the correlatives supporting immune activation after T-VEC therapy even prior to addition of anti-PDL1.\n6) For the gene expression profiling, lack of single cell resolution or spatial context is a limitation. It would be helpful even if for select cases to perhaps look at spatial expression to better support findings. *The authors unfortunately do not have remaining tumor tissue to perform these analyses.", "reviewer_3": "The revision has sufficiently addressed/clarified the statistical concerns.", "reviewer_4": "Significant changes and improvements have been made to the manuscript in response to my comments and suggestions.", "reviewer_6": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter