Citations

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: Breast cancer remains the leading cause of cancer-related mortality in women globally, with metastatic disease accounting for >90% of deaths. Current staging and histopathological features inadequately predict individual recurrence risk. Approximately 20-30% of early-stage patients develop distant metastases despite optimal therapy, while others are overtreated. Circulating microRNAs (MIRNAs) represent a paradigm shift toward liquid biopsy, offering real-time tumor assessment through minimally invasive blood sampling. These stable molecules reflect dynamic tumor behavior and metastatic potential, making them ideal for personalized surveillance strategies. Methods: This prospective proof-of-concept study included 46 breast cancer patients from Sri Shankara Cancer Hospital, India: 37 with metastatic disease (plasma collected at progression, 2024) and 9 disease-free controls (≥3-year follow-up). MIRNA isolation used MIRVANA method with samples processed within 2 hours, stored at -80°C. Expression levels of MIR-21, MIR-30d, and MIR-425 were quantified by TaqMan qRT-PCR, normalized to MIR-16 control. Logistic regression analysis of the transcript levels yielded a probability of predictive accuracy ranging from 0-1. Cut-off of high expression was chosen based on the best sensitivity and specificity by ROC curve analysis. Clinicopathological parameters (CPP) were correlated with MIR expression(Mann-Whitney U test). A combined model of MIR and CPP was built using logistic regression and ROC curve analysis. Results: Patient demographics(in Table) Difference in median age, subtypes and nodal status are interesting to note. MIR-21 showed elevated expression in metastatic cases, particularly hormone receptor-positive tumors. MIR-30d demonstrated significant correlation with grade 3 and node-positive status (p=0.038 for N0 vs N1, p=0.006 for N0 vs N3), with high expression associated with poor progression-free survival (p=0.0057, HR=3.24). MIR-425 exhibited significant differential expression between metastatic and non-metastatic groups with strong correlation to advanced nodal stages (p=0.0018). Univariate analysis with all clinicopathological parametres with DFS revealed LN status as significant predictor (AUC=0.68). Similarly combined MIR score of MIR-425 and MIR-30d achieved 72% predictive accuracy of DFS (AUC=0.716). Interestingly, multivariate analysis of MIR score and LN with DFS, enhanced the AUC to 0.81. Conclusions: This proof-of-concept study suggests circulating MIR-30d and MIR-425 may serve as potential biomarkers for breast cancer recurrence risk stratification. The integrated signature shows promise for enhancing surveillance strategies. Assay also standardized methods for using plasma to measure circulating MIRNA levels. Validation in larger multicenter cohorts is underway for clinical assay development. Citation Format: A. Korlimarla, V. Sasimouli, L. Krishna, S. Shree, H. P.S, B. Sivaraman, S. Appachu, S. Srihari, G. Guhan, P. Advani, S. B.S. Circulating MIRNA Signature Predicts Breast Cancer Metastasis: A Pilot Study for Non-Invasive Risk Stratification [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 PS4-01-04.

Abstract Background: Currently, the gold standard in cancer diagnosis, prognosis, and treatment is assessing cancer cell mitosis in tumor tissue biopsies, which identifies patients with more aggressive cancer subtypes that may require more tailored treatment regimens. Circulating tumor cells (CTCs), cancer cells that detach from primary tumors, enter the bloodstream, and seed metastatic sites, are a well-known non-invasive blood biomarker which can also be used to aid in stratifying metastatic breast cancer (mBC) patients with highly aggressive subtypes. Initial pilot studies have identified a particular subtype of CTCs which are undergoing mitosis that appears to correlate with very poor survival outcomes compared to non-mitotic CTCs alone. In a multi-institutional prospective study, we isolated CTCs from the blood of n=167 mBC patients to categorize mitotic CTCs and evaluate their association with progression-free survival (PFS) and overall survival (OS) over a 3 year period. Methods: Peripheral blood samples (7.5ml) were collected from n=167 mBC patients progressing on systemic therapies and prior to new systemic therapy induction (i.e. chemotherapy n=102, hormone therapy n=13, immunotherapy n=63, targeted therapy n=62). Samples were processed using CellSieve microfilters to isolate CTCs via size exclusion and fluorescently stained for CD45, Cytokeratin, and DAPI. CTCs were imaged, enumerated, and subtyped based on the presence of ≥1 mitotic event, using previously established visual indicators. PFS and OS were assessed over 3 years by censored univariate and multivariate analyses. Results: CTCs were detected in ∼47% (n=79/167) of patients, while mitotic CTCs were identified in a subset of this group, ∼48% (n=38/79). The presence of any CTC was associated with poor outcomes (PFS HR=0.49, 95%CI=0.3-0.7, p=0.0002 and OS: HR=0.42, 95%CI=0.3-0.7, p=0.0002). However, the presence of mitotic CTCs correlated with significantly poorer outcomes compared to non-mitotic CTCs (PFS: HR=0.3, 95%CI=0.2-0.5, p<0.0001 and OS: HR=0.3, 95%CI=0.2-0.6, p=0.0002). Further, patients with no CTCs had a median PFS (mPFS) of 6 months and median OS (mOS) of 22 months, patients with non-mitotic CTCs had a mPFS of 4 months and mOS of 14.5 months; patients with mitotic CTCs had a mPFS of 2.4 months and mOS of 4.4 months. Notably, 95% of patients with a mitotic CTC progressed within 6 months, regardless of systemic therapy type. Interestingly, there were 5 patients who survived longer than 1 year with a mitotic CTC all of which were receiving specific targeted therapies. Conclusion: In this study, the detection of CTCs indicated poor outcomes in patients. Additional cytological analysis of CTCs further identified CTCs undergoing mitosis which correlated to highly aggressive disease with the poorest survival rates. These results indicate that while the presence of CTCs is a prognostic indicator for survival, subtyping CTCs and identifying mitosis may correlate to therapy response. This study highlights the need for larger, more comprehensive studies to expand upon and validate these findings. Citation Format: A. B. Duffy, M. Cristofanilli, C. Reduzzi, W. V. Williams, G. Del Priore, S. Chumsri, C. Tang, T. Iwase, N. T. Ueno, D. L. Adams. Mitotic Circulating Tumor Cells Correlates with Poor Clinical Outcomes in Metastatic Breast Cancer Patients over a 3 Year Period [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 PS4-01-05.

Abstract Background: Immune checkpoint inhibition (ICI) in combination with chemotherapy is associated with improved outcomes in a subset of patients with triple negative breast cancer (TNBC). However, biomarkers for predicting patient response, such as PD-L1 expression, have limited predictive efficiency. Voabil et al. has shown that ex vivo cytokine profiling of live tumor tissue resections treated with αPD-1 can predict patient response to ICI (Voabil et al., Nat Med. 2021). However, this method requires large amounts of tissue to address intra-specimen tumor heterogeneity, which is not feasible with standard of care (SOC) diagnostic core needle biopsies (CNBs) for breast cancer, where tissue is scarce. We show for the first time that ex vivo cytokine profiling is possible in live tumor fragments (LTFs) generated from CNBs in breast tissue. Methods: We present a cytokine profiling platform to assess response to ICI treatment in LTFs generated from CNBs. CNBs were collected from patients who were identified as potential candidates for SOC ICI (by treating physician) and enrolled in one of two prospective observational clinical trials (NCT05520099 and NCT06349642). CNBs were cut by an automated, proprietary cutting instrument to generate LTFs ofapproximately 300 µm in thickness. To address tissue heterogeneity and mitigate tissue scarcity in CNBs, we used a sequential treatment strategy in which control and ICI treatment were applied to LTFs in the same well, in a phased approach. This strategy provides a solution for the variability observed in cross-well comparisons. LTFs were encapsulated in hydrogel and treated with IgG control followed by αPD-1 (BioXCell). Cytokine production rates were measured longitudinally using multiplex assays assessing>45 cytokines, including 15 cytokines assayed by Voabil et al., and the fold changes of cytokine production rates in response to ICI over IgG control were calculated. Results: Using non-breast tumor tissue, we validated that our platform can measure changes in cytokine production in LTFs following sequential treatment, and that these measurements are comparable to those observed in a cross-well setting. In a small cohort of TNBC specimens, we show changes in cytokine production rates in response to ex vivo sequential control and ICI treatment. Ten cytokines, including IFN-γ and CXCL10, shown to be predictive of patient response to ICI in Voabil et al. were found upregulated in response to αPD-1 in TNBC specimens. Future determination of correlation with clinical response data (pathological complete response for early stage and RECIST 1.1 for metastatic patients) gathered from NCT05520099 and NCT06349642 will be used to validate platform responders and non-responders. To date, 11 patients with confirmed TNBC diagnoses have enrolled in these clinical studies. Conclusion: We report, for the first time to our knowledge, the ability to detect immune checkpoint inhibitor responses in live TNBC tissue obtained via CNBs. This proof-of-concept lays the groundwork for integrating functional tissue responses with clinical correlation data, with important implications for guiding current ICI therapies and advancing this platform for use in next-generation immuno-oncology treatments for TNBC. Citation Format: P. P. Advani, J. Harvey, J. Polley, M. Weidner, A. Arnold, D. Haley, R. Bollam, R. Rao, S. Chumsri, K. Hugghins, M. Acampora, C. Vivelo, H. Hernan, L. Hrycyniak, J. Zweng, H. Gierman, S. Bhattacharya, D. Mukhopadhyay. Ex vivo cytokine profiling of live triple negative breast cancer patient specimens from core needle biopsies illustrates proof of concept to assess tumor response to immune checkpoint inhibition [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 PS4-01-08.

Abstract Background: Antibody-drug conjugates (ADCs) are increasingly used in the treatment of breast cancer due to their superior efficacy compared to conventional chemotherapy. The management of male breast cancer is largely extrapolated from clinical data in females and evidence regarding the efficacy of ADCs in men remains limited. This study aimed to compare survival outcomes between male and female breast cancer patients treated with ADCs across the spectrum of human epidermal growth factor receptor 2 (HER2) expression. Methods: We included total of 2,954 (Male: 34, Female: 2,920 breast cancer samples) from patients who underwent treatment with ADCs. We excluded patients with triple-negative breast cancer status due to the rarity of this subtype in males and the worse prognosis of triple-negative breast cancer, so that the cohorts would be more homogeneous without overrepresentation of triple-negative breast cancer in females. We categorized patients by HER2 status as: HER2-null (if there was 0% stain on HER2 IHC), HER2-ultra low (if HER2 IHC was 0 but with stain 1-10%), HER2-low (if HER2 IHC was 1+ or 2+ with a negative chromogenic in situ hybridization [CISH] assay), and HER2-positive (if HER2 IHC was 3+ or 2+ with a positive CISH assay). Real-world median overall survival was derived from insurance claims and calculated from the start of first ADC with either trastuzumab deruxtecan (T-DXd) or trastuzumab emtansine (T-DM1) or sacituzumab govitecan (SG) to last contact using Kaplan-Meier. Statistical significance was assessed using chi-square and Mann-Whitney U. Results: The distribution of sex by HER2 expression group was: HER2-null: 8 males vs 612 females; HER2-ultra low 6 males vs 467 females; HER2-low 11 males vs 903 females; HER2-positive 9 males vs 938 females. Median survival from treatment initiation with either T-DXd or SG in the HER2-null cohort was 21.31 months in males vs 15.72 months in females (HR 0.82, 95% CI 0.33 - 2.01, p=0.67); in the HER2-ultra low cohort was 17.73 months in males vs 14.47 months in females (HR 0.84, 95% CI 0.26 - 2.63, p=0.76); in the HER2-low cohort was 11.08 months in males vs 18.62 months in females (HR 1.53, 95% CI 0.63 - 3.7; p=0.34). In the HER2-positive cohort, 5/9 males (55.5%) and 544/938 females (58%) were treated with T-DXd. Median survival from treatment initiation with either T-DXd or T-DM1 in the HER2-positive cohort was 36.55 months in males vs 46.52 months in females (HR 1.77, 95% CI 0.84 - 3.74, p=0.12). Conclusions: In this study, survival appears to be comparable between males and females with breast cancer across the spectrum of HER2 expression when both are treated with ADCs, although the survival in the HER2-positive cohort appears numerically lower in males, consistent with data on HER2-positive MaBC. An important limitation is the small sample size of the male breast cancer cohorts, and the lack of a categorization of HER2 in HR+ vs HR-, for the small MaBC sample size. However, to our knowledge this is the first analysis evaluating outcomes of men treated with ADCs across the spectrum of HER2 expression. Citation Format: D. Trapani, S. Deshmukh, S. Wu, J. Xiu, N. Lin, G. Curigliano, P. Spanheimer, S. Gandhi, S. Chumsri, S. Graff, M. Lustberg, G. Sledge Jr, S. Tolaney, J. Leone. Comparative efficacy of antibody drug conjugates (ADCs) in male versus female breast cancer across the spectrum of HER2 expression [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 PS4-01-13.

Abstract Background: Microtubule inhibitors remain a standard chemotherapy in the management of HER2-negative metastatic breast cancer (MBC). CALGB (Alliance) 40502 was a phase 3 randomized study involving 799 patients with MBC receiving first-line chemotherapy, to determine the optimal chemotherapeutic agent among paclitaxel, nab-paclitaxel, or ixabepilone (with or without bevacizumab). Secondary analysis suggested inferior PFS with nab-paclitaxel specifically among patients with hormone receptor positive (HR+)/HER2-negative breast cancer. Correlative analysis demonstrated significant association of stromal tumor infiltrating lymphocytes (sTILs) with improved progression-free (PFS) and overall survival (OS) in CALGB40502. We hypothesized that immune activation would be associated with differential benefit among distinct microtubule agents. To address this, we evaluated the sTILs and RNA-based immune features association with clinical outcomes, including evaluation of nab-paclitaxel versus paclitaxel. Methods: To limit heterogeneity, analyses focused on pre-treatment primary tumor breast samples with central review/pathologist-enumerated sTILs in accordance with International TILs Working Group methods and RNAseq (n = 280). PAM50 molecular subtypes were assigned based on transcriptomic features. RNAseq data were used to generate immune deconvolution estimates from 5 distinct algorithms (CIBERSORT, xCell, ABIS, ConsensusTME, ImmuCellAI). 1,018 curated breast cancer gene expression signatures GES) were derived from previously published studies. Associations between sTILs and GES were evaluated with sTILs being a categorical variable with the thresholds <5% (low) and (≥5%) high. Results: In the evaluable population, basal-like PAM50 subtype was enriched for high sTILs category (p=1e-4), as anticipated. T-cell and B-cell immune signatures and immune deconvolution estimates were among most highly associated with sTILs. In addition to the expected prognostic association of high T-cell GES, high B-cell-related IgG signature score was also significantly associated with improved overall survival (OS) among triple-negative breast cancer (TNBC) in CALGB 40502 (HR = 0.50,95% CI:0.30-0.86, log-rank p=0.01) but not in HR+/HER2-. In an exploratory analysis of young patients (age at study entry <50 years), high sTILs were associated with improved OS among TNBC patients but worse OS among those with HR+/HER2- MBC (HR = 2.19, 95% CI:1.10-4.36, log-rank p=0.022). High (above median) RNA-based Xcell total immune score was significantly associated with worse OS in both CALGB 40502 and a validation dataset of primary breast cancers, METABRIC (OS HR = 2.12, 95% CI:1.01-4.45, log-rank p=0.042). To further investigate the clinical trial finding of inferior PFS for nab-paclitaxel compared with paclitaxel in patients with HR+/HER2- MBC, there was a significant enrichment of T-cell signatures associated with poor outcome in patients receiving nab-paclitaxel (Fisher exact p<0.001). Conclusion: In this translational analysis of sTILs and RNAseq from a large phase III clinical trial, sTILs and immune signatures were not uniformly associated with better outcome. Rather, high sTILs or immune signatures were associated with worse OS in young patients with HR+/HER2- breast cancer and higher T-cell signatures were associated with inferior PFS with nab-paclitaxel among HR+/HER2- MBC patients. These data reinforce the importance of context/subtype-specific interrogation of the tumor-immune microenvironment Support: U10CA180821, U10CA180882; https://acknowledgments.alliancefound.org. Bristol Myers Squibb (BMS); Clinicaltrials.gov Identifier: NCT00785291 Citation Format: E. K. Blige, K. V. Ballman, A. Michmerhuizen, M. Vater, L. A. Carey, A. H. Partridge, W. F. Symmans, M. A. Watson, C. M. Perou, D. G. Stover, H. S. Rugo. Rna-based immune features associated with benefit from distinct microtubule inhibitor therapy for metastatic her2-negative breast cancer: a post hoc analysis of the calgb 40502 (alliance) phase iii randomized clinical 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 PS1-13-17.

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 Background: Estrogen receptor (ER) positive breast cancer is commonly treated with endocrine therapy, yet resistance to estrogen deprivation remains a significant clinical challenge. Using custom FISH probes designed to detect tumor-specific genomic alterations, we identified circulating tumor cells (CTCs) of true tumor origin in early-stage luminal breast cancer patients. Longitudinal analysis revealed that 80% of patients had FISH-positive CTCs after completing therapy and while on antiestrogen treatment, despite no clinical evidence of disease. This finding suggests that standard endocrine therapy is insufficient to eradicate all malignant clones, indicating the persistence of therapy-resistant tumor cells. Methods: Primary tumor DNA was analyzed using the Oncoscan microarray platform to identify somatic copy-number alterations (SCNAs). Custom FISH probes targeting these alterations were applied to peripheral blood samples before and after therapy. Immunofluorescence characterization of CTCs was performed to assess epithelial and mesenchymal phenotypes as well as ER expression status. Genomic profiling focused on identifying drivers of proliferation, known ER resistance genes, and ER transcriptional regulators. Results: Immunofluorescence revealed that CTCs were highly heterogeneous, displaying both epithelial and mesenchymal phenotypes. ER expression among CTCs was variable: while some CTCs were ER-positive, a significant proportion showed reduced or absent ER staining. This heterogeneity suggests that loss or downregulation of ER is a key mechanism of resistance, allowing tumor cells to persist despite antiestrogen therapy. Genomic profiling of parent tumors demonstrated multiple, overlapping mechanisms supporting both proliferation and endocrine resistance. All tumors harbored several genomic alterations known to drive proliferation, most commonly involving MCL1, NTRK1, SDHC, SDHD, CHEK1, ATM, and BCL6. Every tumor exhibited at least one established mechanism of ER resistance, with frequent alterations in ATM and PIK3CA, and additional recurrent changes in AKT3, MTOR, and ARID1A. Several tumors also had alterations in genes directly involved in ER transcriptional regulation, notably ARID1A and SPEN. Conclusions: The persistence of FISH-positive CTCs in the majority of patients after therapy—despite no clinical evidence of disease—highlights the insufficiency of current endocrine regimens. The underlying genomic complexity, characterized by multiple drivers of proliferation, redundant ER resistance mechanisms, and disruption of ER regulatory pathways, underpins the survival and persistence of CTCs. These findings underscore the need for therapeutic strategies that address the multifaceted and overlapping nature of endocrine resistance in luminal breast cancer. Citation Format: K. Baldwin, K. von Maltzan, A. Zulli, M. Richard, J. Abdulsattar, D. Maroni, P. T. Greipp, R. Knudson, Mayo Cytogenetics Core, S. P. Thayer. Fish-positive circulating tumor cells persist after effective therapy in estrogen receptor positive breast cancer: insights into genomic mechanisms of endocrine resistance [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 PS2-11-03.

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.