Tumor-Infiltrating Lymphocytes and Survival Outcomes in EarlyERBB2-PositiveBreast Cancer 10-Year Analysis of the ShortHER Randomized Clinical Trial

MariaVitriiacasniDtfnartliniDlhirnuiiCaa AntoninMusolinhDranescoGiottaMDnitaimantiMD;rellGarroneD;naertonD KatiaCagossiMD;amantaartiMD;ntnellaFerMD;edericoiacentinihD;nricorvietoMD Melindaanders,MD;FdericaMigliettaPD;DavideMassaMD;araalduzziD;iefrancoontM; Roberto D'Amico,PhD; Valentina Guarneri, PhD

IMPORTANCEForpatients withearlyERBB2(formerlyHER2)-positivebreast cancer,thereisa needtoidentifybiomarkerstoguidetreatmentde-escalation.

OBJECTivETo evaluate the associationof tumor-infiltrating lymphocytes(TILs) withdistant disease-free(DDFS)and overallsurvival(OS)for patients with ERBB2-positive early breast cancer.

DESIGN,SETTING,ANDPARTICIPANTSTheShortHERrandomized clinicaltrialwasa multicentrictrialinItalythatenrolled patients withERBB2-positivebreastcancerfrom December2007toOctober2013.Patientsreceived9weeksor1yearofadjuvant trastuzumab combined with chemotherapy.Tumor samples were evaluated for TILs.Herein, patientswereevaluatedatamedianfollow-upof9years,anddatawere analyzed from February2023toAugust2024.

INTERVENTioNFour cyclesof anthracycline-based chemotherapyfollowed by4 courses of taxanes combined with trastuzumab for1year (long arm) or3 courses of taxanes combined with trastuzumabfor9weeksfollowed byreduced-dose anthracycline-based chemotherapy for 3 courses (short arm).

MAIN oUTcoMES ANDMEASUREsTheassociationof TILswithDDFS and OS was assessed with Cox models.

RESULTs Of1253patients enrolled in theShortHERtrial,866 women(median[IQR]age,56 [48-64]years)had evaluableTILs.In Coxmodelswith relevant factors,each 5% TIL increment was associated withimprovedDDFS(hazard ratio[HR],O.87; 95% CI,0.80-0.95; P=.001_{.} and OS(HR,0.89; 95% CI,0.81-0.98; P=.01_{*} .The10-yearOSratewas 91.3% for patients with TILs 20% or higher, 93.3% for patients with TILs 30% or higher, and 98.1% for patients with TILs 50% or higher,resulting higher vs lower TIL counterparts.Patients with TILs lower than 20% showed a better outcome with the long vs short treatment(10-year DDFS, 88.7% VS 81.0% .whereas patients with TILS 20% or higher showed the opposite (10-year DDFS, 87.1% VS 92.2% 专 P for interaction \mathbf{\Sigma}=\mathbf{\Sigma} .01). Similarly, patients with TILs 20% or higherhad a10-yearOSrateof 89.3% in the long arm vs 93.1% in the short arm (HR,0.36; 95% CI, 0.10-1.36); patients with TILs lower than 20% had a10-year OS rate of 91.3% in the longarmvs 86.9% in the short arm (HR,1.36; 95% CI, 0.82-2.23; P forinteraction \mathbf{\sigma}=\mathbf{\sigma} .06).

CONCLUSIONS AND RELEVANCEThis follow-up analysis of theShortHERrandomized clinical trial is,toourknowledge,thefirst demonstrationof anindependenteffectofTILsintermsof OSforpatients with ERBB2-positive early breast cancer treated with adjuvant chemotherapy andanti-ERBB2therapy.PatientswithTILs 20% orhigherwhode-escalatedtrastuzumab durationand chemotherapydose were notexposed toanexcessriskofdistantrelapse or death.

AuthorAffiliations:Author affiliations are listed at the end of this article.

TRIALREGISTRATIONEudraCT:2OO7-O04326-25

Corresponding Author:Maria
VittoriaDieciMD,Departmentf Surgery, Oncology and
Gastroenterology(DisCOG),
University of Padova,
via Gattamelata64,35128,
Padova, Italy (mariavittoria.dieci@ unipd.it).

utcomes for patients with early ERBB2 (formerly HER2)-positive breast cancer have improved in recentdecadesbecause ofneweffectivetreatments.The current abundance of drugs and strategies that play a rolein this disease,alongwith theconsideration that mostofthe patients reach optimal survival outcomes at long term,mandatesforcarefultreatment personalization based ontheindividual risk-benefit ratio. In such scenarios,there is an ongoing need toidentify biomarkers that could help refine prognostic stratification andguidetreatment de-escalation.

TheShortHER trial was a phase 3 noninferiority randomized clinical trial comparing chemotherapy combined with 1 year(long arm) vs 9 weeks(short arm)ofadjuvant trastuzumab in patients with ERBB2-positiveearlybreast cancer.While the primary disease-free survival(DFS)analysis did not allow for the noninferiority of the short arm,therecentlypublished 10-year overall survival (Os)update shows quite superimposable outcomes for the 2 arms,especially for patients with no or limited nodal involvement.2

Key Points

QuestionIstheleveloftumor-infiltratinglymphocytes(TILs) associatedwithsurvivaloutcomesinpatientswithERBB2 (formerlyHER2)-positive earlybreastcancer,and canitguide adjuvanttreatmentde-escalation?

FindingsInthis10-yearfollow-up analysisof the ShortHER randomized clinical trial, among 866 patients with available TILs there was astatistically significant associationineach 5% increase in TILs and improved overall survival. Patients with TILs 20% or higher were not exposed to excess risk of death when treated with de-escalated chemotherapy and trastuzumab.

MeaningTILscanserve asabiomarkertoidentifypatientswith ERBB2-positive early breast cancer who may safely undergo de-escalated adjuvant therapy without compromising long-term survival outcomes.

Previously,we reported on the possible prognostic role of tumor-infiltrating lymphocytes (TILs) in the ShortHER trial at a median follow-upof6years,showinga positive,independent associationbetweenTILdensityand distant diseasefree survival(DDFS) (10% increments:hazard ratio[HR],O.73; 95% CI, 0.59-0.89).3 Notably, we observed a statistically significantinteractionbetweenTILlevels at a 20% orhighercutoff and treatment arm, as patients with low TILs showed a statisticallysignificantbenefitfromthelongertreatment(short vs long: HR, 1.75; 95% CI,1.09-2.80), whereas those with high TILs had an excellent prognosis regardless of treatment duration, with even numerically better outcome in the short arm (short vslong:HR,0.23; 95% CI, 0.05-1.09).

Our previous analysis added to a growing body of evidence supporting the prognostic value of TILs in ERBB2- positivebreast cancer in both the neoadjuvant4-9and adjuvant setting,10-12 where TILs are strongly associated with surrogate end points such asincreased rates of pathological completeresponses and reduced riskof relapse.However,to our knowledge,no single study has demonstrated an association between TILs and OS,the most important survival end point in the early setting.Herein, we present the updated analysis ofthe possibleprognosticroleofTILsin the ShortHER trial and assess theimpact ofthisbiomarker onsurvival outcomes, including OS, at a long-term follow-up.

between treatment arms. In the long arm, patients received doxorubicin, 60~mg/m^{2} plus cyclophosphamide, 600~{mg/\Omega} {m}^{2} ,or epidoxorubicin, 90mg/m^{2} pluscyclophosphamid \scriptstyle.=\mathbf{e} 办 600mg/m^{2} administered once every3weeksfor4coursesfollowed by paclitaxel, 175mg/m^{2} ,ordocetaxel, 100mg/m^{2} once every 3 weeks for 4 courses;in the short arm,chemotherapy consisted of docetaxel, 100mg/m^{2} onceevery3weeksfor3 courses followed by fluorouracil, 600~{mg/m^{2}} epidoxorubicin 60, mg/m^{2} ,and cyclophosphamide, 600mg/m^{2} administered once every 3 weeks for 3 courses.

The primary end point of the trial was DFS. OS was analyzed as a second primary outcome.

At the primary DFS analysis(median follow-up of6 years), thestudydid notdemonstratethe noninferiorityoftheshort arm,showing a5-yearDFS of 88% in thelong arm and 85% in the short arm,with the upper border of the CI for the HR of 1.13 90% CI, 0.89-1.42)crossing the upperlimit of1.29,which waschosenasthenoninferioritymargin.Atthemorerecent median follow-up of 9 years,the 10-year DFS was 77% in the long arm and 78% in the short arm (HR,1.06; 90% CI,0.86- 1.31); the 10-year OS was 89% in the long arm and 88% in the short arm (HR, 1.15; 90% CI, 0.85-1.56).2

This trial was approved by local ethical committees and conducted in compliance with the principles of Good Clinical Practiceand theDeclaration ofHelsinki.Patientsprovided writteninformedconsentfortumorsampleuseforresearchpurposes.The trial followed the ConsolidatedStandards of ReportingTrials(CONSORT)reportingguidelines.

Methods

StudyDesign,Patients,andTreatments

The ShortHERtrial(EudraCT:2007-004326-25;Clinical Trials.gov Identifier:NCT00629278)was a multicenter, phase 3 noninferiority study conductedinItaly.Detailsof thetrial design,statisticalpower,andeligibilitycriteriahavebeenpre viously published (see alsoSupplement1).1

The trial randomized 1254 patients with ERBB2-positive earlybreast cancertoreceive 9 weeks(short arm;experimental)vs1year (long arm)ofadjuvant trastuzumab combined with chemotherapy.Notably,the chemotherapy regimen differed

Pathology

TILs werecentrally assessed ona singlehematoxylin-eosinstained slide of the primary tumor tissue according to guideline recommendations.7Details on the TIL assessment,including concordance between the 2 investigators (M.v.D.and M.S.) involved in scoring, have been previously reported.3

ERBB2 and hormone receptor status were assessed locally.Hormone receptor-negative status was defined as estrogenreceptor andprogesteronereceptorexpressionless than 10% ，

Figure1.CONSORTFlowchartfortheTumor-lnfiltratingLymphocytes (TILs) Substudy of the ShortHer Trial

FFPE indicates formalin-fixed paraffin embedded.

Statistical Analysis

The populationfortheTIL analysisisbased on thetotal numberof patients withcentralized tumorsamplessuitableforthe analysis(866 per protocol).Therefore,noformal sample-size calculation was carried out.

DDFSwascalculatedfrom thetimefromrandomization until distant relapse or death,whichever occurred first.13 OS wascalculatedfromthetime ofrandomizationuntil thelast follow-up or death,whichever occurred first.Patients without events were censored at the time of the last follow-up.

For survival analysis,TILs were primarily considered as a semicontinuousvariable (5% increments),and the Coxregression model was used tocalculate HRs and 95% CIs.The KaplanMeiermethod was usedto estimate survivalcurves,and the log-rank test was used to compare survival curves, adopting different arbitrary cutoffs to define patients with low or high TILS >=20% 费 230% and >=50% 0.TheinteractionbetweenTILs and treatmentwasexplored atthe 20% orhighercutoffto definehigh-vs low-TIL groups,to maintain consistency with the previously published analysis.3At that time, this cutoff was arbitrarily chosen to identify a group of patients with a DDFS rate at 5years of at least 95% ,which was considered a clinically acceptable definition oflow-riskpatients.To study the interaction between treatment arm and TILs, we used a Cox model,including treatment arm, TILs, and the interaction term.

At a medianfollow-upof 9.02years 95% CI, 9.00-9.04 years),the total number ofevents was107for DDFS (24 more comparedtothepreviouslypublishedanalysis?)and 74events for OS.The HR for the DDFS comparison between the short vs long arms was 1.26 ( 95% CI, 0.92-1.72) in all randomized patients and1.44 95% CI,0.98-2.10)in the cohort with TILs.The HR for the OS comparison between the short vs long arms was 1.15 95% CI,0.80-1.65) in all randomized patients.2 The HR for OS in the cohort with TILs was similar (1.11; 95% CI, 0.71-1.76).

Data were analyzed fromFebruary 2023 toAugust 2024. Statistical analyses wereperformed using SPSS,version 29 (IBM).All tests were 2-sided,with P<.05 indicating statistical significance.

TILs and Prognosis

The level of TILs was statistically significant and independently associated with both DDFS and OS.In Cox modelsincluding relevant variables(age,menopausal status,anatomic stage,histologic grade,and hormone receptor status),for every 5% increasein TILs there was a 13% reduction in the risk of a DDFS event (HR,0.87; 95% CI,0.80-0.95; P= .001) and a 11% reduction in the risk of death (HR, 0.89; 95% CI, 0.81-0.98, P=.01⟩ .Theother variablethat maintained anindependent association was pathologic stage(Table).

Next,patients werestratified accordingtoTILlevels,and survival curves were compared forpatients with high and low TILs,according to different cutoffs .<20% VS 220% <30% VS 230% and <50% VS >=50% 0.Figures 2 and 3 report KaplanMeier curvesforDDFSand OS according to these cutoffs.In terms of DDFS,patients withhigh TILs experienced a statistically significant better outcome compared to patients with lower TILs ateachconsideredcutoff.The absolute difference in both 8-year and10-year DDFS rates was cutoff dependent, reaching 8.9% and 12.0% in DDFS and OS,respectively,at the 50% cutoff point.The10-yearDDFSratesfor patients in the high-TILgroups reached 89.8% for patients with TILs 20% or higher, 91.7% for patients with TILs 30% or higher, and 96.9% for patients with TILs 50% or higher.Similarly,there was a numericallybetter OSforpatients with highvslowTILs ateach consideredcutoffpoint,reachingstatisticalsignificanceforpatients withTILs 30% or highervs lower(HR,0.38; 95% CI, 0.16- 0.95, P=.04) .Similarlytowhatwasobserved forDDFS,the absolute difference in both 8-year and 10-year OS rates progressivelyincreased at higher cutoff points,reaching 6.1% and 9.3% in DDFS and OS,respectively,atthe 50% cutoff point.The 10-year OS ratesfor patientsin thehigh-TILgroups were cutoff dependent, reaching 91.3% for patients with TILs 20% or higher, 93.3% for patients with TILs 30% or higher, and 98.1% for patients with TILs 50% or higher.

Results

Patients'Characteristics andFollow-Up

Patients characteristics havebeen previouslypublished.3Briefly, 866women(median[IQR]age,56[48-64]years)withavailable TILs were included inthe analysis(TIL cohort), constituting 69% ofallpatients randomizedinthe ShortHER trial(Figure1). Clinicalpathologicalfeaturesofthecohort withTILswererepresentative ofthe broaderstudy cohort and similartopatients without available TILs, exceptforaslightlyyoungerage inthe cohort withTILs.3 Median (IQR)TIL density was 5% (1%-15%)

InteractionBetweenTILsandTreatmentArm

Therewas a statisticallysignificant associationbetweenTILs as a semicontinuous variable( 5% increase) and DDFS and OS in patients from the short arm (DDFS:HR,0.82; 95% CI,0.72- 0.94; P=.003 ；OS:HR,0.86; 95% CI, 0.75-0.99; P=.04, ， though notin patients from thelong arm(DDFS:HR,0.93; 95% CI,0.84-1.03;OS:HR,0.94; 95% CI,0.84-1.05),with nonstatistically significant interaction tests (DFFS: P for interaction \mathbf{\Sigma}=\mathbf{\Sigma} .122;OS:P for interaction \mathbf{\varepsilon}=.39 0.At this updated analysis, the observation of a statisticallysignificantinteraction betweentreatmentarm andTILs wasconfirmed at the 20% cutoff for DDFS {\bf\nabla}_{P} for interaction \mathbf{\Sigma}=\mathbf{\Sigma} .01), with patients with low TILs showing a statistically significant better outcome with the long vs short treatment (10-year DDFS, 88.7% VS 81.0% P=.006 ,whilepatientswithhighTILsshowed anexcellent outcome irrespective of treatment arm,with even numerically improved DDFS when treated in the short vs long arm (10-year DDFS, 92.2% VS 87.1% 电 P=.09_{*} 。

Kaplan-Meier curves are shown inFigure 4A and B. The results of the interaction analysis considering OS asthe clinicaloutcomepointedtowardthesamedirection(Figure4Cand D).For patients with TILs lower than 20% there was a numerical advantageforthelongvs short treatment(10-year Os, 91.3% VS 86.9% ；HR,1.36; 95% CI,0.82-2.23; P=.23⟩ .Patients with TILs 20% orhigher showed a numericallybetter OS in the short vslong arm (10-year rate, 93.1% VS 89.3% HR, 0.36; 95% CI, 0.10-1.36; P= .13), with Pfor interaction of.06).

InteractionBetweenTILsandStage

Tofurtherexplore the independent roles ofTILs and stage,as these were the only 2 factors emerging as having a statistically significant association with DDFS and OS in Cox models (Table),theassociationbetween TILs and survival wasevaluated in stage-defined subgroups.Results showed a statistically significant impactofTILs onlyforpatientswithstageII disease,forboth DDFS and OS.TheHRforDDFSfor each 5% increment in TILs was O.98( 95% CI, 0.88-1.09)for stage I, 0.73 95% CI, 0.59-0.91; P=.004) for stage II, and 0.89( 95% CI, 0.78-1.03; P= .11) for stage III {}^{p} for interaction \mathbf{\varepsilon}=.23\mathbf{\dot{\varepsilon}} .For OS, the HR for each 5% increment in TILs was 1.01( 95% CI, 0.91- 1.12) for stage I, 0.69( 95% CI, 0.51-0.95; P=.02⟩ for stage II, and 0.88( 95% CI, 0.74-1.05) for stage III( (P_{\l} for interactior 1=.07, .Kaplan-Meier OScurvesforpatients with TILs 20% or higher vs TILs lower than 20% in different stage groups are shown in the eFigure in Supplement 2.

Discussion

This updated analysis from the ShortHER trial provides,to our knowledge,thefirstevidenceofanindependenteffectofTILs onOSinpatientswithERBB2-positiveearlybreastcancer treated with adjuvant chemotherapy and trastuzumab.We demonstrated atamedianfollow-upof9yearsthattherewas a statisticallysignificantassociationbetweenincreased TIL density and improved OS and DDFS.Moreover,these data suggest that patients with high TILs at the 20% or higher cutoff whode-escalatetrastuzumabduration andchemotherapy dose are, at least,not exposed to an excess risk of death, indicating that TILs mightbe used toidentify asubgroupofpatients who can safely receive lessintensive treatment.

Theprognosticrole ofTILsinearlyERBB2-positivebreast cancer is supported by growing evidence.14,15 In the neoadjuvant setting,high levels of TILs are associated with increased rates of pathological complete responses and reduced risk of relapse.4-9 In the adjuvantsetting,TIL densityis associated with improved outcomes,though previous studies have focused on surrogate end points rather than Os.10-12 Recently, a large individual data meta-analysis by the Early Breast Cancer Trialists'CollaborativeGroup,including4168patients from5randomizedpivotaltrialsofadjuvanttrastuzumabwithchemotherapyvs chemotherapy alone,confirmed the important prognosticrole ofTILs in terms oftime tofirst recurrence,but no OS data were reported.16 The present study extends these findings by demonstrating a direct independent association between TILs and the most valuable survival end point, strongly reinforcing the role of TILs in patients with early ERBB2-positive breast cancer.

The question about the optimal TILcutoff remains open,sinceinthis study(andothersi6)thereisnoevidence of a natural cutoff that maximizes the possible prognostic potential of TILs.TILs are associated with survival as a continuous variable; therefore,the higher the cutoff point, thehigher the ability of thehigh-TILdefinition toidentify patients with an excellent outcome.Moreover, in this study, theslopeof the curves forlow vshigh TILlevels aredifferent, especially when using higher cutoffs, suggesting that the time course of the disease is fundamentally altered rather thanjust delayed.Althoughthe absenceof a clear prognostic cutoff might put into discussion the translation of TILs into clinical practice, 2 arguments can be raised. First, it is important to define the cutoff that best supports clinicians in a specific treatment choice, which is not necessarily the best prognostic cutoff. This question is optimally addressed in trialstesting de-escalation approaches,as in the ShortHER trial.Second,TILs could be considered as a continuousvariableinintegratedprognostictools.

Regarding the first point,we have demonstrated a statistically significant interaction between TILs at the 20% or higher cutoff and treatment arm, as patients with low TILs showed abetteroutcomewiththestandardlongtreatment, while patients with high TILs not only had an excellent prognosis regardless of treatment duration,but also the outcome was numerically improved in case of treatment in the short arm.This observation suggests that TILs can help identify patients who may be optimally treated with de-escalated treatment.Although the benefit of standard de-escalation overtreatment in patients with low TILs,and therefore at worse prognosis,is more easily explainable,the reasons for observing an apparent improved survival for patients with high TILs receiving the shorter therapy areless straightforward.One could speculate that,in presence of high TILs at baseline, a full treatment, including higher doses of immunogenic drugs (anthracyclines and trastuzumab),might run therisk of exhausting theimmune response,with potential detrimental effects at long term. However, this remains a hypothesis tobe verified.

Figure2.KalanMeCuvefDistatDisasFeSurvivalDDiPatietsWithisLwr-Ifitratinh

Highvaluesarethoseatthecutofforhigherandlowvaluesarethosebelowthecutoffindicatehazardrati

Figure3.Kaplan-MeierCurvesforOverallSurvivalinPatientsWithHighvsLowTumor-lnitratingLymphocytes
Hialtlt hazard ratio.

Previous adjuvant studies have produced mixed results regardingthepredictiveroleof TILsfor trastuzumab benefit.10,11 The recent Early Breast Cancer Trialists’ Collaborative Group meta-analysis did not find any interaction between TILs and trastuzumab treatment,meaning that the benefit of adding adjuvant trastuzumab to chemotherapy was independent from the level of TILs.16 The present data require external validation;therefore,we cannot conclude that high TILs arepredictiveof a betterefficacy of treatment de-escalation.However,with a positiveinteraction test and given the demonstrated effect of TILs, it is highly unlikely that the outcome of patients in the high-level TIL group receiving de-escalation would have,inthe end,worse outcomes than those of patients with high TILs receiving standard treatment.For this reason, we conclude that patients with TILs 20% or higher receiving a reduced anthracycline dose and trastuzumab duration in this study were at least not exposed to an excess risk of distant relapse or death.Fitting with the topic of anthracycline de-escalation/omission, the TRAIN-2 trial showed no difference in pathological completeresponserateandinvasiveDFsbetweenneoadjuvant treatment with anthracyclines and taxane-containing chemotherapy vs anthracycline-free chemotherapy, combinedwith trastuzumab and pertuzumab.5Although there wasnointeractionbetweenTILs andtreatmentarm in terms of invasive DFS,it is worth mentioning that patients with TILs lower than 14% (which is close to the 220% cutoff used for interaction in this study) showed a numerically better outcome if treated with the anthracycline-free regimencompared totheanthracycline-containingregimen (invasive DFS rate at 3 years, 96.0% VS 92.6% .These data, althoughbased on a lownumber of patients and derived from a trial dedicated to a different setting and addressing a different clinical question than ShortHER,show points of convergence with our work.

Figure4.InteractionBetweenTumor-InfiltratingLymphocytes(TILs)and Treatment Arm

Kaplan-Meier curves for distant disease-free survival(DDFS) compare the 2 treatment arms for patients with TILs lower than 20% (A) and patients with TILS 20% or higher (B) (P forinteraction \mathbf{\Sigma}=\mathbf{\Sigma} .01). Kaplan-Meier curves for overall survival(OS) compare the 2 treatment arms for patients with TILs lower than
20% (C) and patients with TILs 20% or higher(D)(Pfor interaction \mathbf{\sigma}=\mathbf{\sigma} .06). Dotted lines indicate the 8- and 10-year survival time points. HR indicates hazard ratio.

The best approachforprognostic patient stratification involvestheintegration of multipleparameters.Currently,the clinical management of patients with ERBB2-positive early breast cancer relies on clinical-pathological staging,17 with limiteduseofbiomarkersforriskstratificationbesidesERBB2 status.18 De-escalation of adjuvant therapy is already a reality for most patients with stageIERBB2-positive breast cancer.In this study,the effect of TILs was predominantly observedfor patients with stage II disease,for whom the implications oftreatmentde-escalationaremoredebated. The present findings advocate for the inclusion of TILs in integrated prognostic tools in this setting,providing a reproducible,19 cost-effective, and readily worldwide available measure contributingtotheidentification of patients with an excellent prognosis whomay derive no harm from deescalationofadjuvanttreatment.

Strengthsand Limitations

Strengths andlimitations of thisstudyhavebeen acknowledgedpreviously.3Thestrengthofthepresentupdateliesin the long-term follow-up. Limitations include the exploratory nature of the analysis, which was not prespecified in the protocol.

Conclusions

ThisupdatedanalysisoftheShortHERrandomizedclinical trial is,to our knowledge,the first report of a strong independent association of TILs with OS for patients with ERBB2-positive earlybreast cancer.Validation is needed to confirm that patients with high TILs can safely undergo treatment de-escalation. These findings support the integration of immune-related features into prognostic tools for ERBB2-positive early breast cancer, moving toward personalizedtreatmentstrategiesbeyond clinical-pathological features.

Accepted forPublication:December10,2024. Published Online:February13,2025.
doi:10.1001/jamaoncol.2024.6872
Open Access:Thisis anopen access article
distributed underthe terms of theCC-BY License. ⊚ 2025 Dieci MVet al.JAMA Oncology.
AuthorAfflitions:DepartmentofSurgry
Oncology and Gastroenterology (DisCOG),
UniversityfPadovaPadva,talyDieciMglitt Massa,Conte,Guarneri);Oncology2,Venet
Instituteof ncologyIOV-IRCCS,Padova,Italy
(Dieci, Miglietta, Massa, Guarneri); Department of Oncology and Advanced Technologies,Azienda USL-IRCCS, Reggio Emilia, Italy (Bisagni);
Department of Nervous System Medical Oncology. IRCCS Istituto delle Scienze Neurologiche di
Bologna,Bologna, Italy (Bartolini);S.Anna
University Hospital, Ferrara, Italy (Schirone);
InternalMedicineandOncologyClinicaPiacenza, Piacenza, Italy(Cavanna);Medical Oncology,Breast and GYNUnit,IRCCSIstitutoRomagnolopelo
Studio dei Tumori(IRST)“Dino Amadori," Meldola, Italy (Musolino); Department of Medical and
Surgicalciencesniversityfolognaolna Italy (Musolino); IRCCS Istituto Tumori “Giovanni Paololl" Bari, Italy (Giotta); Carlo Poma Hospital, ASST Mantova,Mantova,Italy (Rimanti);Medical Oncology Unit, Fondazione IRCCS Ca' Granda
OspedleMaggioreoliclinicoMilano,taly
(Garrone);Medical Oncology.S.Anna Hospital
Torino, Italy (Bertone); Breast Unit Ausl Modena, Ramazzini Hospital, Carpi, Italy (Cagosi); IRCCS Istituto Romagnolo per lo Studio dei Tumori(IRST) “Dino Amadori,Meldola, Italy (Sarti);Rete Clinica Senologica-Oncologia Medica S.Chiara,APSS,
Trento,Italy (Ferro);Department of Medical and Surgical Sciences for Children and Adults,
University Hospital of Modena, Modena, Italy
(Piacentini, D'Amico);Azienda
OspedalirnivrsitariadiModenaodenaal (Piacentini); Pathology Unit, Ulss 5 Polesana,
Rovigo,Italy(Orvieto);Pathology.Microbiologyand Immunology.VanderbiltUniversityMedicalCenter, Nashville, Tennessee (Sanders); University of
Modena and Reggio Emilia, Modena, Italy (Balduzzi) Author Contributions:Prof Diecihad fullaccessto all of the data inthe study and takes responsibility fortheintegrityof thedata and the accuracyof the data analysis.
Conceptanddesign:Dieci,chirone,Cagossi,Conte D'Amico, Guarneri.
Acquisition,analysis,orinterpretationof data:Dieci BisagniBartoliniavannaMusolinit
RimantiGarrone,ertone,CagossiartiFrr PiacentinivitandersMiglittaas
Balduzzi, Conte,Guarneri.
Drafting of the manuscript:Dieci Cavanna,
MusolinrtnrvietoglttaMss
Conte,D'Amico,Guarneri.
Criticalreviewof themanuscript forimportant
intelectualcontent:DieciBsagniartolin
Schirone, Giotta,Rimanti, Garrone, Cagossi, Sarti, Ferro,Piacentinianders,Miliettaali

ARTICLEINFORMATION

Conte,Guarneri.
Statistical analysis: Dieci,Cagossi.
Obtained funding:Dieci, Conte.
Administrative,technical,ormaterialsupport: Giotta,Cagossi,Piacentini,Orvieto,Sanders, Miglietta,Conte.
Supervision:Dieci, Bisagni,Schirone, Garrone, Ferro,PiacentiniSanders,alduzzi,ont, D'Amico, Guarneri.

Conflictof Interest Disclosures:Prof Dieci reported personal fees from Eli Lilly, Novartis, Pfizer,,ildanahi AstraZeneca, MSD, and Exact Sciences; nonfinancial support from Daichi Sankyo, Roche, and Eli Lilly; and a patent for EP20382679.7 licensed to Universita di Padova.Prof Musolino reported grants from Roche and Lilly, as well as personal fees from AstraZeneca, Pfizer, Daiichi Sankyo, Novartis, MSK, and Gilead. Dr Garrone reported personal fees from MSD, Pfizer, Novartis, Lilly, Gilead, Daichi Sankyo, AstraZeneca,andIpsen.DrFerroreported personal fees from Pfizer, Novartis, Daichi Sankyo, Eli Lilly. and AstraZeneca.Dr Miglietta reported personal fees from Roche, Novartis,Seagen/Pfizer, AstraZeneca, Gilead, and Menarini, as well as grants from Gilead. Dr Massa reported nonfinancial support from Eli Lilly. Prof Conte reported a patent for HER2Dx with royalties paid. Prof Guarneri reported personal fees from Eli Lilly, AstraZeneca, Gilead,Menarini Stemline,Novartis,Exact Sciences, MSD,DaichiSankyentivalemaOncly Pfizer, and Pierre Fabre. No other disclosures were reported.

Funding/Support: This work was supported by Agenzia Italiana del Farmaco (grant FARM62MC97). the Italian Association for Cancer Research (project MFAG 2014-15938 to Prof Guarneri; 5 per mille 2019 [ID22759 program; group leader, Prof Guarneri]; and project IG27152 to Prof Dieci), funding from the University of Padova's Department of Surgery, Oncology and Gastroenterology DOR 2023 (to Profs Guarneri and Dieci and Dr Miglietta), and the Veneto Institute of Oncology (5x1000 program to Prof Dieci.

Role of the Funder/Sponsor:The funders had no role inthe design and conduct of the study; collection, management, analysis, and interpretation of the data;preparation,review,or approval of the manuscript; and decision to submit the manuscript for publication.

DataSharingStatement:SeeSupplement3.

REFERENCES

1. Conte P, Frassoldati A, Bisagni G, et al. Nine weeks versus 1 year adjuvant trastuzumab in combination with chemotherapy: final results of the phase Ill randomized Short-HER study. Ann Oncol. 2018;29(12):2328-2333. doi:10.1093/annonc/mdy414

2.Conte P,Bisagni G,Piacentini F,et al. Nine-week versus one-year trastuzumab forearly human epidermal growth factorreceptor 2-positivebreast cancer:10-year update of the ShortHER phase IlI randomized trial.J Clin Oncol. 2023;41(32):4976- 4981.doi:10.1200/JCO.23.00790

3.Dieci MV,Conte P, Bisagni G,et al.Association of tumor-infiltrating lymphocytes with distant disease-free survival in the ShortHER randomized adjuvant trial for patients with early HER2 ^+ breast cancer. Ann Oncol. 2019;30(3):418-423. doi:10. 1093/annonc/mdz007

4. Solinas C, Ceppi M, Lambertini M, et al. Tumor-infiltrating lymphocytes in patients with HER2-positive breast cancertreated with neoadjuvant chemotherapy plus trastuzumab, lapatinib or their combination:a meta-analysis of randomized controlled trials. Cancer Treat Rev. 2017;57:8-15. doi:10.1016/j.ctrv.2017.04.005

5.Liefaard MC,van der Voort A,van Seijen M, et al. Tumor-infiltrating lymphocytes in HER2-positive breast cancer treated with neoadjuvant chemotherapy and dual HER2-blockade.NPJBreast Cancer. 2024;10(1):29. doi:10.1038/s41523-024- 00636-4

6. Fernandez-Martinez A, Pascual T, Singh B, et al. Prognostic and predictive value of immune-related gene expressionsignatures vstumor-infiltrating lymphocytes in early-stage ERBB2/HER2-positive breast cancer: a correlative analysis of the CALGB 406O1 and PAMELA trials. JAMA Oncol. 2O23;9(4): 490-499.doi:10.1001/jamaoncol.2022.6288

7. Ignatiadis M, Van den Eynden G, Roberto S, et al. Tumor-infiltrating lymphocytes in patients receiving trastuzumab/pertuzumab-based chemotherapy:a TRYPHAENA substudy.J Natl Cancer Inst. 2019;111(1):69-77. doi:10.1093/jnci/djy076

8.Salgado R,Denkert C,CampbellC,et al. Tumor-infiltrating lymphocytes and associations with pathological complete response and event-free survival in HER2-positive early-stage breast cancer treated with lapatinib and trastuzumab:a secondary analysis of the NeoALTTO trial. JAMA Oncol. 2015;1(4):448-454. doi:10.1001/jamaoncol.2015.0830

9.Denkert C, Loibl S, Noske A, et al. Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer.J Clin Oncol. 2010; 28(1):105-113. doi:10.1200/JCO.2009.23.7370

10. LoiS,Michiels S,Salgado R,et al. Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial. Ann Oncol. 2014;25(8):1544- 1550.doi:10.1093/annonc/mdu112

11.Perez EA,Ballman KV,Tenner KS,et al. Association of stromal tumor-infiltrating lymphocytes with recurrence-free survival in the N9831adjuvant trialin patients with early-stage HER2-positive breast cancer. JAMA Oncol. 2O16;2 (1):56-64.doi:10.1001/jamaoncol.2015.3239

12. Kim RS, Song N, Gavin PG, et al. Stromal tumor-infiltrating lymphocytes in NRG Oncology/NSABPB-31adjuvant trial for early-stage HER2-positive breast cancer. J Natl Cancer Inst. 2019;111(8):867-871. doi:10.1093/jnci/djz032

13. Gourgou-Bourgade S, Cameron D, Poortmans P. et al. Guidelines for time-to-event end point definitionsinbreastcancertrials:resultsofthe DATECAN initiative (Definition for the Assessment of Time-to-event Endpoints in CANcer trials). Ann Oncol. 2015;26(5):873-879. doi:10.1093/annonc/ mdv106

Tumor-lnfiltrating Lymphocytes and Survival Outcomes in Early ERBB2-Positive Breast Cancer

Original Investigation Research

14. Dieci MV,Miglietta F,Guarneri.Immune infiltrates in breast cancer: recent updates and clinical implications. Cells. 2021;10(2):27. doi:10. 3390/cells10020223 15. EI Bairi K, Haynes HR, Blackley E, et al; International Immuno-Oncology Biomarker Working Group. The tale of TILs in breast cancer: areport from the International Immuno-Oncology Biomarker Working Group.NPJ Breast Cancer. 2021; 7(1):150. doi:10.1038/s41523-021-00346-1 16. Hills RK, Bradley R, Braybrooke J, et al. Do tumor infiltrating lymphocytes (TILs) predict

benefits from trastuzumab therapy for HER2 positive breast cancer? meta-analysis of individual patient data from 4097 women in 5 trials. J Clin Oncol. 2023;41(16)(suppl):508. doi:10.1200/JCO. 2023.41.16_suppl.508

17.Loibl S,Andre F,Bachelot T,et al.Early breast cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up. Ann Oncol. 2024;35(2):159-182. doi:10.1016/j.annonc.2023.11. 016

18. Andre F, Ismaila N, Allison KH, et al. Biomarkers for adjuvant endocrine and chemotherapy in

early-stagebreastcancer:AScOguideline update. J Clin Oncol. 2022;40(16):1816-1837. doi:10.1200/ JCO.22.00069
19. Kos Z, Roblin E, Kim RS, et al; International Immuno-OncologyBiomarker Working Group. Pitfalls in assessing stromal tumor infiltrating lymphocytes (sTILs) in breast cancer. NPJ Breast Cancer. 2020;6(1):17. doi:10.1038/s41523-020- 0156-0