Cell Reports Medicine

Phase 2 trial of perioperative chemo-immunotherapy for gastro-esophageal adenocarcinoma: The role of M2 macrophage landscape in predicting response

Graphical abstract

Authors

ThierryAlcindor,JamesTankel, Pierre-Olivier Fiset,..., Sui Huang, VeenaSangwan,LorenzoFerri

Correspondence

thierry.alcindor@mcgill.ca (T.A.), lorenzo.ferri@mcgill.ca (L.F.)

In brief

Inthisnon-randomizedtrialof neoadjuvantdocetaxel,cisplatin, fluorouracil,andavelumabforlocally advancedesophagealadenocarcinoma, Alcindor et al. find adverse events, complete pathological response, and two-year disease-free survival among 40% ， 14% ,and 67.5% of patients, respectively.M2-tumor-associated macrophage proliferation occurs among non-responders suggesting a potential mechanismoftreatmentresistance.

Highlights

NeoadjuvantavelumabandDcFaresafeandefficaciousin
this group of patients
pCR occurs in 14% of patients, and 2-year DFS is 67.5%
Pre-treatmentM2-TAMproliferationisassociatedwithpoor
treatment response
MIF and CD86 expression among M2-TAMs may explain this
trend

Article Phase 2 trial of perioperative chemo-immunotherapy for gastro-esophageal adenocarcinoma: The role of M2 macrophage landscape in predicting response

Thierry Alcindor, ^{1,2,\star} James Tankel, ^{3,8} Pierre-Olivier Fiset, ^{4,8} Sanjima Pal, ^{3,8} Touhid Opu,2 Michael Strasser,5
Mehrnoushehghani,Nicholaseros,DongmeiZuCamenuelleJonathanCoolsLartigue,MarcHicen,
VictoriaMarcus, ^3 SophieCamilleri-Broet,4AlanSpatz,4Gertruda Evaristo,4Mina Farag,4GiovanniArtho ^7 Arielle Elkrief,1
Ramy Saleh, ^{1,2} Swneke Bailey,3Morag Park,2 Sui Huang,5 Veena Sangwan,3and Lorenzo Ferri3,9,\*
1Department of Medicine,McGilUniversity HealthCentre,Montreal,QC,Canada
2CenterforInnovative Medicine,McGillUniversity HealthCentre,Montreal,QC,Canada
3Department of Surgery,McGilniversityHealthCentre,Montreal,QC,Canada
DivisiofatlatfCliicaaratodiGiiestyathetMtreaa
5lnstitute forSystemsBiology,Seattle,WA,USA
6Department of Nuclear Medicine,McGillUniversity Health Centre,Montreal,QC,anada
7Department of DiagnosticRadiology,McGilUniversityHealthCentre,Montreal,QC,anada
8These authors contributed equally
9Lead contact
\*Correspondence: thierry.alcindor@mcgill.ca (T.A.), lorenzo.feri@mcgill.ca (L.F.)
https://doi.org/10.1016/j.xcrm.2025.102045

SUMMARY

We present the clinical results ofaphase 2trial combining neoadjuvant docetaxel, cisplatin,5Flourouraci and the PD-L1 inhibitor avelumab in locally advanced gastro-esophageal adenocarcinoma (GEA). Fifty-one patients receive neoadjuvant therapy with 50 proceeding to surgery. Grade 3-4 adverse events occur in 40% ; complete/major pathological response is found in 7/50 (14%) and 9/50 (18%) , with 2-year diseasefree survival of 67.5% .There is no correlation between tumor regression and PD-L1 or mismatch repair (MMR) status. Multiplex immunohistochemistry and longitudinal single-cell transcriptomic profiling revealaterations in certain innate immune cellpopulations, particularly noting an M2-tumor-associated macrophage (M2-TAM) proliferation in non-responding tumors.These findings describe the effective nature of this treatment regimen forGEA and reveal associated features of the inflammatory milieuxassociated with response to chemo-immunotherapy. The specific character of the inflammatory environment in non-responders may, in the future, help personalize treatment. This study was registered at ClinicalTrials.gov(NCT03288350).

INTRODUCTION

Gastro-esophagealcancerranksseventhinincidenceandsixth inmortalityamongcancersworldwidewithgastro-esophageal adenocarcinoma(GEA)being themostcommonsubtypeinthe Westernhemisphere.ForpatientswithlocallyadvancedGEA, randomizeddatahaveshownthatneoadjuvanttreatmentoffers better survival outcomes compared with surgery alone.2,3 Althoughneoadjuvantchemoradiotherapyisasuitableoption, asevidencedbytheFLOT4trialneoadjuvantdocetaxel-based chemotherapyhasrapidlybecomeapredominant standard of care.4However,despitetheimprovedefficacyofneoadjuvant docetaxel, approximately 40% of primary lesions will fail to respond to systemic treatment.5

Followingthedocumentedsurvivalbenefitassociatedwiththe additionofimmunecheckpointinhibitiontochemotherapyin recurrent or metastatic GEA,68 support for this therapeutic modality in the neoadjuvant setting is emerging.Both single-arm andrandomizedtrialshavedemonstratedthattheadditionofneoadjuvantanti-PD-L1therapywasassociatedwithimpressiverates ofmajorpathologicalresponse(MPR)andpathological complete response (pCR) of 33%-70% and 15%-45% ,respectively.9.10 However,moreconservativepCRratesofbetween 3.7% and 11.5% associated with otheranti-PD-L1orwith anti-PD-1agents suggest that the ideal regimen has yet to be defined.11-13 This is alsoreflectedinsurvivaloutcomesasneoadjuvantpembrolizumabfailedtoimproveevent-freesurvivalwhencombinedwith cisplatin-based chemotherapy in the KEYNOTE-585 trial.14 In comparison,adjuvantnivolumabimproveddisease-freesurvival (DFS) following neoadjuvant chemoradiotherapy and esophagectomyintheCheckMate577trial.15

Avelumab(healthcarebusinessofMerckKGaA,Darmstadt, Germany）is anintravenously administered，fully human anti-PD-L1 immunoglobulin G1 monoclonal antibody. By blockingtheintercellularinteractionbetweenPD-L1ontumorcells and PD-1on T cells and other immune cells, immunosuppressionis curtailed and effectorTcellslysisof tumor cells encouraged.16Although the survival benefit of avelumabhasbeen demonstratedinpatientswithadvancedurological anddermatological malignancies,17-19 its role in GEA is underexplored, particularly in the neoadjuvant seting.

Figure1.ACoNSORTdiagramshowingpatientinclusioninthe study Fifty-one patients were enrolled of which50 underwent surgery.There were37 patients who continued to receive adjuvant therapy.

Here wereport the resultsof a single-arm phase 2 trial investigatingsafety,efficacy,andimmunologicalcorrelationsofneoadjuvantdocetaxel,cisplatin,and5FUcombinedwithavelumab (aDCF) in patients with resectable, locally advanced GEA.The primary objective of this study was to describe the association ofpreoperative aDCFwithpCRasthisoutcomehasbeenlinked toexcellentsurvival inothertrialsofchemo-immunotherapy.The secondaryobjectivesweretodescribethetwo-yearDFSand incidenceofgrade3/4adverseevents(AEs)associatedwith thisspecificregimen.AposthocanalysisofthetissueinflammatorymicroenvironmentwithaspecificfocusonM2tumor-associated macrophages (M2-TAMs) is also described.

RESULTS

Patientandtumorcharacteristics

Aflowdiagramofpatientinclusionintothestudyisdisplayedin Figure1.BetweenFebruary2018andApril2022,51patients wereenrolled.Onepatientwithdrewconsenttopartakeinthe studyfollowingtheadministrationofneoadjuvantaDCF,leavinganeffectivepopulationof50whounderwentsurgery.The demographic,clinical,andpathological dataof the51patients enrolledinthisstudyaredescribedinTable1.Withamedian age of 64 years (range 18-79),45 (88%) were males. There were35 (69%) with an ECOG (Eastern Cooperative Oncology Group)performancestatusofO,and themedianCharlsoncomorbidity index was 4(2-7). Most of the tumors were in the esophagus or gastro-esophageal junction(Siewert \mathsf{I}/\mathsf{I}\mathsf{I}=41/ 51, 80% with5/51 (10%) ineachofthecardiaandsubcardia/stomach.Eighty-eight percent of patients have cT3lesions, and 61% wereclinicallylymphnodepositive (C N+) on pre-treatment staging investigations. Signet ring cells were found in \boldsymbol{\mathsf{10/}} 51 (20%) ofpatients,and 26/50 (51%) hadpoorlydifferentiated orundifferentiatedlesions.

A total of 49/51 (96%) patients received all 4 preoperative cycles of aDCF withtwo patients not completing neoadjuvant treatment dueto seriousAEs.One patient suffered from pneumonia (grade 4),and another from immune-related hepatic failure and sepsis (grade 3).

Safetyandfeasibility

Thedataforthe51patientsincludedinthesafety-evaluable analysis are presented inTable S1.Using theNational Cancer Institute-CommonToxicityCriteriaforAdverseEventsversion 3.0 (CTCAE),2° grade _{3/4} treatment-related AEs occurred in 20/50 (40%) of patients.The most common grade 3-4 AE was neutropenia (20/51, 20% .Common grade1-2 side effects included fatigue (75%) alopecia (33%) ,and constipation (29%) In four cases, dose reductions of the neoadjuvant therapy were required.Thisincluded5FUforgrade3diarrhea(1/51)andgrade 3 oral mucositis(1/51),docetaxelforgrade 2fatigue(1/51),and cisplatinforgrade3hypomagnesemia(1/51).Theadministration of neoadjuvant aDCFwas delayed in 17patients due to grade 3-4 neutropenia (10/51, 20% )，grade 2 diarrhea (1/51),grade 1-2 arthralgia (5/51),and grade 3 myalgia (1/51).There was no mortality secondary to treatmentwith aDCF.

Surgical outcomes

Afterpreoperativeimmunochemotherapy，noradiological disease progression was observed on the preoperative restaging investigations, and all 50 patients proceeded to surgery.The typeofoperativeprocedure,incidenceofpostoperativecomplications,and dataregarding the adjuvant arm of systemic therapy are described inTable S2.For the 50patientswho underwentsurgery,themediantimebetweenfinishingtreatmentand surgery was53days(22-158days).Themostcommonsurgical procedureperformed wasanlvorLewisesophagectomy(40/50, 80% followed by total gastrectomy (4/50, 8% )and left thoracoabdominal esophago-gastrectomy, McKeown esophagectomy, and subtotalgastrectomy(2/50, 4% each).There were14/50patients with Clavien-Dindopostoperativecomplications >=3 within 30 days of surgerywith the most common complications being anastomotic leak/conduit necrosis(9/50, 18% andaspiration pneumonia (7/50, 14% with7/50 (14%) patients requiring reoperation.The30-daymortalityratewas 0% ,althoughonepatient died47days aftersurgeryfromsepticcomplicationsresultingin a 90-daymortalityrateof 2% .AnROresectionwasachieved in 48/50 (96%) patients. The median lymph node yield was 36 (13-78)while themediannumber ofpositivelymphnodeswas 1 (0-21).

Therewere 30/50 patients (60%) whoinitiatedadjuvanttherapy and received at least2cycles,and24/50 (48%) completed all 4 cycles of adjuvant aDCF. All 50 patients in the efficacy population had surgery with curative intent.

Efficacyandassociationofputativebiomarkersof response

PathologicalresponseresultsaredetailedinTableS3.Usingthe CollegeofAmericanPathologistsprotocol(version4.1)and the Modified Ryan Scheme for tumor regression score,21 seven patients (14%) showed pCR (tumor regression grade, TRG O) and two (4%) hadnear-completepathologicalresponsewithmicroscopicresidual disease(TRG 1).Therefore,a total of9/50 (18%) had an MPR style(\mathsf{T R G}0/1 .Moderateresponse(TRG 2)occurred in 16/50 (32%) andpoorresponse(TRG 3)occurred in25/50 (50%) ：

A mismatchrepair protein deficiency (MMRd) was found in 8/47 (17%) of samplestested,and two of thesepatients (25%) hadpCR.Therewere26/50 (52%) ofpatientswithypT3disease and 48% ， 26% B 12% and 14% with ypN stage 0, 1, 2, and 3, respectively.WithregardstoPD-L1status,27/45 (60%) patients had a combined positive score (\mathsf{C P S})>=5 and17/45 (38%) had CPS >=10 .HER2 positivity was found in 3/46 (7%) patients. As showninTable2,noneofthesebiomarkerscorrelatedwiththe grade of pathological response.Using a conservative cutoff of 70% for Tumor Proportion Score(TPS)expression,based on datacomparingimmunohistochemistry(IHC)assaysanddata among patients with lung cancer,22 no association with pathological response wasfound.

Figure 2displays a waterfallplotofobjective FDG(18F) flourodeoxyglucose)-PET (PositonEmisssionTomography) responseasdefinedbythepercentagechangeinthemaximum standardized updatevalue (\mathsf{S U V}_{\mathsf{m a x}}) between thepre-and posttreatment scan.The majority of patients had a reduction in \mathtt{S U V}_{\mathsf{m a x}} after neoadjuvant aDCF.Inkeeping withprevious literature of a clinically relevant PET-CT response among patients treated with neoadjuvant chemotherapy,23 a decrease of at least 35% in the maximum \mathtt{S U V}_{\mathtt{m a x}} was noted in33/50patients (66%) ， However, this reduction in maximum \mathsf{s u v}_{\mathsf{m a x}} was not associated with pathological response (Table 3).

Survival outcomes

Betweenthedateofhistologicaldiagnosisandtheclosureofdata collection on the 16^{th} of May 2023,the median follow-up for the cohortwas26.5months(5-74).ThemedianOSforthecohort wasnotmet.Thetwo-yearDFSandOSfortheentirecohort were 68% (27/40 patients)and 78% (31/40),respectively. The mediantimetodiseaserecurrencewas13months(range \scriptscriptstyle7- 44).Therewas a trend toward improved two-year DFS and OS when stratifying the cohort by pCR status (6/6, 100% VS. 21/34, 62% ， p=0.091 and 6/6 100% VS.25/34, 74% B p=0.175 respectively)(Figures 2Bi-2Biv). Conversely, MPR status was associatedwithasignificantimprovementin2-yearDFS(8/8, 100% Vs. 19/32 ， 59% p=0.029) (Figure S2i).A trend toward improvedtwo-yearOSwhenstratifying accordingtoMPRstatus was noted (8/8, 100.0% VS.23/32, 72% p=0.100) (Figure S2i). OS was also not affected by mismatch repair (MMR) status (Figure S2i).

Multipleximmunohistochemistryandspatialanalysis: Linkbetweenpathologicalresponseand altered immune cell density

ToinvestigatehowaDCFinfluencesthedynamicbehaviorand spatial distribution of immune cells,we gathered tissue specimens at varioustimepoints during treatment.Pre-treatment biopsiesreferredtotreatment-naivesampleswhilemid-treatment biopsieswereretrievedendoscopicallyafterthecompletionof2 of the4roundsofneoadjuvantaDCF.Conversely,theposttreatment samples were taken from the surgical specimen at the time of resection.For the purposes of this post hoc analysis, andduetotherelativelysmallnumberofpatientswithpCR, sampleswithMPR(TRG0/1)werecompared withmoderate responders(TRG2)and poorresponders (TRG3).Once stratified by pathological response, C D8^{+} T cell,M2-TAM,and cytotoxic T lymphocyte (CTL) cell density (cells/mm²) were determined in pre-treatmentandpost-treatmentsamples.Therewere42pretreatmentand36post-treatmentspecimensavailableformultiplex immunohistochemistry (mlHC) spatial analysis. As shown inFigure3A,arepresentativeselectionofcompositeOPAL-6- stainedmicrophotographsofpairedpatient-matchedpre-treatmentandpost-treatmentsampleswasusedtoassesschanges inthetumorimmunemicroenvironmentinrelationtotreatment response.

The mean percentage changes in these cell densities (cells/ \mathsf{mm}^{2} arepresented inFigure3BiandTableS4i.Whencompared tobaseline M2-TAMdensity onthe treatment-naive sample, aDCF treatment was associated with reduced M2-TAM density inallMPRpatients'resectiontissues.Presentedasmeanwith the 95% confidence interval (Cl),a significant difference was noted in themeanpercentagereductionof M2-TAM densitybetweenpre-andpost-treatmentMPRsampleswhencompared to the same of TRG3 samples (in MPR 70% reduction, 95% CI-87 to -54 vs.in TRG3 102% increase, 95% CI-106to318,two-way ANOvA,Tukey's correctionformultiple comparison, p=0.038) ， A general increase in the total density of {\mathsf{C D8}}^{+} T cells during neoadjuvant treatment was observed with improved pathologicalresponse (Figure 3Bi).Similarly,a trend toward a reduction in the mean percentage increase of total {\mathsf{C D8}}^{+} T cell densitywhencomparingMPRwithTRG3wasalsonoted 304% increase, 95% CI 68 to 675 vs. 105% increase, 95% CI 0.4 to 210).

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Prognosticspatialdistributionofpro-andanti-tumor immunecellsintreatment-naivespecimens

Differencesinthespatialdistributionofselectedimmunecells relativetothetumorcorewereanalyzedusingHalo.Thetumor core wasdefinedbyanexpertpathologist(P.-O.F.)onthehematoxylinandeosinslideof thebiopsyspecimenandconfirmedby staining positively for CK7.Once stratified by pathological response，the densities of intra-tumoral M2-TAM \mathsf{(C D68^{+}} CD163+)，CTLs (\mathsf{C D8^{+}G N Z B^{+}}) ，and TREG (Foxp3+)were calculatedinthepre-treatmentsamples(Figure3Bi; Table S4i. All samples from patients that would subsequently haveanMPRfollowingtreatmenthadanM2-TAMdensityof <500 counts *\mathsf{m}\mathsf{m}^{2} within tumor cores on their corresponding treatment-naivebiopsy.Theintra-tumoralM2-TAMdensityof MPRtreatment-naivesampleswassignificantlylessthanthat of TRG3 samples(212.8 counts \scriptstyle\prime\min^{2} B 95% CI 116.2-452.9 vs. 397.0 counts /\mathsf{m}\mathsf{m}^{2} 95% CI 180.3-780.0, Welch's unpaired two tailed t test, p=0.024) .Only 1/14 (7.1%) patient with anM2- TAM density in the top quartile (>500 per \mu\mathsf{m}^{2}) had an MPR whose lesion was also MMRd (Figure 2). As noted in Figure 3C, thereseemedtobenodifferencebetweentheincidenceofM2- TAM density >500\ \mathsf{c e l l s}/\mathsf{mm}^{2} when comparing patients with, andwithout,clinical lymphnodemetastases (8/19\mathsf{c N}+ versus 11/22\mathsf{c N-}) ：

Taking advantage of the spatialresolution data,proximity analysis(seemethod details)was performedto quantify numbers and average distance of M2-TAM， TREG， and CTLs withina 50~{\upmum} proximityoftumorcellsintreatmentnaive specimens (see Figure S3).M2-TAMs were significantly closertotumorcellsinpatientswithTRG3comparedtoMPR samples (Figure 3Bii, 15.2~\upmum 95% CI12.87-18.9 vS. 24.2~{\upmum} 95% CI 19.78-35.05, Welch's unpaired two tailed t test, p~=~0.005) .This suggests a mitigation of the responsetoneoadjuvantaDCFifM2-TAMsareaccumulated ingreaternumbersandcloserproximitytotumorcells.Overall,the data implied a potential for increased intercellular communicationbetweenimmune-suppressiveM2-TAMand cancer cells in tumors that responded poorly to neoadjuvant aDCF.

To further assess the relationship between M2-TAM, TREG, andCTLsubpopulations,a nearest-neighboranalysiswasalso performedontreatment-naive specimens(TableS3ii).There wasaninverserelationshipbetweentheproximityof \mathsf{F o x P3+} TREG andM2-TAMsuchthatpoorerresponsewasassociated withclosercell-cellproximity(Spearmancorrelation {\sf r}=0.968 \mathsf{p}=<0.001-0.007) .Conversely,for CTL,a constant proximity toM2-TAMwasmaintainedirrespectiveofsubsequentdisease response.

A diagram proposing a prognostic scheme foraDCF treatment based on the intra-tumoral density and proximity to tumor cells of M2-TAM, CTL, and TREG is shown in Figure S4.

Single-cell transcriptomicsofbiopsysamples

Weobtainedfreshtissuesamplesfrom11/50patientsforsinglecellRNA transcriptomicsequencing(scRNA-seq)onthe 10x GenomicsChromiumplatform.Thisyieldedatotalof20samples,10pre-treatmentsamples,5mid-treatmentsamples,and 5 post-treatment samples. Stratified by pathological response, there were 2 TRG1 samples,4 TRG2 samples,and 14 TRG3 samples.Allsampleswereconfirmedhistologicallytohave beentakenfrom anarea thatcontained esophagealadenocarcinoma(EAC)bypathological assessment.Afterqualitycontrol (see methods),44,118cellswithusable transcriptomes were identified.To align the scRNA-Seq analysis with the IHC results, we focused on identifying CTL,M2-TAM, and TREG cells,based on the same markers as used for the IHC.Thus,after batch correctionanddimensionreductionofcelltranscriptomes(see methods)andmapping eachcelltoa 2Dplane(YMAP),we labeled the cells for these three cell types (Figure 4A),which encompassed 5,237 cells, with 2,702 CTLs (\mathsf{C D8^{+}G Z M B^{+}}) ， 2,105 M2-TAMs (\mathsf{C D68^{+}C D163^{+}}/\mathsf{C D206^{+}}) ，and 425 TREGs (\mathsf{C D4^{+}F O}\mathsf{X P3^{+}}) .The top 10 differentially expressed genes in eachof thesecelltypeswereidentifiedwithaviewtofurther describethetranscriptomicidentityandfunctionoftheseclusters(Figure 4B).For instance,C1QA,which hasbeen associated withcancerprogressionand Tcell exhaustion,wasexpressed by the M2-TAM among many samples.

Locationof these three celltypes on theUMAPspacerelative to all the cell types ispresented inFigureS5i-S5ili.When these mapswerefurtherstratifiedbyresponsetoneoadjuvant therapy (Figure S6),it becomes evident that the CTL cells of samples of the TRG3response group hadtranscriptomesdistinct from those of the TRG1 sample(Figure S7).The absolute cell counts stratifiedbysampleandpathologicalresponseareshownin TableS5.Motivated byour findings in IHCof differential cell type abundance across response groups, we first examined the changes in cell typeproportions over the course of treatment.Thechangingcellproportions aredisplayed inFigure4C, including the absolute cell countof eachsample.Thepercentageof M2-TAMwithinTRG1pre-treatmentsamples appears to be lower with a higher proportion of CTL in comparison to treatment-naiveTRG2/3samples.Thisisinkeepingwiththe findingof M2-TAMenrichment innon-responders described earlier on immunohistochemistry.We note that the bulk of the cellsintheanalysisoriginatedfrompost-treatmentsamplesin keeping with thehigher tissue yield from theresection specimens (Figure 4D).

Characterizing transcriptomesofM2-TAMofpoor responders

Considering the association with the changing density and proximity of M2-TAM seen onthe spatial analysis earlier and pathological outcome,the changing transcriptomes in the M2-TAM

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throughout thetreatment cycleof TRG3sampleswasthen explored.AcomparativeanalysiswithMPRsampleswasnot possiblesincenoMPRpost-treatmentsampleswereavailable foranalysisbecauseoflackofaresidualtumormass.

WhenconsideringTRG3patientsonly,thebulkof thecellsin the analysis originated from post-treatment samples most likely because these samples were harvested from the surgical specimen rather than endoscopic biopsy (Figure 5A).Therefore, to correctforvastlydifferentcellnumbers,geneexpressionanalysiswasperformed asapseudobulk(summingofallthecellsfollowedbynormalizationforcellnumber)forcomparingtreatment-naiveandpost-treatmentTRG3samples.Differential expression analysis with differentialexpression analysis(DESeq) is shown in the volcano plot in Figure 5B.After filtering (see method details),， 49 differentially expressed genes remained thatwere statisticallysignificantwithanadjusted p valueof <0.05 .Theheatmapdisplayingthedifferentialexpressionof these genes in theM2-TAM cells in TRG3 samples throughout the treatment cycle is displayed inFigure 5C.Therewere 3patientsforwhomsamplesateachtimepointwereavailable.The top10 differentially expressed genes,based on adjusted p value, fromtheanalysisofthecohortasawholewereidentified,andthe log-normalized count of these genes plotted for each of these three patients stratified by the time the biopsy was taken(FigureS8).Thesedatasuggestthatthetranscriptomicprofilefound amongpatientswithTRG3waspresentthroughout thetreatmentcycleincludingwithin treatment-naive specimens.

Treatment-naiveTRG3M2-TAMswereenrichedwithproinflammatory andpro-migratorygenesthathavebeenassociatedwithpoorsurvival incancer(C19orf33,migrationinhibitory factor [MIF], MRPL12, GDF15, CLDN3,and CLDN4). In comparison, post-treatment samples were enriched in genes for macrophage infiltration(CRTAPand SELL),activationof inflammasome complexes andsecretionofcytokines(IL1RAP, LRRK2, and FOSL2),malignant cellinfiltration and proliferation (ADIPOR1, SAP30, RGCC, and FKBP5), and antioxidant propertiesthatreduceoxidativestressandpreventcellulardeath (GCH1). The enrichment profile of treatment-naive vs. posttreatment TRG3 samples was further interrogated using DEnrichPlot. This revealed upregulation of adhesion proteins and signaling pathways including P13K-Akt, Rap1,and Ras. Conversely, estrogen and retinol metabolism pathways were downregulated (Figure 5C).

Pathologicalresponse-associatedvariation in intercellularcommunication

Finally,onthelimitedsetofsamplesavailable,weassessedcellcellcommunicationbetweencelltypesusingtheexpressionof ligandsandtheircognatereceptorsindistinctcelltypepatterns inscRNA-seqdata.Specificallyweaskedwhethercell-cell interaction pairs were altered between the response groups. Using theCellPhonealgorithms,24weidentified53differentiallyexpressed ligand-receptor pairs with a majority involving M2- TAM(Figure5E).Manyof thesealteredcommunicationchannels were driven by MIFand were enriched in M2-TAMof TRG3 samples(Figure5Fi),suggestingincreased signalingbetweenmacrophages and all othercelltypes.Of note,a transcriptional signaturesuggestiveofactivationof anintercellularsignaling axis based onMIF(onmacrophages)and CD74(onTREG)was found. This has been implicated in promoting tumor-infitrating TREG and modulatingresponse to checkpoint inhibitors and was enhanced in theTRG3samples.25Conversely,we detected adecreasedinteractionoftheM2-TAMandTcellsviaCD86(on macrophage)and thecoactivatorCD28(onTcells)intheTRG3 samples(Figure5Fi)basedonthereductionofbothrespective transcripts in these cell types.Thisreduction of theT cell costimulatoryreceptor molecule CD28engagement onTcells, includingTREG,byitsligandCD86onmacrophagessuggested a global lowering of T cell activation.While this seems to contradict theobserved accumulationofTREG,it maybeopposed by the decreased communicationof CD86with thenegativeregulator CTLA-4 in these non-responding patients, thereby unleashing thefeedback control of TREG population.26

Insummary,scRNA-seqfindingsreplicated theenrichment in thepoorresponders(TRG3samples)ofTREGandM2-TAM,as observedinIHC.Inaddition,cell-cellcommunicationnetwork analysis supports the concept of a mutual interaction between M2-TAMand TREG thatpromotes tumor-infiltrating TREG and maycontributetoimmunosuppressionanddampenthe response to immune checkpoint inhibitors.27,28

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Figure.nlecelanalyiglightindiferetlexessinandceladistritongseifstofret Aserfas the treatmentcycle,(i)samplenumber,(iv)responsetoeoadjuvant therapy,and(celltypeallocation:TL C D8+G Z M B+) ,TREG (\mathsf{C D4+F O}x\mathsf{P3+}) ,and M2- TAM (\mathsf{C D68^{+}C D163+}/\mathsf{C D206}+) cells from all collected samples (n=20) ， (B) A heatmap showing the top 10 genes (weighted by lowest adjusted p value)per clusterof M2-TAM,TREG, andCTLcell clusters among allsamples (n=20) ， baisss (treatment pre \begin{array}{r}{{\boldsymbol{n}}=10}\end{array} treatment mid n=2 and treatment post \scriptstyle n=5 with the corresponding numberof cellsper sample. (Dtrassiionofthaedahwng theidcelldsbtioncrsallsalstratiiedbtandrpntt

DISCUSSION

In this study, we have shown the feasibility of combined immune checkpointinhibitorandchemotherapy(aDCF)inthetreatment of GEA and favorable oncological efficacy.The study reached itsstatisticalprimaryendpointintermsofachievingpCR,thus corroboratingthepromisingefficacyofaDCFasanexperimental perioperativeregimenforthe treatment of locally advanced GEA. These results suggest an added benefit of combining the anti-PDL1monoclonal antibody,avelumab,withDCFsince the 14% rate ofpCRdescribedhereishigherthanthe 7% describedwithDCF alone as reported previously in our institutional cohort.29 Multiplex imaging analysis also suggests an association of the accumulation of M2-TAM and regulatoryT cells with poorresponse to the combined therapy. Cell-cell communication network analysis affordedbyscRNA-seq,whichidentifiesputative pairsof interacting cell typesbased onexpression of transcripts of ligands and their cognatereceptorintherespectivecells,alsosuggeststhatM2- TAMs,generally thought tobeimmune suppressive,represent a cell-cell communication hub, signaling to many stromal cells. In turn,thismayaccountforatumorimmunemicroenvironment (TIME)thatpromotesimmune evasion.

It is well established that tumor cell expression of PD-L1 interacts with PD-1 on Tlymphocytes inhibiting their cytotoxic ability. Thismolecularinteractionenablestumorstocircumventhostimmune surveillance.However,anothercriticalimmunecheckpoint involvesCTLA-4thatcompeteswithCD28forbindingtoCD80 and CD86 on antigen-presenting cells including macrophages. This interaction delivers co-stimulatory signals also essential foractivatingTlymphocytes.Duringimmunosuppressivestates, thegreater affinity of CTLA-4forCD80/CD86is thoughttodownregulate the stimulatory effect of CD28.30 This explains why CTLA-4 inhibition, which removes the CD80/86 binding competition allowing proinflammatory CD28 upregulation, has been identifiedasapromisingalternative treatmentparadigmin gastrointestinal malignancies.31

AmongtheTRG3samplespresentedhere,wefoundreduction inCD86/CTLA-4andCD86/CD28interactionsacrossvarious T cell populations, including TREG, and macrophages.These disruptionsimpairTcellproliferationandcytotoxiccapabilities and builds onprevious evidence describing the immunosuppressive environment found in the TIME of gastric cancer.32 Importantly,this“immune-cold”environmenthasbeen associated withpoorresponseto systemic therapy and impaired survival outcomes among patients withlocally advanced gastric adenocarcinoma.3Althoughfurtherresearchisneededonthis topic,we suggestthat this characterizationof theTIMEinpart explainspoortumoralresponsetoaDCF.

Additionally,theM2-TAMsfound inpoorresponderswerealso found tohave accentuated activation of themacrophageMIFCD74 pathway.The release of the chemokine MIF by M2-TAM, whichhasbeenfound during cancerprogression,is thought to promoteintra-tumoralaccumulationofTREGs,whichspecifically overexpress CD74.34 In addition to promoting the polarization of macrophagesinto theM2phenotype,MIF/CD74 interactionenhancestheproductionof immunosuppressivemolecules byM2-TAMandreducestheircapacitytoactivateCTLsencouraging anti-tumor immunity.Importantly,M2-TAMshave been foundtocontributetoTcelldysfunctionviathesecretionofspecific cytokines and metabolites affecting the therapeutic effect of anti-PD-L1 treatment.35,36 High M2-TAM concentration has also beenassociatedwithincreasedPD-L1expressiontherefore providinganother means of M2-TAM-derivedresistanceto anti-PD-L1 therapy.37,38 Our data also reveal that M2-TAMs interactwithotherelementsoftheTIMEviainteractionwith TNFRSF14，apotentproinflammatorytransmembraneprotein expressed onmultiple cells in theTIME.Downregulationof MIF interactionwithmembersofthetumornecrosisfactorsuperfamily has not been observed in the seting of neoadjuvant therapy resistance in gastrointestinal carcinoma.39

Withregardsto thesafetyof aDCF,theincidenceofgrade3-4 treatment-relatedAEs of 40% described herewashigherthanthe 20% describedinbothtrialandreal-worlddescriptionsofother taxane-based regimens.4.40 while the incidence of neutropenia anddiarrheawassimilartothatdescribedforflourouracil,leucovorin,oxaliplatin,docetaxel(FLOT),itisconsiderablylessthanthe 78% of grade 3 orworse AEs found when combining perioperative FLOTwithpembrolizumabin theKEYNOTE-585trial.14 Interestingly,inthistrial,theincidenceof treatment-related complications didnotvarybetweenthearmsimplyingthattheAEprofileisdriven bythesystemicchemotherapyandlesssobytheadditionof immunotherapy.Itisalsointerestingtonotethat,incomparison tothedatapresentedhere,only 33% ofpatientshadgrade _{3/4} AEs when camrelizumab, apatinib, nab-paclitaxel, and S-1 were combined.1° Additionally, in this study too, the addition of immunotherapydidnotseemtoaffecttheincidenceof treatmentrelated AEs. Therefore, we suggest that the side effect profile described here is in keeping with other studies based on taxane-basedsystemictherapyamongthisspecificcohortofpatients.Furthermore，whether adjuvant immunotherapy alone couldoffsetAEswhilestilldeliveringpotentialpathological and survivalbenefitsneeds tobeunderstood.

Figure5.Exploringsingle-cellcharacteristicsofM2-TAMsamongpoorresponderstoneoadjuvanttherapy
Fpa each time point in the treatment cycle (treatment pre, n=6 treatment mid, \begin{array}{r}{{\boldsymbol{n}}=3}\end{array} and treatment post \begin{array}{r}{n=5)}\end{array} ：
(B)A volcano plot of an unmatched comparison of differentially expressed genes among M2-TAM(C \mathsf{;D68^{+}C D163+/C D206+}) cells of patients with TRG3lesions between thetreatment pre (n=6) and treatment post (n=5) samples using a LogFold change cutoff of +1-1.5 and p valueof <0.05
(C)A heatmap of differentially expressed genes of M2-TAM (\mathsf{C D68^{+}C D163+}/\mathsf{C D206}+) among patients withTRG3lesions,comparing treatment pre (n=6) and treatment mid (n=3) with treatment post (n=5) with a LogFold change cutoff of +1-1.5 and p valueof <0.05
(）Funtiolepfifnlxdgeoftrisreala downregulated (red) genes.
(E)Aplotstrainad-rdifentilexreneMandothMelinea [n=14) ： (F)A dot plot comparing (\mathfrak{j}) MIF and (i) CD68 expression among M2-TAMs stratified by TRG status (MPR \begin{array}{r}{{\boldsymbol{n}}={\boldsymbol{1}}}\end{array} ,TRG 2 n=2 andTRG3 n=3!

Despite these knowledge gaps, that 96% of patients recruited to the study completed allpreoperative cycles and ultimately underwentsurgeryfurtherhighlightsthefeasibilityofthisregimen. Finally,theincidence of surgical complications amongpatients treatedwithaDCFwasalsoequivalenttopreviouslypublished data describing our institutional experience.41

Prospectivedata frombothsingle-arm andrandomizedtrials reveal a variation in the rate of pCR and MPR achieved. Among several single-arm phase 2 trialsinvestigating therole of neoadjuvantchemotherapyandanti-PD-L1therapyinthetreatmentof locallyadvancedGEA,pCRandMPRoccuratratesbetween 17%-45% and 55%-70% respectively.9.42-45 Emerging data from several randomized trials confirm this trend. The recently published NEOSUMMIT-01,inwhichtoripalimabwasadded to neoadjuvantSOX,describedapCRandMPRof 22.2% and 44.4% ，respectively.46TheDANTE trialfound that the addition ofatezolizumabtoFLOTwasassociatedwithapCRof 24% VS. 15% \left(\boldsymbol{p}=0.032\right) .47Similarly,basedonFLOTanddurvalumab,thephase3MATTERHORNtrialalsodescribedanincrease in pCR 19% VS. 7% ， \mathsf{p}=<0.001 andMPR 27% VS. 14% ， \mathsf{p}=<0.001_{,} .48Reflecting the greater efficacy of FLOT over alternative chemotherapeutic agents when pembrolizumab wasadded tocisplatin-based chemotherapy,pCRwas more conservative 12.9% versus 8% p=<0.001_{.}^{} .14 Furthermore, whencamrelizumabandapatinibwereusedincombination withnab-paclitaxel,anon-significanttrendtowardhigherpCR was found( 16.3% Vs. 6.0% p=0.094_{.} .10

Whileanequivalentoncologicalefficacyhasbeendescribed when comparing FLOT and DCF,41 the 14% pCR and 18% MPRfound inthepresentcohort arelowerthanthoseof thedocetaxelandanti-PD-L1regimensdescribedearlier.Despite thesevariations,it seems that survival outcomes among all patients who achieve pCR are similar irrespective of the regimen chosen.Moreover,whencomparing theDFSand OSof the entirecohorttotheaforementioned trials,theresultsare similar.

Theintra-trialvariationintheefficacyofdifferentanti-PD-L1 therapies interms ofpathological responsereflects,inpart,the differingexperienceofthepredictivevalueofpre-treatment PD-L1 expression in terms of response to treatment. For instance,a \mathsf{C P S}>1 wasassociatedwithimprovedoverallsurvival inpatientswithmetastaticGEAtreatedwith nivolumabor sintilimabinthe CheckMate 649^{6} and ORIENT 16^{49} trials. In an exploratory analysis,using a similar cutoff data from the MATTEHORN studyseemstoshowapositiverelationshipwithpathological response.DatafromtheDANTEtrialsuggestthatpCRwashigher if theCPSscorewas >=10 Similarly,MPRwasdoubledwitha C P S>5% among those treated with camrelizumab and apati\mathsf{n i b}^{10} and with toripalimab and chemotherapy.46

Conversely,CPSwasnotpredictiveofpathologicalorsurvival outcomesamongrecipientsofneoadjuvantpembrolizumaband FLOT14 or atezolizumab when combined with neoadjuvant chemoradiotherapy.50 Specifically among patients with MMRd lesions, \mathsf{C P S}>1 wasalsonotassociatedwithpathological responsefollowing treatmentwithneoadjuvanttremelimumab and durvalumab.51Despite the documented poor sensitivity of TPS in the setting of gastric cancer,52evenwhena threshold of 1% is used todefinepositivity,thelackofcorrelationbetween CPS and TPS described in the current studyfurtherunderscores theneedtoidentifyalternativebiomarkerspredictiveofpathological response to anti-PD-L1 therapy with a view to personalizing treatment.53 Such a discovery is crucial as the achievement of pCR following the administration of combination therapy seemstobeassociatedwithimprovedsurvivaloutcomes.

By questioning the role of PD-L1as a marker predictive of pathologicalresponse,effortshavefocusedonfindingbiological markersthatpredicttreatmentoutcomeswithaviewpersonalizingtreatment.Interesthasturnedtowardprognosticfeatures of theTIME,includingM1/M2-TAM,whicharecloselyassociated with tumor progression and response to systemic therapy.54 Specifically, in the setting of gastric cancer, although a higherlevel of {\mathsf{C D68}}^{+} macrophageinfiltrationhasbeenfound amongresponders toneoadjuvanttreatment，response to PD-L1 blockage plus chemotherapy is characterized more specificallybyanelevatedratioofM1 \scriptstyle(\mathsf{C D68^{+}C D11b^{+}F4}/ 80\substack{+\mathsf{C D}206-}) toM2 (\mathsf{C D68^{+}C D11b^{+}F4}/80^{+}\mathsf{C D206^{+}}) macrophages.55 The finding that there is a negative correlation betweenpro-tumoral M2-TAMdensity and response to therapy is welldocumented.56

The increased density of \mathsf{C D68^{+}C D206^{+}} M2-TAM in close proximity to tumoral cells has also been previously described, whichinturn is associated with improved survivabilityof cancer cells.57,58 M2 polarization during co-culture with gastric cancer cellsled to CxCL5 expression engendering chemoresistance viatheactivationofPI3K/AKT/mTORpathways.59Similar pathwayupregulationwasfound inthepathwayenrichment analysis described earlier. Unsurprisingly， the proximity of \mathsf{C D68^{+}C D163^{+}C D206^{+}} T2-TAM to the tumor core has therefore been negatively associated with patient survival,6 particularly if there is PD-L1 enrichment of \mathtt{C D163^{+}} M2-TAM.61 This is likely because M2-TAMs are thought to increase resistance to antiPD-L1 therapybybinding toPD-1and CTLA-4 on the surface ofcytotoxicTcells,blockingthelymphocytes'adaptiveimmune responseandreducingtheanti-cancereffectofimmunotherapy.62 Chen et al. identified that increased \mathsf{C D68^{+}S T I N G^{+}} densitywasnegativelyassociatedwithobjectiveresponseto anti-PD-L1 therapy among patients with gastric cancer.63 However,unlikeintheresultsdescribed inthepresentstudy,these findings were not correlated with pathological response.

WefailedtofindarelationshipbetweenMMRdandpathologicalresponse to treatment,afindingalso described elsewhere.14,46 Understanding why there is a lack of association betweenMMRdstatusandfavorablepathologicalresponse described in this cohort is challenging due to the limited data availablecomparingtheoutcomesofavelumabtherapyinpatientswithMMRdversusMMRplesions.Ina limitedstudyofpatientswithadvancedendometrialcanceralsotreatedwithavelumab,the presence of MMRd was not associated with the incidence of objective response to treatment.64Additional data fromthisstudyfoundthatJAK1andB2Mmutationscharacterize MMRdlesionsthatfailedtorespondtoavelumab.

This contrastsrecent randomizeddatadescribingan enhancedpCRrateof 58.6% amongMMRdpatientstreated withneoadjuvantnivolumabandipilimumabaspartofthe NEONIPIGA study.65 Improved pathological response among MMRd lesions hasalsobeendescribed within otherrandomized trial and single-arm studies including the INFINITY trial.14,51,66 In patientswithcolorectalcancer,MMRdstatushasbeenassociatedwithsubtlechangesintheTIMEincludingahigherdensity of multiple infiltrating lymphocyte lineages,67 production of more neoantigens,68 and modulators of systemic inflammation.69 The enhanced inflammatory response inherent encompassing thesetumors mayexplainwhy,inpart,subgroup analysesoflargecohortsofpatientswithearly-stageGEAhave reported no benefit in patients with MMRd tumors receiving chemotherapy alone.7° Although higher than in other recently published cohorts, the 14% incidence of MMRd within the study cohortiswithintherange of 4%-24% incidencewithinGEA described in a recent review.71 However, absence of samples prevented a single-cell analysis of this specific subgroup.

ConsideringthecomplexityofusingtheTIMEtopredict response to anti-PD-L1 therapy,interest has also turned to clinicaltools that are more readily available.Changes in tumor metabolism on PET/CT have been demonstrated to be a strong predictorofpathologicalresponsefollowingneoadjuvant chemotherapy.72 In the setting of esophageal squamous cell carcinoma，asimilarrelationshiphasalsobeendescribed following treatment with neoadjuvant chemo-immunotherapy.73 Conversely,among patients with adenocarcinoma treated with neoadjuvant chemo-immunotherapy,changes in the SuV and totallesionglycolysisduring treatment donot correlate with pathologicaloutcomeswithahighincidenceoffalsenegatives reported.9The results presented here further highlight the limitation of this imaging modality among this specific group of patients in terms of discerning disease response to neoadjuvant therapy.

Taken together,these findings alludeto the complexmodulatory relationship that M2-TAMs have withthepathological responseofGEAtoneoadjuvanttherapies.Assuch,theneed toidentifymorerobustandreliablebiomarkerstoguideprecisiontreatmentdecisionsandprognosticateresponsetoneoadjuvant systemic therapy is desperately required.

Overall，theactivityandsafetyofperioperativeaDCFare promising,and we have alluded to the fundamental role that M2-TAM and TREGplay in the microenvironment and the associationwithpathologicalresponsetoneoadjuvantchemo-immunotherapy.Whilethesurvivaloutcomesof thisstudyrequire longer-term data to robustly describe,we postulate that M2- TAMandTREGdensityandspatiallocationoftheM2cellsare potentiallyuseful inhelping guide precision systemic chemoimmunotherapyinpatientswithlocallyadvancedGEA.

Limitationsofthestudy

There are some limitations to this study.The small sample size and the single-center and non-randomized nature of this study mayaffect thepowerof the study,particularlywithregards to thehigh-dimensionaldata,notablyduetothelowavailability of samples for single-cellanalysis for patients with pCR.As a result, wewereunabletocomparebetweenpathologicalresponses andperformsubsetanalysisonpatientswithMMRdandHER2 positivity.Thisisparticularlypertinentconsideringthatanumber ofpatientsinthistrial wereMMRd (n=8) ,which hasbeen associatedwithadistinctimmuneprofileandresponsetoimmunotherapy. As such,the treatment and translational results for this specific cohort of patients may vary to those who are not

MMRd.Unfortunately,we did not have a sufficient number of MMRd patients to extensively differentiate the immune microenvironment between MMRd and MMRp tumors.By combining cohortswithotherstudies,thiscomparisoncouldbeexplored further in future work. The single-cell transcriptome data are furtherlimitedbythepresenceofsomeepithelial cellmarkersexpressed within the identified clusters implying potential contaminationatthesampleprocessingstage.Thelimitedavailabilityof single-cellsamplesalsoprecludedathoroughanalysisofvariablesbeyondTRGthatmayhaveaffectedelementsofM2- TAMfunction.Extendingtothedensityandspatialanalyses, the limited number of patients with non-cT3 lesions prevented comparison within the TIME across lesions with varying depth ofinvasion.Selectionandtreatmentbiasareinherentinthe studyduetothe single-centernatureof thestudy.Finally,the immaturity of thesurvival data limitsfurther analysisofsurvival outcomesstratifiedbyvariablesidentifiedintheanalysesin this manuscript.

RESOURCEAVAILABILITY

Lead contact

Requestsforfurtherinformation andresources should bedirected to and will be fulfilled by the lead contact, Dr LorenzoFerri(Lorenzo.Ferri@mcgill.ca).

Materialsavailability

This study did not generate new unique reagents.

Data andcodeavailability

·The RNA single-celldata havebeen deposited at the SRA repository as PRJNA1052389andarepubliclyavailableasof thedateofpublication. ·Multipleximagingdatareported inthispaperwillbesharedby thelead contact upon request. ·Thispaperdoes notreport any original code. ·Anyadditionalinformationrequired toreanalyzethedatareported inthis paper is available from thelead contact upon request.

ACKNOWLEDGMENTS

This research was financially supported by EMD Serono, Mississauga,Ontario,Canada,abusiness ofMerckKGaA,Darmstadt,Germany,and waspre viouslyconductedunder analliancebetween thehealthcarebusinessof MerckKGaA,Darmstadt,Germany,andPfizer.The authors alsoacknowledge the support of theMcGillUniversity Health CentreFoundation and theMontreal GeneralHospitalFoundation,Canada.Some of the samples used in this study are based upon work supported by the Department of Defense, U.S. Army Medical Research Acquisition Activity, under grant number W81XWH-21-0623 (CA200572).

The funding source did not have anyrole ondata analysisorinterpretationof results.The finding source did not assist with drafting of the manuscript.

ThehealthcarebusinessofMerckKGaA,Darmstadt,Germany，andPfizer reviewed themanuscript formedical accuracyonlybefore journal submission. The authors are fully responsible for the content of this manuscript,and the views andopinions described in thepublicationreflectsolely thoseof the authors.

AUTHORCONTRIBUTIONS

Conceptualization: T.A.,J.T., P.-O.F., S.P.,M.S., N.B., S.B., S.H.,V.S., and L.F. Methodology: T.A., J.T., P.-O.F., S.P., T.O., M.S., N.B., D.Z., C.M., J.C.-L., M.H., V.M., S.C.-B., A.S., G.E., M.F., G.A., A.E., R.S., S.B., M.P., S.H.,V.S., and L.F. Software: J.T.,M.S., and S.B.Validation: J.T., P.-O.F., S.P., T.O., and M.S. Formal analysis: T.A., J.T., P.-O.F., S.P., T.O., M.S., M.D., N.B., D.Z., C.M., J.C.-L., M.H., V.M., S.C.-B., A.S., G.E., M.F., G.A.,

A.E.,R.S., S.B., M.P., S.H., V.S., andL.F. Investigation:T.A., J.T.,P.-O.F.,S.P., M.S., M.D., N.B.,G.E.,S.B.,and L.F.Resources:T.A.,P.-O.F.,S.P., M.D., N.B., and S.B. Data curation: S.P., M.D., and S.B.Writing - original draft: T.A., J.T., P.-O.F., S.P., T.O., M.S., N.B., D.Z., C.M., J.C.-L., M.H., V.M., S.C.-B., A.S., G.E., M.F., G.O., A.E., R.S., S.B., M.P., S.H., V.S., and L.F. Writing -review and editing: T.A., J.T., P.-O.F., S.P., M.D., N.B., S.B., and L.F. Visualization: J.T., S.P., M.S., and M.D. Supervision: T.A. and L.F. Projected administration: M.D. and N.B.Funding application:T.A. and L.F.

DECLARATIONOFINTERESTS

T.A.hasreceivedresearchfunding from EMD Serono andconsultancyfeesfor BMS,Roche,Merck,Novartis,and Tiaho.Therest of the authors declare no competinginterests.

STAR★METHODS

Detailed methods are provided in the online version of thispaper and include the following:

·KEYRESOURCESTABLE
·EXPERIMENTALMODELANDSTUDYPARTICIPANTDETAILS oEnrolled patients
●METHOD DETAILS 0 Primaryand secondary endpoints 0 Treatment 0 Pathological assessment and response grading 0 Tissue collection and sample processing 0 OPAL 6 and spatial analysis 0 Singlecelltranscriptomics o Single-cell RNA sequencing oSingle cell quality control and processing
·QUANTIFICATIONANDSTATISTICALANALYSIS
·ADDITIONALRESOURCES

SUPPLEMENTALINFORMATION

Supplemental information canbe found online at https://doi.org/10.1016/j.
xcrm.2025.102045.

Received:July 18,2024
Revised:November28,2024
Accepted:March 6,2025
Published: April 15, 2025

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STAR★METHODS

Cell Reports Medicine Article

EXPERIMENTALMODELANDSTUDYPARTICIPANTDETAILS

Enrolled patients

Thestuyisreisterednwww.clinicaltralsgovNC8350andasreceivdapprovalfrmHalthCandaandthpitl ReearchEthicBardllpatientsgaveinformedandwrttenconsent prortenrolmntTheullstudyprotocolisavalab from the authors on request.

Fity-onepatients Maland6Fmalewererolled in thestudyPatientswerecharacterizedbabsenceof primaligna andauto-imuedieasendadquatehsilgicareeetotleratbthsystemicthrayndsurgicaroedur graphics of enrolled patients is included in results above and Table 1.

METHODDETAILS

Primary andsecondaryendpoints

Thisisainl lehaesttatthieritlnMnlanaa versityafliadtiarycentrsareferaentforpatentsfrmthrughutQubewithhaggastriccacThpary endpointof thisstudywastodesribetheassociationof preperativeaCFwithRasthisoutcoehasbeenlinkedtexellent survivalitherrialfchem-mtherahecondarnintwreteribthtw-adiese-fresuivalD and incidenceof grade _{3/4} adverse events(AE)associated with thisregimen.We also described a post hoc analysis of the tissue inflammatory microenvironment with a specific focus on M2 tumor associated macrophages.

Considerationsregarding sample size are discussed below.

Screeningroceduresforstagingoutielynlddcontrastifuecmutdtmgraphyofthehest,admenandpliad whole-body-FDG positronemotiontomography.Endscopiculrasoundwasselectivelyused anddiagnosticaparoscopyonl performed for sub-cardia gastric and Siewert type Ill gastro-esophageal junction tumors.

Inclusionandexclusioncriteria

Patientstghttlbrlusnnthstuyereeridacrdinthfolwinglsirtratitst beenearrlanadbipyfmdaencarcimofthhagumachrgstrohagetiolal stagedIItoIB(AJCC8thedition,74includingT1b, \N+Mx orT2-4,Nany,Mx)that wasdeemed tobe surgicallyresectable;aWorld Health Organization performance status score of 0{-}1 ;normal fullblood count, liverandrenaltest values.Patientswere excluded from thestudyif theyhad a dailyintakeofprednisonegreaterthan 10\mathsf{m g} (orequivalent)orrecent use ofotherimmunosuppressive medicationswithdyfenrolmentintthtrialseriousatomueorftiousdisasesthatcouldeacerbatdbym therapyrevisldoratralatatioonrainatintanfthchthrataentethprtl systemictherayfoatientswerealexcluedifthyhadkwseverhyerensitityreactionsmnoloalanti orthelentsofthrtlicallsignifantcadiasuleaestrkeriosdingartmiaad drug oralcohol abusedisorder.

Treatment

Theexpermntalerioperativeregimnsistedof4cyclfaCFgivneveryweekbefreandafugeryndaf premedication(dexamethasone12mgintravenously(IVaprepitant25mgorall)ocetaxelwasgivenatthedoseof 40mg\mathsf{N}/m2 over1h;cisplatin was administered at the doseof 40mg\mathsf{N}/\mathsf{m}2 ；5-fluorouracilwasinfusedover 48\mathsf{h} at thedoseof 1000mg/m^{2}/d ay. On day 4,avelumab was injected IV at the dose of 10mg/kg after oralintake of 650mg acetaminophen and IV administration of 50mg ofdiphenhdramihrtwasthnmnddtalwadiisratifallcancerdgndatarinwithaab Thesefphctifgastatthetfthstiatprtihladdaftr duetoneutropeniaor anepisodeof febrileneutropenia.

Toxicitywas graed according toNationalCancernstituteCmmonoxicityCriteriaforAdverseEvents (CTAE), version3 Administratflattelaafdiitifraret bocytopenia <75,000/\upmu L .Forneutropenia <1500/\upmu L ,chemotherapy could be withheldoradministered withG-CSFaccording to the investigator's judgment.Cisplatin dose was reduced by 50% for decreased creatinineclearanceto50-60 mL/minand was discontinuedi \mathsf{f}<50\mathsf{m L/\min} Docetaxel wasreduced by 25% incaseof CTCAEgrade4neutropeniapersisting longerthan7daysoraccompanied by feverand thrombocytopenia of CTCAE grade 3or greater.The dose of docetaxelwas reduced by 25% in case of CTCAE grade4neutropeniapersisting longerthan7daysoracompaniedbyfeverand thrombocytopeniaofCTCAEgrade3orgreater. Docetaxelwasdntid inthcaefeverehersensititybibirthnrf peripheraratf CTCAEgrade3or greater.The doseof 5-FU was reduced by 25% at anytimefor CTCAEgrade3orgreaterdiarrhea ormucositis, andCTAEgradrgreateand-fotydrmendruwasdisctidinthventfadelaybygreatrthaweed drug-specific toxicity.

Followingltfthaduant theaifttwapratulituatietsestgdw dedicatedcontrast infusedCTofthecest amenandpelvisandanDGTCTcanprformd5weksaerthendfth last neoadjuvant treatment cyclatientswithouistantmetastasesonrestaging dagnsticmaging nderwent reectionwi thefollowing2weekSurgicalapproachwasdictatedbytumorlocationandpatientperformancestatus.Extendedcelc ymphadenectom D2dissectionwasperformed inalpatients and en-blocmediastinaldissectionforesophageal cancerperformed where aplicable.Mnimall invasive approaches were adopted whendeemed appropriate by the managing surgeon. Post-operativecomlicationweregraddbytheThoracicSurgeryQualtymrovemet Claificationamodificationof t ClavienDindclaificationsystem.Postoperativ treatment wascommenced612weesafteurgicalectionand folwd asmilarasthejatretattsertheraftfolwedclcallyadrailgicallwihtrasd CTofthechet ameandpelseemnthsforthefirstwyears, andeverymnthstil coletiofearsoffolu. Upper endoscopy was performed 6 months for the first 3 years and then yearly for the following two years.

Pathologicalassessmentandresponsegrading

ProcessingofthespecimenandassessmentwasperformedwiththeClegeofAmericanPathologist (CAP)Protocls version4.1) AsperCAPrecommendationsfrphagealcarcinmas,thpre-perativetherapywasgradedaccordingtothdifidyan Scheme for Tumor Regression Score.21

Ascorefavevaacereledtedpatlialtreeiilelare groupsofcancerelereeneanthpreecfreidualacrwithidenttmrgresntmorethal celmeraterenseandifthereasextensivrsidualancerporrepne)coresandtgetherfindmajat ologic response MR)Fhermore, reponderswere defind as patients inwhmTG Owas found whilst on-reponers referred to patients with TRG 3.

Immunohistochemistrywasperformed onformalinfixedparaffinembedded (FFPE)sections with theRocheVentanaBenchmark UtraRohiastiCanada)MathpaiMhisthmistraefdwthrdlddne hMLH1(G168-15, Roche Diagnostics), hMSH2 (G219-1129, Roche Diagnostics), hMSH6(SP93, Roche Diagnostics) and hPMS(ochianstisthprereatetbiMathpairdeficinywasdeindadefinye ormorefthMprtasethtmlatefrrprtinreultfbmarrestinfpcimnfratieti carcinomaofthecolonandrectum(version1.2TumorPD-LexpressionwasdeteminedusingDak73-10cloneAglentch nolgieCanadcsintheaAutstairapparatusAgintchgis)-slesitivontrolsncluddi and placenta.The Combined Positive Score (CPS)and Tumor Proportion Score(TPS) were both assessed.

Tissue collection and sample processing

Tisuasretrivedviandpicbipyatseveralstagsthroughutthpatient'steatmtathwadingtreatetaiv mid-treatent aftetwylofaCFandpst-treatmetpost-resectionsamlsinghig-eiionwhitelighted ensurethffifaleltulienthetdhaddiolaa the samelocation.The formerwasplacedintoacold mediumof RPMIInitrogen), Primocin (nvivgen andgentamycin(nvitrogen) whilsthelattewasformalinfidandparaffinmeded(Ffrformalpatholgicalanalsistcnfimthepreencefadn carcinomawithin the sample.

Ifcarcinmawaspathlgicalfme, thesamlwasroesedforinleceltranscripticanpatialanali diagram describing the experiment workflowis displayed inFigureS1.

OPAL6andspatialanalysis

FFPE sectionsweremathed and analzedusingmuliexmmunoistochmistrywithanOPAL(6-plexkit (N00a Biosciences)The labeling forvariousprimarantiodisinclded K7(Roch,#794462),D8DAKO,#M103),granz (Roche,#7604283),CD68(Roche#790-2931),D163(Roche#760-4437)and FoxP3CST,#9837).Asecondary antibodyand OPALdyswere thnsed andfinlly counterstained withspectralDAPAreference slie with sinle labelswas alsprepae. ALSM710microscoe (Zeisswas sed to capture slide images with 56 regionsof interest per samle for each patient. The HALO software package (Indica LabsSAwasused forimaging analysisHighcelldensitywasdefined as allvalspe \upmum^{2} greater than the median density forthe treatment naive and post-treatment samples separately.

UsingtheHALHighexmduleathlgistsblindlyeviwedtissueanclasiedtissueastumororstmaexcldingnmal and fibroticareasCegmentationandindividualemarkinwerethfomdand thpatiaAalsisMdfL thenuseddtemnethatialrelationhpoftmrcellteithertxielacrophaeorptentialglat Tcells.The proximity algorithm computed the number of cells within a given distance of the tumor cell (</{>}50up\upmum) ，

Singlecell transcriptomics

To achieve single cell disassociation, specimens were dissected minced and digested using 5m L of Advanced DMEM/F12 containing 10\mathsf{m g} Collagenase Type 3Worthington) and 500U Hyaluronidase (Sigma) ina Ctube (Mitenyi using the gentleMACS OctoDissociator(MiteniandresusendedinSandMMfTThesspensionwaspassedthrougha0Oumcelstrainer(Fisherand centrifuged (500x9 at 5 min, 4°\mathsf{C}) . The cells were resuspended in 0.25% Trypsin-EDTA (Invitrogen) and subsequently incubated for 5min at 37°\mathsf{C} The trypsin was then inactivated by adding 10% fetal bovine serum.After being re-centrifuged (500x9 5min, 4°\mathsf{C}) the cell pellet was resuspended in 2.5U Dispase/ 10\upmu\up g DNAse buffer and incubated for 5 min at 37°\mathsf{C} which was then inactivated by adding PBS. The homogenate was then passed through a 40~\upmu\upmu strainer (Fisher) prior to being centrifuged again (500x9 5 min, 4°\mathsf{C}) ： UsingCLnufferGfmnatmeratdeerfaddingndenenifugda (500xg,5 min, 4°\mathsf{C} ,the cell pellet was washed twice with 2% fetal bovine serum in PBS.The substrate was then processed via the 10x Genomics platform.

Single-cell RNA sequencing

TheLighcleReaTeinstrumentRoheandtheAAlbrayqantiationkit Rochwreedtquantiyel libariswittriliateemhequantiicatialedflbraryeranlnq normalization.

Afer10XGenomicslbraryconstruction,theMGIasyUniversalLibrarConversionkitwasusedtoensurealloutputswer compatibwitGsqecrverioirculariesthlbralincaliatontharatnst which individuallyfold intoa tight nanoball whereeach library fragment results ina singleDNAnanoball clone).

The DNA nanoballs werequantified with Qubit ssDNAH assay kit ThermFisher), normalized and loaded intothe sequencing flowcellssingana-oadingmthdsMG0OautderTheflowcelaveafuntional surfacethatcaptureanm mobizesthanball inagridipattw lbrarieweetypcallladdelanforthinlcelsqi using theDNBSEQ-G40ORSPE100MGIkit withApp-A primersonaNBSEQ-G400MGIsequencerSinglecellRNAlibraries weresequnced with28cyclesforRead50yclsforRead2andyclsforthei7index.Colorbalancingwasachived withcolor-balancedsingleindxadapters(xGenmicsweresedforbrarissequencedonMlThesMGrunwereu tiplexedbyfastq-multxhttps://ithub.com/bwnjfastq-multxorfgbiDemxasts(htp:/fulcrumgenmics.githbo/fgbiotos/ latest/DemxFastqhtml.nbothinstancesweusedamismatchof.AferpolA-trimingviacutadapt (3.2)86,reaswere pseudo-alignedtotheGRCh38referencetranscriptome(ENSEMBLrelease96)withkallito0.46.287usingthedefault ke size of31.Thepeu-alignd readswere processed intoacel-by-gene count matrixusing bustols 040.0)Cellbarcds werealtered using thewhitelist (v3)providedby1xGenomics.AllfurtherprocesingwasdoneinSeurat(version4.4).

Singlecellqualitycontrol andprocessing

Seurat \mathsf{v}.4.4^{77} was used forallsinglecelanalyses.Standard pre-processing steps wereperformed starting withqultycontrolon the single celltranscriptomes.Cells with <1000 UMIs or <500 genesdetected wereremoved.Cellswith {>}20% mitochondrial RNA content werealsexldedawoutswere thnpressed asinhnt alusinpe-cellnmalation,folwedbighla iable gene selection h=4000 genes), log1p transform and z-scoring per gene.PCA was computed based onthe z-scored values.A nearest neighbor graph (\mathsf{k}=15) was built onthe first 50 PCs.UMAP embeddings and Leiden-clustering were calculated based onthis nearest neighbor graph.ntegrative analysiswaserformedusingCanonicalcorelationanalysisandceldoubletswerethn removed viaDoubletinderThis yieldd48highqualitytranscriptmes.Thecetpeswere assigned tclustesbasedon expression >1% of CTL {\mathsf{C D}}8{\mathsf{+G Z M B+}} (CTL), \mathsf{C D4+F O}\mathsf{X P3+} (TREG) and \mathsf{C D68^{+}C D163+}/\mathsf{C D206}+ (M2-TAM). Samples were grouped accordingttheirGstatusnfinlpathlyTG/stratibyretralpointinthetreatmntpathwayreatmaive ‘pre';mid-tretntmid;adst-tretnt,sthereweresamsfrmptntwahiedavaila analysis.

DifferetillxssdgenwesteralclatdviaWldsttiakaad to compute and visualisecetp proportions pe samle arosstreatment cycles and across resonse grousDEeqwas usedtoperfmdkdiferential xressionanalyisweepreidandstsaleierentialyxred genes were identified using JLogFold changel >1.5 and an adjusted p valueof <0.05 .Finally, in order to explore gene enrichment, theDEenrichlot packagewasused withtheKEGG_2019_Huma'databasetoidentify thetop5verexpresedandunderexpressed pathways among TRG3‘pre'and‘post’samples.

QUANTIFICATIONANDSTATISTICALANALYSIS

Tomeet theprimary objective and todetect an improvement of pCRfrom the 7% described in historical studies of DCFinpatients withesophagogastric adenocarcinoma toaproposed incidence of 20% ,aSimon2-stageschemewaschosento achieve astatistical powerof 80% .Allowing foran a errorof 5% the first stage of the study was considered successfulif more than one out of the first16 patietsiiladwtiacrualtptahwitst positivfatrethaatietsahiederelrethapattamnthfistnol study would be closed tofurtheraccrual.

Secondary endpoints were the two-year disease-free survival(DFS)and incidence of grade _{3/4} avelumabrelated adverse events (AE)associated withtisgxatonntwearatobased,hythei-generatixpermntThisid using the intensityof-L1 exressionontmorcels Sscoreorcombined tumorand immuecellsCPSscore),epresonf mismatchreairMrotianstatuimhistohemistryandgenrevealdbyinglecetrancripoinl ysis to predict tumor response to the experimental regimen.

CategoricalataisdescribdasabsoluteumbersandpercentagesContinuousdemgraphicdtaspresentedamedianwt range whilst data from the IHC analysis is presented as mean with 95% Cl.Fisher's exact test was used to compare categorical variables including HER2 and MMR statusstratied byTRGcategoryThisincluded the assesment of the percentage changen maximum SUV (\mathsf{S U V}_{\mathsf{m a x}}) which was generated by calculating the change in the post-treatment \mathtt{S U V}_{\mathtt{m a x}} and the pre-treatment \mathsf{s u v}_{\mathsf{m a x}} Using {>}35% change as the cut-off forin this measurement,changes in the \mathtt{S U V}_{\mathtt{m a x}} could be compared betweenTRG categories.

For the comparison of continuousvariablesrelating to demographic data,the Mann-Whitney- U test wasused.To compare the changingdensitywitinthMtwediffeeGatoristratidbyeutyetaywithT tinwalarifeittleifatrt Welch'scorrection was chosen.Samples were excluded from this analysis if theyfellbeyond the 95% CI.Finally, comparison of differentillpredgeewaspuiWa-talaMualuereeneratdwithuivalu comparewitRanMantCoxdianfolwuwcalulatdintevrKalaMethdtain thedateof pathologicaldiagnosis,thedatacutoffdatetocalculatedisease free andoverallsuvivalwasthe 16^{th} of May 2023.

Boxplots areshownwiththehorizontallinedenoting themedian,verticallinedenoting the 95% confidenceintervaland *_{+}, denotingtheatltachdtretsthtitrastnrpi foreachsubgroup.Sinificant differences within these diagrams are highlighted with \*'or\*\*'respectively.Al p values are two-sided with a value of <0.05 being considered statistically significant.Allanalyses were performed inRv.4.1.0with ggplot2 which, along with SPSS version 29, was used to create all plots.

ADDITIONALRESOURCES

Thestudy isregisteredonwww.clinicaltrals.govURL:https://clinicaltrialsgov/t2/sh/CT03288350 (NCT03288350)