LBA538 Background: This phase III trial is a prospective, randomized, double-blinded, multi-center study (NCT05232916) in HLA-A*02 patients at approximately 140 sites in the US and Europe. A third non-randomized arm of approximately 250 non-HLA-A*02 patients is now fully enrolled and preliminary immune response data is presented below. GP2 is a biologic nine amino acid peptide of the HER2/ neu protein delivered in combination with Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) that stimulates an immune response targeting HER2/neu expressing cancers, the combination known as GLSI-100. Methods: After standard of care neoadjuvant and adjuvant therapy, 6 intradermal injections of GLSI-100 will be administered over the first 6 months and 5 subsequent boosters will be administered over the next 2.5 years. The participant duration of the trial will be 3 years treatment plus 1 additional year follow-up. Immune responses to GP2 were measured over time using delayed-type-hypersensitivity (DTH) skin tests and injection site reactions (ISRs). The patient population is defined by these key eligibility criteria: 1) HER2/neu positive and HLA, 2) Residual disease or High risk pCR (Stage III at presentation) post neo-adjuvant therapy, 3) Exclude Stage IV, and 4) Completed at least 90% of planned adjuvant trastuzumab-based therapy. Results: All patients (n=247) were vaccinated with GLSI-100. Injection site reactions and erythema (redness) were assessed at various time points and represent an in vivo immune response in patients. The ISR orthogonal mean was measured 48-72 hours following vaccination with GLSI-100. For GP2 treated patients, there was a significant increase in the percentage of patients experiencing ISRs in the 4th, 5th or 6th vaccination compared to the ISRs from the 1st vaccination. In this preliminary analysis, the frequency of ISRs increased significantly from 20.2% of the patients experiencing an ISR after the first vaccination to 55.3% of the patients experiencing an ISR after the 4th, 5th or 6th vaccination (McNemar p < 0.001). The study is ongoing and data collection and cleaning continue, so final results may vary. Conclusions: Preliminary injection site reaction data comparing vaccination over time in GLSI-100 treated non-HLA-A*02 patients showed a significant increase in immune response. Future studies may explore the use of immune responses to assess correlation of DTH to ISRs, immunogenicity of GLSI-100 by specific HLA type, timing of boosters to sustain immunity, clinical site performance, and the discontinuation of treatment for non-responders. Funding: This trial is supported by Greenwich LifeSciences. Clinical trial information: NCT05232916 .

Purpose ESR1 mutations are commonly acquired on endocrine therapy (ET) and contribute to therapeutic resistance. The impact of increasing degrees of polyclonality - including dynamics of ESR1-mutant clones, co-occurring mutations, and treatment outcomes - is not well understood. Methods We conducted a retrospective, descriptive review of patients with estrogen receptor-positive (ER+) metastatic breast cancer (MBC) and >10 concurrent ESR1 variants on circulating tumor DNA (ctDNA) using either Guardant360 or Tempus xF between August, 2020 and July, 2025, defined as extreme ESR1 polyclonality. Patient and tumor characteristics, frequency of ESR1 variants, co-occurring mutations, and clonal changes in ESR1 in response to various treatments were explored. Results Nine patients with ER+ MBC and extreme ESR1 polyclonality were identified; 5 were post-menopausal at diagnosis. Five had received adjuvant chemotherapy and 6 received adjuvant ET for a minimum of 3 years (range, 3-7). Patients received a median of 1 line of ET in the metastatic setting before their first ctDNA test, which showed 0-31 ESR1 clones on initial ctDNA (mean, 6). The most frequent ESR1 clones were Y537N, Y537S, and D538G. The most frequent co-alterations included TP53 and RB1 mutations and CCND1 amplification. Tumor mutational burden (TMB) at the time extreme ESR1 polyclonality emerged ranged from 6.7-56.5 mutations per megabase (mut/Mb); all assessable tumors were microsatellite stable (MSS). Capecitabine and olaparib were associated with a decline in ESR1 polyclonality on ctDNA after radiographic progression. Other cytotoxic agents did not have the same observed effect. Increased ESR1 polyclonality was observed after progression on the oral selective estrogen receptor degrader (SERDs) elacestrant and imlunestrant. Among the 4 patients treated with elacestrant, the median progression-free survival was 6 months (range, 4-6). At data cutoff, 3 patients had died from progressive disease, 1 had transferred care, 1 was in hospice, and 4 remained on active treatment. Conclusions This is the first case series to explore extreme ESR1 polyclonality. Characterizing the mutational dynamics associated with this phenomenon may improve our general understanding of ESR1 mutations and related impact on treatment resistance.

e13103 Background: CDK4/6 inhibitors (CDK4/6is) are a mainstay of treatment for hormone receptor–positive breast cancer but can cause adverse events that require prompt recognition and management to maintain adherence by patients. We developed an online Interactive Decision Support Tool (www.clinicaloptions.com/CDK46AEtool) for healthcare professionals (HCPs) with case-specific recommendations for managing adverse events associated with CDK4/6i therapy. Methods: To use the online tool, HCPs entered the type of adverse event, symptom grade/severity, and their planned management strategy. The tool showed the management recommendation for that adverse event based on the prescription information and expert guidance and then asked if the recommendation changed HCPs’ intended management approach. Results: From April 2025 to January 2026, 80 HCPs entered 90 case scenarios into the tool; 76% treated > 10 patients with breast cancer/month, and 36% of HCPs practiced in the United States or Europe. Among the 90 cases, 41% were scenarios associated with ribociclib use, 40% with abemaciclib use, and 17% with palbociclib use. The most common adverse events entered regardless of specific CDK4/6i were cytopenias (39%), gastrointestinal toxicity (22%), and hepatotoxicity (18%). The predominant adverse events entered by HCPs varied with the specific CDK4/6i: abemaciclib, gastrointestinal toxicity (47%) and cytopenias (33%); palbociclib, cytopenias (71%); and ribociclib, hepatobiliary toxicity (35%) and cytopenias (30%). HCPs self-reported uncertainty or a difference in adverse event management strategy compared with recommendations based on the prescribing information and expert guidance for grade 1-3 ANC decrease/neutropenia (38%, n = 39); grade 2 diarrhea (38%, n = 13); grade 3 hepatotoxicity (67%, n = 12), and QTcF > 480 ms to ≤500 ms (83%, n = 6). Conclusions: These data suggest that some HCPs are challenged to optimally manage select adverse events associated with CDK4/6is and may not be managing their patients in concordance with consensus recommendations based on prescribing information and expert guidance. A detailed analysis of HCP CDK4/6i adverse event management vs recommendations, including by type of adverse event and severity, will be presented.

1011 Background: Approximately one-quarter of patients with node-positive, triple-negative breast cancer (TNBC) did not experience a complete nodal response to neoadjuvant chemo-immunotherapy in KEYNOTE-522. We conducted a randomized trial to evaluate whether no-, low-, or high-dose preoperative radiotherapy (RT) to the breast primary tumor combined with pembrolizumab (pembro) enhances tumor T-cell infiltration (TCI) and pathologic response in non-irradiated lymph node (LN) metastases (NCT04443348). Methods: Between 2021-2025, 55 patients with cT1c-T4c, cN1-3, cM0, ER/PR < 10%, HER2-negative breast cancer and biopsy-proven, clipped axillary LN metastasis were randomized to no RT (0 Gy), low-dose RT (9 Gy) or high-dose RT (24 Gy) to the primary tumor with concurrent pembro, followed by an on-treatment tumor biopsy at 2 wks. Stratification factors included cT stage (T1c vs. T2-4) and cN stage (N1 vs. N2-3). Patients subsequently received pembro (200 mg q3w or 400 mg q6w) with 12 wks of paclitaxel/carboplatin, followed by four cycles of doxorubicin-cyclophosphamide (q2w or q3w) with pembro, surgery and adjuvant therapy. Primary endpoints were 2-wk tumor TCI and nodal pathologic complete response (ypN0) at surgery. TCI was assessed by multiplexed immunofluorescence (panCK/CD3/CD8) using a rank-based Immunoscore. 2-wk TCI for each treatment arm was compared to a common reference cohort of pretreatment TCI measurements. The study was powered to detect an increase in upper-quartile TCI from 25% to 55%. Statistical comparisons used two-tailed Fisher’s exact tests. ypN0 and secondary endpoints pCR (ypT0/TisN0) and Residual Cancer Burden (RCB) 0/1 were underpowered for a statistical test. Results: Among 55 enrolled patients, 48 were evaluable for TCI and 51 for ypN0. Median age was 50 years (range 28-77). 81.8% had cT2-4 disease, 20% had cN2-3, and 87.3% had grade 3 tumors. The proportion of tumors with upper-quartile TCI was significantly increased with RT plus pembro, but not pembro alone, when compared to the pretreatment cohort: 44% (0Gy; p = NS), 80% (9Gy, p < 0.0001), and 82% (24Gy; p < 0.0001). ypN0 rates were 73.3% (0Gy), 88.2% (9Gy) and 78.9% (24Gy). pCR/RCB 0-1 rates were 66.6%/66.6% (0Gy), 76.5%/82.4% (9Gy) and 68.4%/84.2% (24Gy). Tumors with upper-quartile TCI at the 2-wk timepoint had a significantly higher ypN0 rate relative to tumors without upper-quartile TCI (94% [31/33] vs. 46% [6/13]; p = 0.0009). Conclusions: The addition of preoperative RT to pembro significantly increased 2-wk TCI and yielded high rates of ypN0 and pCR after chemo-immunotherapy, with the numerically highest ypN0 rate observed in the 9Gy arm. 2-wk TCI correlated strongly with surgical ypN0 status. A larger study that tests whether preoperative RT and pembro increases pCR should be conducted in node-positive TNBC. Clinical trial information: NCT04443348 .

e23373 Background: Multiple biomarker-driven therapies are approved for ER+/HER2- mBC. Oral selective estrogen degraders (SERDs) are approved for ESR1 mut ER+/HER2- mBC, but ESR1 mut may co-occur with other actionable biomarkers, including somatic PIK3CA/AKT1 / PTEN mut , germline BRCA1/2 mut , and HER2-low disease. Optimal timing of matched therapies remains uncertain in patients (pts) with multiple targetable mutations. This study evaluated institutional prescribing patterns, focusing on the first biomarker-matched therapy selected following elacestrant approval, and associated clinical characteristics in pts with ER+/HER2- ESR1 mut mBC with other actionable variants. Methods: Pts with ESR1 mut mBC were included if they had ≥1 additional targetable biomarker and received a matched agent after elacestrant approval (Jan 27, 2023). Genomic and pathology data were assessed for PIK3CA/AKT1/PTEN mut , HER2-low status (IHC 1+ or 2+/FISH negative), and g BRCA1/2 mut . Variants were considered actionable if a matched therapy was available as standard of care or through a clinical trial at treatment selection. Clinical factors annotated included age at therapy start, ECOG, hemoglobin (HgB), HgB A1c, creatinine (Cr), bilirubin, and visceral disease. Comparisons were performed across treatment groups defined by the first matched therapy received (oral SERD vs PI3K/AKTi vs Trastuzumab deruxtecan [T-Dxd]) using Kruskal–Wallis and Chi Square tests. Results: 87 pts with ESR1 mut mBC had ≥1 additional targetable variant at treatment selection (Table 1). Oral SERDs were prescribed first for most pts with concurrent PIK3CA mut , and evenly with T-Dxd for pts with HER2-low mBC. If ≥2 PI3K/AKT pathway mutations were present, a PI3K/AKTi was selected. Median age differed significantly, with older pts more likely to receive oral SERDs (SERD 73, PI3K/AKTi 64, T-Dxd 62 years; p = 0.001). Cr levels differed with lower values in T-Dxd and PI3K/AKTi groups (p = 0.034). In a subanalysis, excluding pts who received a matched agent pre-elacestrant approval (n = 55), median age was higher in oral SERD recipients (75 vs 66 vs 59 years; p < 0.001). All groups had median HgB A1c < 6.5 and no significant difference in Cr. Conclusions: Guidelines for initial biomarker-driven treatment selection in ER+/HER2- ESR1 mut mBC are not well defined. This work shows variability in real-world treatment practices and potential associations with toxicity-related factors. Larger analyses are planned to validate these findings and to evaluate treatment sequencing patterns in this setting. Mutation Profile SERD first (n = 43) PI3K/AKTi first (n = 21) T-Dxd first (n = 23) ESR1 + PIK3CA (n = 28) 20 (71%) 8 (29%) — ESR1 + AKT1 (n = 1) 0 1 (100%) — ESR1 + PTEN (n = 1) 0 1 (100%) — ESR1 + ≥2 PIK3CA/AKT1/PTEN (n = 5) 0 5 (100%) — ESR1 + HER2-low (n = 28) 14 (50%) — 14 (50%) ESR1 + HER2-low + ≥1 PIK3CA/AKT1/PTEN (n = 23) 8 (35%) 6 (26%) 9 (39%) ESR1 + HER2-low + BRCA1/2 + ≥1 PIK3CA/AKT1/PTEN (n = 1) 1 (100%) 0 0

623 Background: In early-stage HER2–positive breast cancer, achieving a pathological complete response (pCR) after neoadjuvant therapy is strongly associated with improved event-free survival (EFS); however, a substantial proportion patients (pts) fail to achieve pCR, requiring subsequent adjuvant systemic therapy and remaining at higher risk for recurrence. Pembrolizumab (pembro) is well-tolerated, invigorates anti-tumor immunity, and may enhance response of chemotherapy and/or anti-HER2. We previously reported the positive primary outcome of the randomized phase 2 neoHIP trial, showing an improvement in pCR with the addition of pembro to neoadjuvant paclitaxel, trastuzumab, and pertuzumab (4 cycles, THP–pembro, pCR: 67% v. THP: 48%, p=.026). With long-term follow-up now mature, we now report EFS outcomes. Methods: Pts aged ≥ 18 years with stage II–III HER2–positive breast cancer were randomized, stratified by hormone receptor status and nodal status, to receive neoadjuvant THP or THP–pembro. EFS was defined as the time from randomization to progression of disease precluding surgery, local or distant recurrence, or death from any cause. EFS was estimated using the Kaplan–Meier (KM) method and compared between arms using a two-sided stratified log-rank test. Data were analyzed with a cutoff date of January 23, 2026. Results: Among 116 pts (58 per arm) randomized to THP +/- pembro, median follow-up was 37 months (range, 33–43). The 36-month EFS rate was 95% (95% CI, 89–100) for THP and 100% (95% CI, 100–100) for THP-pembro, with a trend towards significance (p=.099). Three EFS events were observed, all occurring in the THP arm, with one death related to recurrence and n=2/3 events occurring in patients with non-pCR. Conclusions: THP + pembro was associated with 100% EFS after a median follow-up of 37 months, whereas recurrences were observed in the THP arm, consistent with historical EFS expectations for neoadjuvant chemotherapy + dual HER2-targeted therapy. Although not powered for EFS, these findings support definitive evaluation of THP +/- pembro in a phase III trial. Clinical trial information: NCT03747120 .

511 Background: Sacituzumab govitecan (SG) is a TROP-2 directed antibody-drug conjugate (ADC) approved for metastatic triple negative breast cancer (TNBC). Pembrolizumab (P), an anti-programmed death 1 monoclonal antibody, is approved for early-stage TNBC and metastatic PD-L1 positive TNBC. However, safety and efficacy of SG+P in early TNBC is not known. We published results of Arm A1 investigating neoadjuvant SG monotherapy in early TNBC (Spring et al. Annals of Onc 2024). Here we present results from Arm A2 of the NeoSTAR study investigating the combination of neoadjuvant SG + P in early-stage TNBC (NCT04230109). Methods: Patients (pts) with early TNBC (tumor size ≥2 cm or node positive) with no prior treatment were eligible. Pts received SG at starting dose of 10mg/kg on days 1,8 of a 21-day cycle for 4 cycles with P 200 mg given on day 1 of each cycle. After trial regimen, pts underwent imaging to determine residual radiographic disease per RECIST v1.1. A biopsy was performed if residual disease (RD) was suspected. Additional neoadjuvant chemotherapy (ANACT) was at discretion of the treating physician prior to definitive surgery. The primary objective was rate of pathologic complete response (pCR) with neoadjuvant SG/P. Secondary objectives included need for ANACT, radiographic response (RR), safety and tolerability (adverse events [AEs] per CTCAE v5.0) and event-free survival. A Simon two-stage design and standard descriptive statistics were utilized, including 95% binomial confidence intervals for all rates estimated. Results: From 5/19/23-8/13/24, 50 pts were enrolled (median age: 57 years, range 23-77). Clinical anatomic stage was II in 48 pts (96%) and III in 2 pts (4%). 64% of pts were node negative at diagnosis. 44 pts (88%) completed the trial regimen (5 pts had toxicity, 1 pt progressed on treatment). In interim analysis, 5/15 had pCR and so the remaining 35 were enrolled. The pCR rate per protocol (pts with pCR at surgery directly after SG/P without ANCT) was 16/50 (34%, 95% CI 19.5-46.7). The RR rate (complete CR or partial response PR) was 66% (95% CI 50-78%), 30% CR and 36% PR. Of 26 pts who received ANACT, 9 experienced pCR (2 biopsy-confirmed RD, 6 negative or non-diagnostic RD biopsy, 1 no biopsy). Overall, 25 (50%, 95% CI 35.5-64.5) pts had pCR at surgery. Of 5 pts with pathogenic BRCA mutations, 3 (60%) had pCR after SG/P, and 1 pt had pCR after ANACT. 20 pts (40%) had grade 3 or higher AEs. The most common AEs were nausea (28, 56%), alopecia (26, 52%), fatigue (23, 46%), and diarrhea (22, 44%). Dose reductions of SG occurred in 4 pts (8%). Updated survival and biomarker data will be presented at the meeting. Conclusions: In the first trial to investigate the SG/P combination in early TNBC, 34% of pts had pCR. Additional research is needed to determine the optimal duration and sequence of neoadjuvant SG/P and chemotherapy for pts with TNBC. Clinical trial information: NCT04230109 .

Immune exclusion inhibits anti-tumor immunity and response to immunotherapy, but its mechanisms remain poorly defined. Here, we demonstrate that Trophoblast Cell-Surface Antigen 2 (TROP2), a key target of emerging anti-cancer Antibody Drug Conjugates (ADCs), controls barrier-mediated immune exclusion in Triple-Negative Breast Cancer (TNBC) through Claudin 7 association and tight junction regulation. TROP2 expression is inversely correlated with T cell infiltration and strongly associated with outcomes in TNBC. Loss-of-function and reconstitution experiments demonstrate TROP2 is sufficient to drive tumor progression in vivo in a CD8 T cell-dependent manner, while its loss deregulates expression and localization of multiple tight junction proteins, enabling T cell infiltration. Employing a humanized TROP2 syngeneic TNBC model, we show that TROP2 targeting via Sacituzumab govitecan (SG) induces an anti-PD1 response associated with broad immune cell activation, and TROP2 is highly associated with lack of response to anti-PD1 therapy in human breast cancer. Thus, TROP2 controls an immune exclusion program that can be targeted to enhance immunotherapy response. This work was supported by DOD/CDMRP Grant BC200924 and by R01CA260890 (to L.W. Ellisen, A. Bardia), by the Tracey Davis Memorial Breast Cancer Research Fund (to L.W. Ellisen), and by NIH/NCI K99CA286969 and The Terri Brodeur Breast Cancer Foundation Fellowship (to B. Wu). Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Background: Treatment of HR+/HER2- MBC often involves an antiestrogen agent and CDK4/6i, and following disease progression, multiple therapies are approved in the second line. Management is increasingly guided by a precision-based approach, including the use of AKTi in tumors harboring an AKT1 or PIK3CA mutation or PTEN loss. However, little is known regarding molecular factors that mediate resistance to AKTi. Results from the TAKTIC trial demonstrated antitumor activity and tolerability of the AKTi ipatasertib with endocrine therapy (ET) +/- palbociclib post-CDK4/6i (Wander et al., 2023). We hypothesize that next-generation sequencing (NGS) of tumors among patients (pts) receiving ipatasertib could inform genomic predictors of response to AKTi. Methods: TAKTIC was a phase Ib open-label trial evaluating ipatasertib in combination with fulvestrant, an aromatase inhibitor, or fulvestrant + palbociclib, in participants with HR+/HER2- MBC who received ≥1 line of prior therapy for MBC and had exposure to CDK4/6i (NCT03959891). An exploratory objective of TAKTIC was to identify genomic biomarkers that correlate with response to an AKTi-based combination regimen. Blood samples for circulating tumor DNA analysis were drawn at routine timepoints and archival tumor tissue was obtained. Mutational profiling was performed using commercially available NGS-based assays (frequently via Guardant360). Progression free survival (PFS) was estimated using the Kaplan-Meier method, and survival analysis was implemented with the Breslow approximation for ties. Univariable and multivariable hazard ratio (HR) and 95% confidence interval (CI) analyses were estimated using a cox proportional hazards model. Results: TAKTIC accrued 77 pts (6/2019 – 2/2022), enrolling 35 on doublet therapy (AKTi + antiestrogen) and 42 on triplet therapy (AKTi, fulvestrant, and palbociclib). Baseline NGS results were available in 58 of 77 pts, and alterations in PI3K/AKT pathway genes were found in PIK3CA (43%), AKT1 (7%), and PTEN (10%). Mutations in ESR1 (29%) were seen at rates consistent with prior studies in MBC post-ET. A subgroup of 20 pts who received the triplet ipatasertib regimen and had baseline NGS data within 60 days of drug start were analyzed. Mutations in PIK3CA (n=7, 35%; n=5 polyclonal) and PTEN (n=4, 20%; n=1 polyclonal) were detected, as were alterations in ESR1 (n=5, 25%), FGFR1 (n=4, 20%), KRAS (n=4, 20%), and ERBB2 (n=3, 15%); no baseline AKT1 mutations were seen in this subgroup. Univariate gene analysis demonstrated that FGFR1 amplification was associated with shorter PFS (HR 5.42, 95% CI 1.3 – 22.1, P=.019), and ERBB2 alteration trended toward worse outcomes (HR 3.38, 95% CI 0.8 – 13.6, P=.086); no significant difference was seen between ESR1 mutant vs. ESR1 wild-type tumors (HR 1.6, 95% CI 0.6 – 4.6, P=.384). Multivariate gene analysis demonstrated that PIK3CA/AKT1/PTEN altered tumors (n=9) had greater response to the AKTi triplet combination compared to tumors without mutations in this pathway (n=11) (median PFS 505 vs. 114 days, HR 0.2, 95% CI 0.1 – 0.7, P=.015). Breast cancers harboring alterations that upregulate RAS pathway signaling (KRAS/NRAS, BRAF, FGFR1/2, ERBB2, EGFR; n=9) showed a trend toward inferior outcomes compared to non-altered disease (n=11) (median PFS 114 vs. 253 days, HR 2.0, 95% CI 0.8 – 5.3, P=.160). Additional analyses at the individual gene and pathway level will be presented at the meeting. Conclusions: Genomic insights using NGS suggest that MBC post-CDK4/6i is more susceptible to an AKTi-based treatment with ipatasertib in the presence of a PI3K/AKT/PTEN pathway mutation, whereas alterations in FGFR1 are associated with worse outcomes. This effort is one of very few studies prospectively evaluating mediators of AKTi response, an area of active interest given changes in the therapeutic landscape. The results presented here are hypothesis-generating; future work is underway to further expand upon these data. Citation Format: Maxwell Lloyd, Geoffrey G. Fell, Elizabeth Scott, Jennifer C. Keenan, Laura M. Spring, Jennifer Shin, Steven J. Isakoff, Lianne Ryan, Sarah Padden, Elizabeth Fisher, Amber Newton, Beverly Moy, Andreas Varkaris, Leif W. Ellisen, Douglas S. Micalizzi, Daniel Haber, Dejan Juric, Aditya Bardia, Seth A. Wander. Genomic predictors of response among patients with hormone receptor-positive (HR+)/HER2- metastatic breast cancer (MBC) receiving the AKT inhibitor (AKTi) ipatasertib combined w/ endocrine therapy & a CDK4/6 inhibitor (CDK4/6i) in TAKTIC trial [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-01-17.

1005 Background: Trastuzumab deruxtecan (T-DXd) is FDA-approved for HER2-low, but not HER2-0 metastatic triple negative (TNBC) and hormone positive breast cancer. Therefore, identifying HER2-low status is of great clinical importance. Prior studies have shown HER2-low status in TNBC is dynamic, but the correlation between the number of successive biopsies (Bxs) conducted and the likelihood of a HER2-low result is unknown. Methods: Patients (pts) were identified from an institutional database including all pts with TNBC treated in a single large academic center between 2017-2022. Only pts with TNBC at diagnosis were included. Bxs without known HER2 status were excluded. Pathological, clinical, and demographic data were extracted. HER2-low was defined as HER2 IHC 1+, or 2+ with non-amplified ISH. The type of Bx was categorized as core Bx, surgical Bx, or metastatic Bx based on the timing and method of Bx acquisition. For the early-metastatic matched analysis, the core Bx was considered the early Bx, unless the core Bx was missing and then the surgical bx was used instead. For cases with several metastatic Bxs the first metastatic Bx was used. Results: 529 consecutive pts with TNBC at diagnosis were included. The proportion of pts with HER2-low result increased as the number of successive Bxs increased (60%, 74%, 83%, 87% and 100% when 1 (192 pts), 2 (235 pts), 3 (52 pts), 4 (38 pts), and 5-9 (12 pts) Bxs were conducted, respectively). In women without a prior HER2-low result, about one third converted to HER2-low with each successive additional biopsy (e.g. 322/529 at 1 st biopsy, 44/131 on 2 nd biopsy, 8/25 at 3 rd biopsy, 3/8 at 4 th biopsy). HER2 status distribution did not significantly vary between the different types of Bx (58%, 63%, and 54% of pts had a HER2-low result in their core, surgical or metastatic Bx, respectively; p=0.2). Among 246 women with matched core-surgical biopsies, one quarter changed their HER2 status (55% from low to 0, 44% from 0 to low, and 1% from low to 3+). Core-surgical HER2 status conversion rates did not differ between women who had neoadjuvant therapy with residual disease and women who had surgery as their primary intervention. Among women with both matched early-metastatic (70 pts) or two matched metastatic Bxs (39 pts), nearly half (44%) converted their HER2 status (68%, 26% and 6% or 35%, 59% and 6% were converted from low to 0, 0 to low and low to 3+ in the matched early-metastatic or the two matched metastatic Bxs, respectively). Conclusions: Our findings show that HER2 status is dynamic in pts with TNBC and support the idea that HER2-low is a spectrum, not a specific entity. We further report the novel finding that for pts with TNBC without a prior HER2-low result, repeat Bxs at progression can increase the chance of obtaining a HER2-low result and provide clinically impactful information. Whether the dynamic HER2 result represents underlying biology or analytic variation remains to be determined.

GATA3 mutations are among the most common alterations in hormone receptor-positive (HR+) breast cancer (BC), yet these have no targeted therapies. MDM2 is an E3 ubiquitin ligase that targets p53 for degradation, and pre-clinical data suggests MDM2 inhibition may effectively treat GATA3mut HR+ BC. The GATA3 co-mutational landscape has been described only in primary BC tissue, and the mechanism of MDM2-driven efficacy is incompletely understood. Circulating tumor DNA (ctDNA) was assessed for GATA3 mutations via targeted sequencing. Associations with co-alterations and clinical/pathologic factors were estimated using Pearson's chi-squared test, two-sample Wilcoxon rank-sum, and multivariable logistic regression. Impact on survival was analyzed using multivariable Cox regression analysis. Tissue-based data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database was evaluated for expression and phosphorylation of GATA3 and associated proteins. Among 609 patients with HR + /HER2− MBC, ctDNA detected non-synonymous GATA3 variants ctDNA in 69 (11

PURPOSE:Antibody-drug conjugates (ADC) harboring topoisomerase I (TOP1) inhibitor payloads have improved survival for patients with metastatic breast cancer. However, knowledge of ADC resistance mechanisms and potential impact on the sequential use of ADCs is limited. In this study, we report the incidence and characterization of TOP1 mutations arising in the setting of ADC resistance in metastatic breast cancer. EXPERIMENTAL DESIGN:Patients with metastatic breast cancer treated with ADCs with available posttreatment plasma-based genotyping were included. TOP1 mutation incidence, mutant allele frequency, and functional characterization were assessed, and incidence was compared with that in patients with metastatic breast cancer not receiving ADC treatment and in The Cancer Genome Atlas. RESULTS:Plasma-based genotyping identified distinct TOP1 mutations (S57C, R364H, W401C, and G359E) in 12.9% of patients (4/31) at the time of disease progression on ADC, compared with 0.7% (3/420) in non-ADC-treated patients with metastatic breast cancer and 0.5% in The Cancer Genome Atlas. The appearance of mutations was associated with clinical cross-resistance, as median duration on the first ADC was 455 versus 52 days for the second ADC. The functional characterization of three novel TOP1-mutant proteins demonstrated that all exhibited reduced enzymatic activity, attenuated covalent DNA binding, and resistance to TOP1 inhibitor ADC payloads SN38 and deruxtecan. CONCLUSIONS:We describe the recurrent emergence of functionally altered, resistance-associated TOP1 mutations in vivo under selective pressure from ADCs and the potential impact on mediating cross-resistance to sequential ADCs. TOP1 mutation may represent a biomarker of resistance in this setting, and additional work is needed to optimize biomarkers and ADC payload design to improve outcomes for the sequential use of ADCs. See related commentary by Gwin and Hurvitz, p. 1824.

Detection of circulating tumor DNA (ctDNA) in early breast cancer (BC) is a prognostic biomarker for disease recurrence. This pilot study evaluates the performance of a tissue-free epigenomic ctDNA assay in pre-neoadjuvant therapy (NAT), on-NAT and post-op plasma samples from patients (pts) with BC. Pre-NAT, on-NAT and post-op plasma samples from 13 pts that received NAT for BC were retrospectively analyzed for ctDNA using Guardant Reveal powered by Infinity, a tissue-free assay that analyzes differentially methylated regions of DNA using an applied bioinformatics filter optimized to detect BC. It results in binary ctDNA detection calls and a quantitative estimate of tumor fraction (TF) in ctDNA+ samples. A total of 27 samples from 14 pts were evaluated, with 96% (26/27) samples passing quality control and included in the analysis (10 pre-NAT, 3 on-NAT, and 13 post-op). ctDNA was detected in 42% (11/26) of samples: 70% (7/10) pre-NAT, 33% (1/3) on-NAT and 23% (3/13) post-op (Table). For post-op samples collected within one year of recurrence, sensitivity was 100% (3/3). 2/5 pts who experienced recurrence, whose post-op samples were collected during adjuvant therapy and were ctDNA-, had a recurrence >15 months after the sample collection. No pts (0%, 0/8) without recurrence were ctDNA+ post-op. A pt with a brain-only recurrence had the lowest TF fraction amongst ctDNA+ post-op samples at 0.9%, with a median post-op TF of 2.5% (range, 0.9-23%) among the 3 ctDNA+ samples. ctDNA was detectable in 70% of pts pre-NAT, 33% on-NAT and 23% post-op, with persistent ctDNA associated with recurrence. In the subsequent full study, ∼ 600 samples will be evaluated at these timepoints. The dataset will provide a rich clinical landscape to understand sensitivity and specificity of methylation-based ctDNA testing, optimal timing and key areas to study therapeutic interception. Arielle J. Medford, Justine Knape, Derek Dustin, Haiyang Zhang, Caroline Weipert, Shivahamy Maheswaran, Irene Kuter, Jeffrey Peppercorn, Seth Wander, Steven J. Isakoff, Beverly Moy, Dejan Juric, Laura M. Spring, Aditya Bardia. Pre- and post-operative analysis of circulating tumor DNA in patients with breast cancer treated with neoadjuvant therapy using a tissue-free, methylation-based approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3360.

Drug resistance is the leading cause of neoadjuvant chemotherapy (NAC) failure to achieve pathological complete response (pCR) in more than 40% of patients with triple negative breast cancer (TNBC). Thus, understanding the potential drug resistance mechanisms in TNBC patients is a critical unmet clinical need. Using quantitative high-throughput metabolomic and proteomic approaches, we systematically analyzed clinicopathological, metabolic, and proteomic data profiled from NAC treatment-naive biopsy samples derived from primary TNBCs. Particularly, we identified that non-pCR TNBC primary tumors are uniquely and highly enriched in metabolic pathways for the TCA cycle and oxidative phosphorylation (OXPHOS) along with nucleotide metabolism relative to pCR TNBC tumors. In addition, sirtuin 5 (SIRT5) as a master regulator of mitochondrial metabolism is dramatically increased in non-pCR TNBC primary tumors. Of note, we found that SIRT5 is frequently overexpressed in TNBC tumors due to copy number gain and amplification. Furthermore, metabolomics and 13C-glucose tracing uncovered that SIRT5 gain of function promotes chemoresistance by diverting TCA cycle intermediates into the pentose phosphate pathway (PPP) for nucleotide replenishing. Moreover, SIRT5 knockout restores chemosensitivity to TNBC cells. We anticipate that blocking SIRT5-mediated metabolic rewiring may reverse chemoresistance in refractory TNBC patients. Zuen Ren, Kiran Kurmi, Tiziano Bernasocchi, Eric Zaniewski, Shakchhi Joshi, Garrett Lam, Chenxu (Vincent) Guo, Ilze Smidt, Preshita Dave, Agustina Maccio, Johannes Kreuzer, Gabrielle Elena Gioia, Veerle I. Bossuyt, Brian N. Dontchos, Gary X. Wang, Shinn-Huey S. Chou, Michael Lawrence, Esther Rheinbay, Laura M. Spring, Wilhelm Haas, Raul Mostoslavsky, Marcia C. Haigis, Leif W. Ellisen. SIRT5 promotes chemoresistance through metabolic rewiring for nucleotide replenishing in refractory triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4449.

Introduction: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in conjunction with endocrine therapies have transformed the treatment landscape for patients with metastatic hormone-receptor positive (HR+)/HER2- breast cancer. Studies exploring the clinical utility of CDK4/6i re-introduction after disease progression on prior CDK4/6i-based therapy have yielded mixed results, including the recent phase III postMONARCH trial (which interrogated the combination of fulvestrant and abemaciclib in the second-line metastatic setting). Here, we explore the clinical outcomes of abemaciclib monotherapy after disease progression on prior combined CDK4/6i and endocrine therapy. Methods: We collected retrospective clinical data at two academic institutions (Massachusetts General Hospital and Barnes-Jewish Hospital) according to site-specific IRB-approved protocols from patients with metastatic HR+/HER2- breast cancer who had received abemaciclib monotherapy after disease progression on another CDK4/6i-based therapy in the metastatic setting. We summarized patient and treatment characteristics and conducted time-to-event analyses. Results: In this preliminary analysis, a total of 16 patients received abemaciclib monotherapy after disease progression on prior CDK4/6i-based therapy. All 16 received prior palbociclib-based therapies. Eleven of these patients continued abemaciclib until disease progression or death, while five patients stopped therapy due to toxicity (three due to gastrointestinal side effects, one due to fatigue, and one due to atrial fibrillation). Patients received palbociclib-based therapy for a median of 16.2 months and abemaciclib for a median of 3.8 months (95% confidence interval, 2.0-7.5 months). Five patients received abemaciclib for >180 days prior to disease progression/death. Conversely, only two patients discontinued abemaciclib prior to 90 days after initiation due to disease progression. Four out of the 16 patients received abemaciclib as the subsequent line of treatment immediately following progression on palbociclib. The median time from progression on palbociclib-based therapies to starting abemaciclib was 10.7 months, with a median number of two intervening lines of therapy. Efforts are underway to combine this cohort with additional patients receiving abemaciclib monotherapy in this setting at other institutions to enhance clinical sample size. Planned analyses will include retrospective review of radiographic images to estimate overall response rate and exploration of genomic sequencing to identify molecular mediators of response and resistance to abemaciclib monotherapy. These ongoing efforts will be presented at the meeting. Conclusions: A subset of patients tolerated abemaciclib monotherapy for > 180 days before progression/death despite progression on prior combination palbociclib and endocrine therapies. This is the first effort to interrogate the utility of abemaciclib monotherapy in this setting and will provide additional insights related to serial CDK4/6-directed therapy in this patient population. These results demonstrate clinical promise in continued CDK4/6 inhibition and raise questions about mechanisms of resistance and the identification of patients that would benefit from abemaciclib monotherapy. Citation Format: Sahar Shahamatdar, Katherine Clifton, Julianna Wu, Annika Putur, Irene Kuter, Aditya Bardia, Dejan Juric, Laura Spring, Katherine Harris, Beverly Moy, Jennifer Shin, Neelima Vidula, Cynthia Ma, Seth A. Wander. Abemaciclib monotherapy after disease progression on prior CDK4/6 inhibitors in patients with metastatic hormone-receptor positive breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-10-12.

6533 Background: A breast biopsy tissue biobank is a valuable resource for studying breast cancer biology and treatment response. However, underrepresentation of patient populations in biobanks limits the generalizability of findings. The aim of this study was to assess potential age and racial/ethnic disparities in the recruitment process for an institutional breast biopsy tissue bank. Methods: Ultrasound-guided (USG) research biopsy cores were collected immediately after routine clinical biopsy from January 2019 to October 2022 at a large academic center. Study eligibility included patients (pts) aged > 18 years undergoing USG clinical breast biopsy with a radiographically evident mass ≥ 0.6 cm in the longest dimension. Eligible pts, identified by a research associate on the day of the biopsy, were invited to participate at the discretion of the radiologist performing the biopsy. Those approached either consented or declined a research biopsy. Demographic and clinical data of eligible pts were extracted from the EMR. Results: 2449 pts underwent USG breast biopsy and 1309 were deemed eligible for a research biopsy. Of the eligible population, 564 (43%) consented to the study, 322 (25%) declined, and 423 (32%) were not approached. Consented pts were younger compared to those who declined or to those not approached (median age 49, 51, and 51 years, respectively; p = .01). Comparison of study groups by age and race categories are shown in the table. Pts > 70 were less likely to be approached compared to pts < 70 (p = .01). However, The likelihood of pts being approached to consent did not differ significantly with age (p=.09 ). Of the eligible pts, 951 (73%) were White, 327 (25%) were non-White (10% Asian, 8% Black, 1% Hispanic, and 7% other race), and race was unknown for 31 (2%) pts. White and non-White pts were equally likely to be approached (p = .3). However, approached non-White pts were significantly less likely to consent compared to White pts (p =.01). Within the non-White population, Black pts were more likely to decline a research biopsy, with only 50% of approached Black pts consenting compared to 66% of White pts, 61% of Asian pts and 55% of pts of other race. Conclusions: We found disparities based on age and race/ethnicity in participation in a breast biopsy tissue biobank. Older pts were less likely to be offered participation but equally likely to consent when invited, while Black pts were equally likely to be offered participation, but less likely to consent when invited, compared to other racial subgroups. Our findings support targeted interventions to increase participation across diverse subgroups of pts. [Table: see text]

PURPOSE:Race/ethnicity may affect outcomes in metastatic breast cancer (MBC) due to biological and social determinants. We evaluated the impact of race/ethnicity on clinical, socioeconomic, and genomic characteristics, clinical trial participation, and receipt of genotype-matched therapy among patients with MBC. EXPERIMENTAL DESIGN:A retrospective study of patients with MBC who underwent cell-free DNA testing (cfDNA, Guardant360, 74 gene panel) between 11/2016 and 11/2020 was conducted. Receipt of genotype-matched therapy targeted at a cfDNA actionable mutation was determined. Pearson χ2 and Wilcoxon rank-sum tests were used to compare categorical and continuous variables between groups. Multivariable logistic regression was used to assess the association of race and receiving matched therapy. RESULTS:A total of 425 patients with MBC and cfDNA results were identified (White: 369, Black: 27, Hispanic: 15, and Asian: 14). White patients traveled further for cancer care than other groups (P < 0.001). White patients had the highest rates of commercial insurance, Black patients had the highest rates of state-supported insurance, and Asian patients had the highest uninsured rates (P < 0.001). Clinical trial enrollment did not differ by race/ethnicity (P = 0.34). The proportion of patients with ≥1 actionable mutation in cfDNA did not vary by race/ethnicity (P = 0.18). The highest rates of matched therapy were observed in White patients (P < 0.001). After multivariable logistic regression adjusting for subtype, commercial versus other insurance, Charlson Comorbidity Index, and distance to center, White patients remained more likely to receive matched therapy (P = 0.024). CONCLUSIONS:Racial/ethnic minority patients were less likely to receive matched therapy. Further research is needed to identify barriers to precision medicine.

Addition of pembrolizumab to neoadjuvant chemotherapy followed by adjuvant pembrolizumab improved outcomes in patients with high-risk, early-stage, triple-negative breast cancer. However, whether the addition of neoadjuvant pembrolizumab to chemotherapy would improve outcomes in high-risk, early-stage, estrogen receptor-positive/human epidermal growth factor receptor 2-negative (ER+/HER2−) breast cancer remains unclear. We conducted a double-blind, placebo-controlled phase 3 study (KEYNOTE-756) in which patients with previously untreated ER+/HER2− grade 3 high-risk invasive breast cancer (T1c-2 (≥2 cm), cN1–2 or T3–4, cN0–2) were randomly assigned (1:1) to neoadjuvant pembrolizumab 200 mg or placebo Q3W given with paclitaxel QW for 12 weeks, followed by four cycles of doxorubicin or epirubicin plus cyclophosphamide Q2W or Q3W. After surgery (with/without adjuvant radiation therapy), patients received adjuvant pembrolizumab or placebo for nine cycles plus adjuvant endocrine therapy. Dual primary endpoints were pathological complete response and event-free survival in the intention-to-treat population. In total, 635 patients were assigned to the pembrolizumab−chemotherapy arm and 643 to the placebo−chemotherapy arm. At the study’s prespecified first interim analysis, the pathological complete response rate was 24.3 NCT03725059 . In this prespecified interim analysis of the KEYNOTE-756 phase 3 trial, pembrolizumab and chemotherapy treatment of patients with high-risk, early-stage, estrogen receptor-positive/human epidermal growth factor receptor-negative breast cancer improved the pathological complete response rate compared with chemotherapy alone.

Optimal timing and dosing of adjuvant cyclin-dependent kinase (CDK) 4/6 inhibitor in early breast cancer is controversial. This prospective phase II clinical trial investigated tolerability and safety of two ribociclib dosing schedules. Patients with stage I–III hormone receptor-positive (HR+)/HER2– breast cancer on adjuvant endocrine therapy (ET) were randomized to two ribociclib dosing schedules: 400 mg continuous vs 600 mg intermittent, with initiation in early (prior ET < 2 years) vs delayed (prior ET ≥ 2 years) setting. Primary objective was to evaluate safety and tolerability of continuous vs intermittent schedule. Primary endpoint was proportion of patients who discontinued ribociclib before completion of all 12 cycles (measured at 12 months). Recurrence free survival (RFS) and circulating tumor DNA (ctDNA) detection were also evaluated. 81 patients were enrolled. Only six serious adverse events occurred, with no significant difference between treatment arms and no subject deaths. Twenty-five patients (31%) discontinued ribociclib before completion of 12 months, with no significant difference between treatment arms. Ribociclib discontinuation was higher in early vs delayed initiation (36% vs 21%). At median follow-up of 20 months, two patients in the intermittent arm (600 mg; Arm 2) experienced disease recurrence (2-year RFS 97%, 95%CI 88–99%), vs none in the continuous arm (400 mg; Arm 1) (2-year RFS 100%). ctDNA was only identified in the two subjects with recurrent disease at median of 7.5 months prior to radiological recurrence. Ribociclib is a safe and well-tolerated adjunct to adjuvant ET in early-stage breast cancer. Delayed initiation of ribociclib at 400 mg continuous dosing was feasible, better tolerated and associated with promising outcomes. ctDNA detection preceded clinical evidence of recurrence and may be considered as a surveillance tool in breast cancer.