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Abstract Background: Endocrine therapy (ET) with aromatase inhibitors (AIs) plus ovarian function suppression (OFS) is a standard endocrine strategy for premenopausal women with ER+/HER2- breast cancer. However, poor tolerability to AI+OFS limits its use, leaving tamoxifen as the only alternative. Prospective studies demonstrated that premenopausal women treated with neoadjuvant ET whose tumors exhibit Ki-67 ≤ 10% following 4 weeks of therapy achieve 5-year distant disease-free survival > 96%. Problematically, only 41% of patients reach this threshold with tamoxifen compared to 78% with AI+OFS (Nitz JCO 2022). (Z)-endoxifen (ENDX) is a potent selective estrogen receptor modulator that dually targets ERα and PKCβ1. At plasma concentrations of 3-5 ng/mL, it inhibits ERα; at ≥ 500 ng/mL, it targets PKCβ1, leading to AKT suppression. The EVANGELINE trial aims to examine endocrine sensitivity in terms of Week 4 Ki-67 in premenopausal women whose baseline Ki-67 > 10%, and objective response rate after 24 weeks of ENDX + goserelin in premenopausal women with baseline Ki-67≤ 10%. Methods: EVANGELINE (NCT05607004) is a multicenter, open-label, Phase 2 study comprised of three parts. Eligible patients are premenopausal women with Stage IIA/IIB ER+/HER2- breast cancer. • Part 1 (PK Run-in): assessed 40mg vs 80mg ENDX (+/- OFS) in 22 patients. • Part 2: patients with baseline Ki-67 >10% were randomized to 40 mg ENDX + goserelin or exemestane + goserelin for 6 months with the primary objective being 4-week ESD rate. Patients with a baseline Ki-67 ≤10% were assigned to a single-arm cohort of 40 mg ENDX monotherapy (no OFS) to evaluate the 24-week endocrine sensitivity disease rate. • Part 3 (replaces Part 2): all patients are treated with ENDX 40mg daily + goserelin every 28 days. A two stage Phase II design was chosen to assess outcomes as follows: Part 3a: Up to 45 patients with baseline Ki-67> 10% to assess Week 4 Ki-67≤ 10% rate is at least 65%. If 9 or more of these patients have a Week 4 Ki-67>10%, enrollment will be stopped due to futility. Otherwise, enrollment will continue. Part 3b: Up to 20 patients enrolled with baseline Ki-67≤ 10% to assess Week 24 objective response rate. Ki-67 levels are determined by central laboratory and images are centrally reviewed. Secondary objectives include safety, tolerability, surgical outcomes, and biomarker analysis using paired biopsies (baseline, week 4) and surgical specimens. Results: The PK run-in (completed Fall 2024) enrolled 22 patients and assessed ENDX doses (40 mg daily vs 80 mg daily (+/-OFS)). The Week 4 Ki-67 ≤ 10% rate with ENDX was 86% and did not differ according to dose. Early safety, PK, efficacy and compliance data supported the selection of 40 mg ENDX daily as the optimal dose. Enrollment in Part 2 began in May 2025. Part 3 will replace Part 2 with simplification of the trial design. Conclusions: EVANGELINE is the first trial to evaluate (Z)-endoxifen + OFS as neoadjuvant therapy in premenopausal ER+/HER2- breast cancer. By targeting both ER and PKC pathways, ENDX may offer a well-tolerated alternative to AI-based regimens and expand endocrine therapy options for this population. Clinical Trial Registration: NCT05607004 Citation Format: M. P. Goetz, V. J. Suman, C. Lopez, H. Erickson, L. Beaulieu, S. S. Hammer, L. Mina, R. Leon-Ferre, K. N. Hunt, M. Piltin, A. Degnim, J. N. Ingle, J. C. Boughey, B. A. Sarah, J. M. Reid, M. Schellenberg, J. R. Hawse, P. Advani, K. Giridhar, F. Batalini, D. Flora, J. M. Jones, N. A. Bagegni, J. Jakub, V. Abramson, V. Dabak, W. J. Irvin, E. Sima, K. Seema, S. C. Quay. A Phase 2 Trial of (Z)-endoxifen + Goserelin as Neoadjuvant Treatment for Premenopausal Women with ER+, HER2-, Breast Cancer (EVANGELINE) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-12-16.

Abstract Introduction: An Our Practice Advisory (OPA), formerly known as Best Practice Advisory (BPA), is an electronic health record (EHR) tool. OPAs identify key parameters to alert providers of potential interventions that could enhance quality of care. Such functionality is increasingly relevant given the rapid pace of new drug approvals. Within the Mayo Clinic system, we previously identified that amongst Mayo Clinic patients (pts) meeting the MonarchE criteria, 28% of pts did not undergo a documented discussion with their provider regarding the role of adjuvant abemaciclib (Premji, SABCS 2024). Further, a Flatiron study identified that only 40% of eligible pts received abemaciclib (Sandoval, Miami Breast 2025). To address this practice gap, in 2024 we began to implement EHR clinical decision support by creating an OPA trigger for pts eligible for an adjuvant CDK 4/6 inhibitor (CDK 4/6i). Here we report on ‘action taken’ versus not for the OPA and the most common reasons for re-prompts. Methods: The Mayo Breast Disease Group designed an OPA including pts who are within 14 months of a clinical or pathologic diagnosis of high-risk early-stage breast cancer. We leveraged structured data captured in the Epic staging forms (Epic Systems) to identify pts with clinical or pathologic T-stage, N-stage, grade, ± Ki-67/ multigene signature to identify ER+/HER2- pts eligible for adjuvant ribociclib or abemaciclib based on FDA approvals. The OPA was triggered by a patient encounter in oncology when endocrine therapy (but not CDK4/6i) was on the active medication list resulting in a hard stop. The OPA gathered limited details about whether adjuvant CDK4/6 inhibitors were discussed and whether the provider followed a link that resulted in prescribing one—considered an indicator of action taken. In this IRB-exempt quality improvement project, we describe the responses captured in the OPA trigger utilizing Epic tools. Results: The OPA went live on 1/10/2025. Over a 5-month period post OPA initiation, we identified 517 OPA alerts among 332 unique pts. Their median age was 62.5 years, and the majority were female (97%). The median time to resolve the OPA was 17.5 seconds (range 2-567 seconds). 12.6% (65/517) of the time there was ‘action taken’ by selecting the hyperlink to potentially start a treatment plan. Providers selected the following OPA pre-set responses: ‘re-prompt in one month’ 248 (48%), ‘patient not a candidate for therapy’ 48 (9%), ‘patient declines’ 69 (13%), ‘outside oncologist will prescribe’ 36 (7%), ‘not discussed’ 44 (9%), and unknown 72 (14%). The most common free text comments among the 517 OPA alerts (summarized) included: ‘revisit in future’ 108 (21%), ‘too early’ 65 (13%), ‘patient declines’ 66 (13%), ‘outside oncologist will start’ 31 (6%), ‘not a candidate’ 26 (5%), and ‘co-morbidities’ 18 (3%). Erroneous OPA prompting occurred in 14 (3%) who previously failed a CDK4/6i due to toxicity and in 18 (3%) for pts currently on a CDK4/6i. 30 (6%) OPA comments noted that they discussed or started therapy that day. Conclusion: The OPA offered clinical decision support and ensured that clinicians considered prescribing CDK 4/6i in all pts identified as eligible, and captured deferrals. Refinements are needed to ensure appropriate timing of OPA and minimize erroneous firing. The OPA remains active and captures the reason for deferral of adjuvant CDK 4/6i prescription. This data pairs real world decision making to better understand clinically meaningful application of improved invasive disease-free survival (iDFS). Citation Format: S. Premji, P. Advani, S. Chumsri, L. Mina, J. Taraba, J. Carroll, J. Hoppenworth, E. Cathcart-Rake, T. Haddad, B. Rudder, G. Choong, D. Idossa, C. O’Sullivan, S. Yadav, K. Ruddy, P. Peethambaram, T. Hobday, M. Goetz, R. Rao, B. Ernst, F. Batalini, R. Leon-Ferre, A. Tevaarwerk, K. Giridhar. Implementing an Our Practice Advisory (OPA) to provide clinical decision support for adjuvant CDK4/6 inhibitors in early-stage hormone receptor positive breast cancer: The Mayo Clinic Experience [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-11-10.

Abstract Breast cancer is the most common female malignancy in the U.S. and the leading cause of cancer-related deaths in women worldwide. Nearly 80% of breast tumors express estrogen receptor alpha (ERα) which drives tumor progression. While CDK4/6 inhibitors in combination with endocrine therapy improve overall survival in the metastatic setting and disease-free survival in the adjuvant setting, many patients eventually relapse/progress with resistant forms of disease. In the post-CDK4/6 inhibitor setting, second line endocrine therapy responses are limited, highlighting the need for alternative treatment approaches. Through a genome-wide CRISPR knockout screen, we identified Lemur Tail Kinase 2 (LMTK2) as the top hit exhibiting synthetic lethality in the presence of the tamoxifen metabolite, endoxifen, in both endocrine-sensitive and -resistant cell line models. We further studied multiple other endocrine resistant models and identified that LMTK2 mRNA and protein expression were both increased in ERα cell lines resistant to estrogen deprivation, tamoxifen, fulvestrant, abemaciclib and palbociclib, as well as in endocrine resistant patient derived models. We then studied LMTK2 in patient samples and identified copy number gain/amplification in 67% of ERα+ human metastatic breast tumors, while only 1% of primary tumors harbored these genomic alterations. From a prognostic standpoint, elevated tumoral LMTK2 expression was associated with worse recurrence-free survival and overall survival in early-stage breast cancer. Through proteomic studies, we identified the MET-PI3K-AKT pathway as overactivated in LMTK2 overexpressing cells. Unlike activating PIK3CA mutations that drive AKT signaling, LMTK2 was found to employ an alternative mechanism to activate AKT signaling by inhibiting PP1α, an AKT-inhibitory phosphatase. Further, LMTK2 overexpression was sufficient to drive endocrine and CDK4/6 inhibitor resistance in vitro and resulted in dramatic increases in AKT and ERα phosphorylation and activity. While knockout of LMTK2 in treatment naïve ERα+ breast cancer cells neither altered AKT/ERα phosphorylation/activity nor endocrine/CDK4/6 inhibitor responsiveness, attempts to knockout LMTK2 in endocrine/CDK4/6 inhibitor-resistant models failed, further confirming its essentiality in this setting. In summary we have uncovered LMTK2 as a novel biomarker of endocrine/CDK4/6 inhibitor resistance that is mechanistically linked to the AKT pathway. LMTK2 may be a novel drug target for the effective treatment of resistant and advanced forms of ERα+ breast cancer. Citation Format: A. M. Detry, S. Marigliano, J. Heinen, A. Bass, C. Jones, R. S. Muthyala, M. P. Goetz, J. R. Hawse. Lmtk2: a novel driver of endocrine resistance in ER+ breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-11-01.

Abstract Background: We have identified two potentially predictive signatures of palbociclib resistance: the RBsig, composed of E2F1/E2F2 dependent genes, which is associated with genetic loss of RB1, and the ratio between the gene expression levels of CCNE1 to RB1 (CCNE1/RB1). Both signatures have been previously analyzed in vitro and in neoadjuvant and metastatic studies with palbociclib. The present analysis aims to explore the role of RBsig and CCNE1/RB1 in the phase III adjuvant PALLAS trial, which randomized patients to palbociclib plus endocrine therapy, versus endocrine therapy alone. Materials and methods: Gene expression data from PALLAS were generated using the HTG EdgeSeq Oncology BM Panel. Of the 87 genes composing RBsig, 46 were available within the EdgeSeq dataset and were used for the analyses; CCNE1 and RB1 were both available. RBsig was calculated as the mean of the Z-score scaled gene expression (log) of the 46 genes; CCNE1/RB1 was computed as the log ratio between the mRNA expression of CCNE1 and RB1. The prognostic/predictive effect of the continuous signatures in terms of invasive disease-free survival (IDFS), distant recurrence-free survival (DRFS), and overall survival (OS) was evaluated using Cox proportional hazard models. Results: HTG data were available from 2,587 of 5,748 patients from the entire PALLAS intention-to-treat (ITT) cohort. As RBsig and CCNE1/RB1 levels are affected by pre-surgical treatments, we limited the analyses to 1612 pre-treatment samples (530 core biopsies and 1082 surgical specimens) (analysis population). Compared with the entire PALLAS ITT cohort, the analysis population was significantly enriched for patients with low clinical risk (41.3% vs 59.4%) who did not receive prior chemotherapy (17.5% vs 55.9%). In keeping with the PALLAS ITT cohort, no significant benefit from the addition of palbociclib was observed in the analysis population. Higher baseline RBsig or CCNE/RB1 values were significantly and independently associated with worse IDFS (hazard ratio [HR] 1.63, 95% confidence interval [CI] 1.3-2.0; HR 1.22, 95%CI 1.1-1.3, respectively), DRFS (HR 1.94, 95%CI 1.5-2.5; HR 1.26, 95%CI 1.1-1.4, respectively) and OS (HR 1.79, 95% CI 1.3-2.4; HR 1.26, 95%CI 1.1-1.4, respectively) in the analysis population. No predictive effect of palbociclib benefit was observed for either signature in the analysis population, nor according to clinical risk or chemotherapy receipt. Interestingly, higher RBsig was strongly prognostic in patients who did not receive adjuvant chemotherapy (HR 1.83, 95%CI 1.4-2.3) but not in those who received it (HR 1.01, 95%CI 0.6-1.6) (interaction p=0.02) suggesting a potential predictive role of RBsig for chemotherapy benefit. Conclusions: In the PALLAS trial, higher baseline RBsig or CCNE1/RB1 values are associated with worse prognosis, but are not predictive of benefit from palbociclib in the adjuvant setting. RBsig might be associated with differential benefit from chemotherapy. Further study of these biomarkers may strengthen their role in risk stratification.https://acknowledgments.alliancefound.org Citation Format: L. Malorni, D. Hlauschek, E. Mayer, M. Benelli, L. Biganzoli, I. Migliaccio, A. Kermanidis, G. Pfeiler, E. P. Mamounas, A. M. Brufsky, M. Bellet-Ezquerra, K. Clifton, G. Rubovszky, T. Foukakis, M. Goetz, S. Lee, M. Ruiz-Borrego, W. Symmans, K. V. Ballman, F. Liu, J. Machacek-Link, C. Denkert, A. DeMichele, M. Gnant. Prognostic and predictive role of RBsig and CCNE1/RB1 gene-expression signatures for patients with early breast cancer treated with endocrine therapy with or without palbociclib in the PALLAS trial (ABCSG-42, AFT-05, BIG 14-03) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-07-13.

Abstract Background: BTX-9341 is a first-in-class bifunctional degrader of cyclin dependent kinase (CDK)4 and CDK6 that also inhibits the transcription of CDK2 and Cyclin E. Preclinical data highlight its superiority compared with approved CDK4/6 inhibitors (CDK4/6i) in inhibition of phosphorylation of retinoblastoma (p-RB), cell cycle arrest, and in vivo efficacy in breast cancer (BC) xenografts. BTX-9341 is active in CDK4/6i-resistant models with p-RB half-maximal inhibitory concentrations (IC50s) of 1-15 nM and can overcome key mechanisms that drive CDK4/6i resistance. Here we report early monotherapy data from the first-in-human study of BTX-9341 in patients (pts) with advanced and/or metastatic HR+/HER2- BC. Methods: BTX-9341-101 is a multicenter, nonrandomized, open-label trial to assess the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of orally administered BTX-9341 in pts with RB1 wild type advanced and/or metastatic HR+/HER2- BC who received prior CDK4/6i therapy. An accelerated titration and Bayesian Optimal Interval design (BOIN) were used. The primary objective of dose escalation is to determine the maximum tolerated dose (MTD) or maximum evaluable dose (MED) of BTX-9341 monotherapy and in combination with fulvestrant. Secondary objectives include the characterization of BTX-9341 on PK and efficacy; exploratory objectives include the assessment of BTX-9341 on PD and PK/PD relationships. Results: As of June 9, 2025, 12 pts were evaluated at the following BTX-9341 once daily (QD) dose levels: 50 mg (dose level [DL]1), 100 mg (DL2), and 75 mg (DL3) monotherapy, and 50 mg in combination with fulvestrant (DLA). Pts received up to 4 lines of therapy in the metastatic setting prior to enrollment. No dose-limiting toxicities (DLTs) were observed at any dose levels during the 28-day DLT period. The most common adverse events (AEs) during the DLT period were decreased neutrophil and white blood cell counts (Grades 1-4). There were no dose reductions in DL1. The starting dose was reduced in all pts in DL2; DL3 is currently enrolling. At least 2-fold accumulation was observed after QD dosing, and steady-state trough concentrations were in the range of in vitro IC50 values at DL1 and higher dose levels. Serum thymidine kinase activity (sTKa), a clinical biomarker for tumor response to CDK4/6 inhibition, showed >50% reduction at Cycle 2 Day 1 (C2D1) compared with baseline for 7 of 8 evaluable monotherapy pts, with 6 pts below the sTKa detectable level. “On target” PD reductions in CDK4, CDK6, CDK2, and Cyclin E levels were observed in peripheral blood mononuclear cells. Of the 8 evaluable monotherapy pts, the best overall responses in pts with measurable disease included 1 pt with a confirmed partial response (PR) and 3 pts with stable disease ([SD] first assessed at 8 weeks; i.e., the first time point for assessment in the protocol), and in pts with non-measurable disease, 2 pts had non-complete response (CR)/non-progressive disease. For all pts, the longest duration on treatment was 10 cycles. A clinical benefit rate (defined as a best overall response of CR, PR, or SD for pts with measurable disease or non-CR/non-progressive disease for pts with non-measurable disease, per RECIST v1.1 at Week 24) of 66.7% was observed in the completed cohorts (DL1 and DL2). Conclusion: BTX-9341 monotherapy shows a favorable safety profile, with the most common AEs being decreased neutrophil and white blood cell counts, at doses that show encouraging preliminary PK/PD and efficacy, enabling further evaluation of monotherapy and in combination with fulvestrant. The trial is actively enrolling with anticipated completion of dose escalation enrollment by the end of 2025 (Clinical trial: NCT06515470). Citation Format: A. Patnaik, M. Block, M. Cristofanilli, Q. Liu, H. Majeski, A. Shakya, P. Rajagopalan, C. Deshpande, T. Marmon, S. Padam, D. Powell, A. H. Chourasia, R. Urbanski, M. Goetz, R. M. Layman. First-in-human Phase 1 study of BTX-9341, a CDK4/6 bifunctional degrader, as monotherapy and in combination with fulvestrant in patients with advanced and/or metastatic HR+/HER2- breast cancer - first emerging data [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-06-04.

Abstract Significance: Emergence of resistance to endocrine therapies represents a major challenge in theclinical management of estrogen receptor positive (ER+) breast tumors. CDK4/6 inhibitors(palbociclib, ribociclib, abemaciclib) have improved survival when combined with endocrine therapy. Nonetheless, resistance to CDK4/6 inhibitors eventually occurs. One of the emerging mechanisms ofresistance toward endocrine therapy and CDK4/6 inhibitors is characterized by the enrichment ofbreast cancer stem cells (CSCs) that show stem-like features such as self-renewal capacity, highCD44 expression and ALDH activity. We have demonstrated that Aurora-A kinase (AURKA) has apivotal role in promoting cancer cellular plasticity and clonal expansion of endocrine resistantCD44high/ALDHhigh CSCs. Our preclinical studies on AURKA-driven tumor stemness provided thetherapeutic foundation for a Phase-II clinical trial (TBCRC041) of the AURKA inhibitor, alisertib, whichdemonstrated clinically meaningful activity in patients with endocrine and CDK4/6 inhibitors resistantER+ metastatic breast cancer (MBC). Despite such activity, alisertib resistance finally occurred. Therefore, we hypothesize that up-regulation of IL6R/STAT3 and PR stemness oncogenic pathwaysmay sustain the clonal expansion of CD44high/ALDHhigh CSCs after tumor progression on AURKAinhibitors. Experimental Design: We used variant MCF-7 breast cancer models that show resistance toendocrine therapy (letrozole and fulvestrant) and alisertib (MCF-7 LR/FR, MCF-7 LR/FR-AlisR) todefine in vitro and in vivo the efficacy of stemness-targeted therapies to enhance alisertib activity byimpairing the enrichment of hormone refractory CD44High/ALDHHigh CSCs. Parental MCF-7 cells wereused as control. We employed exclusive PDX-derived 3D-Mammospheres (MPS) that wereestablished from the Phase-II clinical trial TBCRC041 to test the efficacy of stemness-targetedtherapies in enhancing the therapeutic activity of alisertib. In Vitro Studies: Immunofluorescence andimmunoblot assays were used to assess the expression of breast cancer stemness biomarkers. Enrichment of ALDHHigh CSCs was measured using Red-Aldefluor kit. Cell proliferation and Red-Annexin-VHigh apoptotic cells were quantified in real-time using IncuCyte S3. In Vivo Studies: 1x106breast cancer cells were injected into the mammary fat pad of NSG-female mice and treated withfulvestrant, alisertib and tocilizumab as monotherapy or in combination. Control groups were treatedwith saline solution placebo. Drug Treatments: Tocilizumab and Onapristone were used to inhibit theIL6R/STAT3 and PR stemness pathways, respectively. Results: Our study demonstrates for the first time that nuclear STAT3/PR complex mediatesAURKA-induced expression of KLF4 stemness gene and enrichment of endocrine resistantCD44high/ALDHhigh CSCs. Alisertib resistant MCF-7 LR/FR cells showed increased expression ofIL6R, T288-AURKA, S727-STAT3 and S294-PR compared to alisertib naïve cells. MCF-7 LR/FR-AlisR-derived 3D-MPS had increased KLF4 and CD44 expression. Alisertib resistant PDX modelsalso showed high levels of CD44, IL6R/STAT3 and KLF4 expression. Remarkable, pharmacologicalblockade of PR and STAT3 transcriptional regulatory networks enhanced the efficacy of alisertib byreducing the enrichment of CD44High/ALDHHigh CSCs. Conclusions: These findings provide the strong preclinical rationale for the development of novelcombinatorial therapeutic strategies targeting IL6R/STAT3 and PR stemness pathways to enhancethe efficacy of AURKA inhibitors that will result in the selective eradication of CD44High/ALDHHighCSCs with consequent benefits on the overall survival of patients with advanced ER+ breast tumorsrefractory to endocrine therapies and CDK4/6 inhibitors.輀 Citation Format: A. B. D'ASSORO, M. Drago, T. Haddad, M. GOETZ, K. Giridhar, K. Kalari, L. Manzella, P. Vigneri, F. Drago, C. Vancheri, C. Lange, J. Ingle. Stemness-targeted therapies to enhance the efficacy of AURKA inhibitors in endocrine resistant er+ breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-11-08.

Abstract Introduction Serial assessments of circulating tumor cells (CTCs) in metastatic breast cancer (MBC) provide independent prognostic significance for progression free survival and overall survival. A low CTC burden (<5 CTCs / 7.5mL blood) at baseline is favorable prognostically, and reduction of CTCs during treatment indicates response to treatment (Janni, CCR 2025). Despite increasing evidence supporting its clinical validity, real-time serial CTC enumeration has not been utilized broadly in clinical practice in the U.S. The ACT-MBC study is a prospective observational study to evaluate the impact of serial CTC testing on treatment decisions and response assessment (NCT05662345). Provider (physician or advanced practitioner) perception of CTC testing is measured with questionnaires at baseline (PQ1), and for each pt during the first restaging visit (PQ2), and at the end of the study (PQ3). We previously reported PQ1 results (Giridhar, SABCS 2024). Here, we report on results of PQ2. Methods ACT-MBC prospectively enrolled patients (pts) at Mayo Clinic Rochester and Mayo Clinic Health Systems sites (Mankato, Albert Lea and Eau Claire). Pts with MBC were enrolled at time of progressive disease during any line of therapy (TNBC) or ≥2nd line metastatic therapy for ER+/HER2- disease. Pts were tested for CTCs using the FDA-cleared CellSearch® assay which included enumeration and biomarker testing on CTCs for ER, HER2, and PD-L1. The treating clinician determined the regimen and timing of restaging scans. CTC testing was performed once per cycle (every 3-4 weeks) with results returned to the treating provider within 7 days. We descriptively summarized PQ2 responses from providers who had at least one on-treatment CTC result available by the first restaging visit. We report the proportion (90% CI) of providers who agreed or strongly agreed that assessing CTCs enhanced their ability to determine response to therapy and that the CTC results helped guide their decision to continue or change therapy (co-primary endpoints) while accounting for within provider correlation. Results As of June 25, 2025, 15 providers enrolled a median of 2 pts each (range: 1 to 15 pts) and completed PQ2 surveys on 40 pts. 7 providers (47%) practiced in the community setting and 4 (27%) were nurse practitioners. Most pts were female (98%), White (98%), and had ER+/HER2- MBC (80%). Baseline (C1) CTC results were elevated (≥5 CTCs) in 24 (60%) pts (CTC range 0-609). At C2 (n=39), CTC values remained undetected in 7 pts, decreased in 21 pts, increased in 8 pts, and remained unchanged in 3 pts. At PQ2 (n=40), based on clinician interpretation of radiographic findings, results showed 2.5% of pts with complete response, 20% with partial response, 25% with stable disease, 17.5% with mixed response, and 35% with progressive disease. Clinicians reported that CTC results were concordant with imaging results in 34 (85%) responses, discordant with imaging in 7.5% of responses, and unclear in the remaining 7.5% of responses. Most clinicians (82.5%) agreed or strongly agreed that assessing CTCs enhanced their ability to determine response to therapy (90% CI: 68.3%, 96.7%). Further, 70% of providers agreed or strongly agreed that the CTC results helped guide their decision to continue or change therapy (90% CI: 50.8%, 89.2%). 32.5% of the time, CTCs results influenced the timing of the first restaging scans. In 11/25 pts without progressing disease, CTC results influenced the timing of future scans. In the 7/8 pts with a mixed response at first restaging scans with decreasing CTCs, 4 remained on the same systemic therapy. Conclusions Oncologists in academic and community practice responded that serial CTC assessments enhance ability to determine response of therapy, guide decisions to continue or change treatments, and influence timing of radiographic assessments. Citation Format: K. V. Giridhar, D. Zahrieh, M. Hanna, A. Singh, E. Al-Hattab, P. Schumacher, J. Carroll, J. Hoppenworth, T. Haddad, A. Tevaarwerk, B. Rudder, G. M. Choong, S. Basu, R. A. Leon-Ferre, T. O'Brien, A. Lewis, F. de Snoo, L. Stork-Sloots, F. K. Kuhr, S. Lazaro, R. Prendergast, D. S. Spinner, M. P. Goetz. Assessing the impact of real-time serial circulating tumor cell results on the management of patients with metastatic breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-03-10.

Abstract Background Invasive lobular carcinoma (ILC) differs from invasive ductal carcinoma (IDC) in its estrogen receptor (ER) signaling pathways, response to systemic therapy, and imaging features. We evaluated differences in early change in the proliferation marker Ki67 and features on magnetic resonance imaging (MRI) after novel endocrine based neoadjuvant therapy in lobular versus non-lobular cases on the I-SPY2 EOP Trial. Methods EOP is a prospective, randomized NET trial enriched for patients with ILC. Patients with stage II-III ER positive HER2 negative breast cancer with low molecular risk (Mammaprint low or Mammaprint Hi1 plus high Sensitivity to Endocrine therapy Index) were randomized to one of several endocrine therapy-based arms. Treatment arms include novel oral SERDS with or without CDK4/6 inhibitor, aromatase inhibitors, and SERMs with or without ovarian function suppression in pre-menopausal patients. Patients undergo repeat core needle biopsy with central Ki67 assessment after 3 weeks of treatment and serial breast MRI. Here, we compare endocrine therapy responsiveness as measured by change in Ki67 at 3 weeks and functional tumor volume (FTV) on pre-surgical MRI in lobular versus non-lobular cases. Results We evaluated 253 patients of whom 148 (58.5%) had non-lobular cancer (93.9% IDC), and 105 (41.5%) had lobular cancer (17.1% mixed ILC/IDC). Overall, mean age was 53.3 years, 63.8% of tumors were grade 2, and 86.5% were Mammaprint (MP) Low with the remaining being Hi1, with higher rates of MP Low in the lobular versus non-lobular cohorts (92.4% versus 83.4%, p=0.0358). At baseline, tumors in the lobular cohort had significantly lower mean Ki67 than those in the non-lobular cohort (11.2% [SD 12.8] versus 16.4% [SD 14.4%], p=0.0038). Consistent with this, the proportion with baseline tumor Ki67 <10% was significantly higher in the lobular cohort than the ductal cohort (60.4% versus 39.3%, p=0.001). At 3-week biopsy, mean Ki67 remained lower in the lobular versus non-lobular group (3.4% versus 5.4%, respectively, p=0.0415). The proportion of patients with Ki67 <10% at 3 weeks did not differ significantly between lobular and non-lobular cases (88.6% versus 79.7%, respectively, p=0.08). Among the 40 lobular cases and 88 non-lobular cases with baseline Ki67 ≥10%, week 3 Ki67 decreased to <10% in 90% of lobular cases and 72.7% of non-lobular cases (p=0.07). On MRI, mean FTV at baseline was similar among lobular and non-lobular cases (21 cc3 versus 16.3 cc3). On pre-surgical MRI, patients in the lobular cohort had significantly higher mean residual FTV than those in the non-lobular cohort (5.7 cc3 versus 3.8 cc3, p=0.034). Interestingly, when splitting the lobular cohort into mixed lobular and pure lobular, mean residual MRI FTV was 7.5 cc3 in mixed cases and 3.8 cc3 in pure lobular cases, reflecting that FTV decreased the least in mixed lobular cases (decrease of 51.1% in mixed cases, 69.7% in pure lobular cases, and 73.9% in non-lobular cases, p=0.0255). Conclusions These results highlight differences in lobular versus non-lobular breast cancer. Lobular cases had lower baseline Ki67, and similar rates of decrease in Ki67 after 3 weeks of NET than non-lobular cases. Despite this, mean FTV after completion of 6 months of NET was higher in lobular tumors. These findings may reflect differences in early versus late indicators of response to NET, or differential response patterns by histology. Alternatively, standard response measures such as Ki67 and FTV may need lobular-specific modifications. Ongoing work includes central assessment of Ki67 from surgical specimens, analysis of changes in gene expression signatures, and evaluation of surgical outcomes such as breast conservation rates by histologic subtype. Citation Format: R. Mukhtar, P. Norwood, A. Borowsky, S. Alkhafaji, K. Giridhar, C. Vaklavas, A. Elias, M. Wei, M. Goetz, N. Onishi, O. Olopade, L. Van 't Veer, L. Huppert, W. F. Symmans, L. Brown Swigart, C. Wu, C. Yau, D. Yee, M. Magbanua, E. Price, N. Hylton, L. Esserman, J. Chien. Differential early response to neoadjuvant endocrine therapy in lobular versus non-lobular breast cancer on the Endocrine Optimization Pilot (EOP) of the I-SPY2 Trial [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-02-01.

Abstract Background: Invasive lobular carcinoma (ILC) accounts for up to 15% of breast cancer (BC) cases and has distinct clinical and biological features compared to invasive carcinoma of no special type (IBC-NST). Prior studies, including PENELOPE-B, suggested greater benefit from adjuvant palbociclib (palbo) in ILC. We assessed the prognostic and predictive value of histology in patients (pts) treated with endocrine therapy (ET) ± palbo. Methods: PALLAS is a global, phase III trial in which pts with stage II-III, HR+/HER2− BC were randomized to 2 years (y) of adjuvant palbo + ET or ET alone. Pts with IBC-NST or pure ILC per local pathology assessment were included. Endpoints included invasive disease-free (IDFS; primary), distant recurrence-free (DRFS), locoregional recurrence-free (LRRFS), and overall survival (OS). Prognostic value was assessed using univariable and multivariate Cox models adjusted for age, T and N stage, grade, and progesterone receptor. Predictive value was assessed using histology-by-treatment interactions. Results: Among 5,748 pts in the ITT population, 4,982 (87%) were included (3,838 IBC-NST [67%], 1,144 ILC [20%]); median follow-up 6.9y. Compared with IBC-NST, ILC was associated with older age (median 54 vs 51 y), higher T stage (T3/T4: 41.7% vs 20.4%), fewer grade 3 tumors (12.7% vs 33.5%), higher mastectomy rates (74.5% vs 58.4%), less (neo)adjuvant chemotherapy (72.2% vs 86.2%), and less tamoxifen as initial ET (28.8% vs 34.5%) (all p<0.001). ILC prevalence varied by country: lower in Asia (Japan 10.5%, Korea 0%, Taiwan 2.8%) and higher in Europe (23.1%) and North America (24.0%). When evaluating the predictive value, palbo appeared to confer greater benefit in ILC for all endpoints (HRs ranging from 0.41 to 0.86) and not in IBC-NST (HRs ranging from 0.97 to 1.01), although the histology-by-treatment interaction was significant only for LRRFS (Table). In the overall population, 7-year IDFS was 79.3% for IBC-NST and 75.8% for ILC. While the difference was significant in univariable analysis (HR 1.17, 95% CI 1.01-1.35), it was not in multivariable analysis (HR 1.06, 0.91-1.24). Notably, the proportional hazards assumption was violated, and time-dependent modeling demonstrated that worse outcomes for ILC on IDFS, DRFS, and other endpoints emerged predominantly beyond 3 years. Conclusions: In this large, randomized dataset of pts with early HR+/HER2− breast cancer with long-term follow-up, ILC demonstrated distinct clinicopathologic features and geographic distribution. While ILC was not an independent prognostic factor over the entire follow-up, time-dependent modeling revealed worse long-term outcomes which only emerged after 3 years. Palbo appeared to be associated with more favorable outcomes in ILC than in IBC-NST across endpoints. These findings suggest a potential benefit of palbo in ILC that warrants further investigation in other adjuvant trials evaluating CDK4/6 inhibitors. Citation Format: G. Nader-Marta, C. Desmedt, D. Hlauschek, A. DeMichele, G. Zoppoli, E. Blondeaux, E. de Azambuja, M. Bellet-Ezquerra, O. Metzger-Filho, J. M. Suga, G. Pfeiler, M. P. Goetz, M. Ruiz-Borrego, S. Loibl, J. Meisel, A. Ring, K. Van Baelen, E. P. Mamounas, N. Zdenkowski, E. Agostinetto, M. Lambertini, E. Gauthier, L. Soelkner, A. C. Dueck, M. Gnant, E. L. Mayer. Long-term prognostic and predictive value of lobular histology in the PALLAS trial [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-11-06.

Abstract Background: Tumor-infiltrating lymphocytes (TILs) in HER2+ and triple-negative breast cancer (BC) are prognostic and associated with therapy response; however, there are few data regarding distribution and significance of TIL values in ER+ HER2-negative (HER2-) MBC patients (pts) treated with CDK4/6 inhibitors (CDK4/6i). CDK4/6 inhibition may promote antitumor immunity by production of type III interferons and tumor antigen presentation, and by inhibition of regulatory T cells (Goel, Nature 2017). Further, the addition of immune checkpoint therapy to palbociclib numerically prolonged PFS in the 2nd line (2L) setting (Mayer, JCO 2024). We aimed to investigate the prognostic role of TILs derived from fresh metastatic tumor biopsies in pts with ER+HER2- MBC prior to starting CDK4/6i and endocrine therapy (ET) in the first line (1L) and 2L setting. Methods: PROMISE (NCT0281902) is a multicenter, prospective study that enrolled women commencing the CDK4/6i palbociclib (Pb) with letrozole (1L) or fulvestrant (2L) for HR+HER2- MBC. The primary goal was to identify novel genomic variants and pathways associated with PFS. Here, we performed a secondary analysis to quantitate stromal TILs using protocol mandated pretreatment tumor biopsies and determine their association with PFS. Using the Kaplan-Meier method, we reported median PFS (mPFS) and 3-year PFS percentage (%) (95% confidence intervals [CIs]) by TILs [≥20% vs <20%] separately within 1L and 2L. As sample TIL values were generally concentrated at zero (27/58 = 46.6%), coupled with wide spread of sample TIL values ≥20% [range: 20%- 80%], we applied a ≥20% threshold for PFS analysis (high vs low TIL group) as described by Luen, Salgado et al. Lancet Oncol 2017. Statistical significance was based on the Wilcoxon test of the null hypothesis that there was no difference in the PFS curves by TILs. PFS is reported at median follow-up of 21 months. Results: Pretreatment tumor biopsies from 58/68 (85.3 %) pts were evaluable (42 in 1L and 16 in 2L). Median [Q1, Q2] TIL values were higher in 1L (5% [0, 10]) vs 2L (0% [0, 15]). The most common biopsy sites were bone (44.8%), lymph node (17.2%), liver (17.2%) and breast (12%). Overall, TILs frequency (>20%) by the 4 most common biopsy sites were bone (2/20, 10%), lymph node (4/7, 57%), liver (2/6, 33%), and breast (1/7, 14%). In 1L, although PFS was not significantly different (P = 0.237), mPFS (44 vs 34 months) and 3-year PFS 75.0%, [95% CI: 50.3%, 100%] vs 46.4% [95% CI: 31.0%, 69.5%]) were longer in pts with high versus low TILS, respectively. In 2L, the 3-year PFS was numerically similar comparing high and low TILs groups, (25.0%, [95% CI: 4.6%, 100%] vs 22.5% [95% CI: 6.7%, 76.1%]) with no significant difference in PFS (P = 0.761). Conclusions: In pts with ER+HER2- MBC treated with Pb and ET, PFS was numerically longer comparing high vs low TIL values in 1L but not 2L settings. Future studies evaluating TILs in larger datasets are needed to validate these findings. Genomic analysis (DNA/RNA) and its association with TIL levels is underway and will be presented at the meeting. Funding: The Center for Individualized Medicine, Mayo Clinic; Mayo Clinic Breast Specialized Program of Research Excellence (SPORE P50CA116201); Mayo Clinic Cancer Center (CA15083-40A2); TEMPUS (https://www.tempus.com/) and Conquer Cancer Foundation of ASCO (Career Development Award 2018, Dr. O’Sullivan) Citation Format: C. C. O'Sullivan, A. M. Moyer, A. Nassar, D. M. Zahrieh, V. Suman, X. Tang, K. J. Thompson, R. C. Hentz, J. J. Harrington, A. Moreno-Aspitia, D. W. Northfelt, T. C. Haddad, S. Chumsri, P. P. Peethambaram, K. J. Ruddy, K. V. Giridhar, R. A. Leon-Ferre, R. Weinshilboum, L. Wang, K. R. Kalari, M. P. Goetz. Tumor-infiltrating lymphocytes in advanced ER-positive, HER2-negative breast cancer prior to treatment with palbociclib and endocrine therapy: a retrospective analysis of the PROMISE study [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS1-10-22.