Abstract Purpose: Multiple antibody drug conjugates (ADCs) are approved for the treatment of human cancers, yet there remains significant potential for improvements through iterative learnings from nonclinical and clinical experience. ZW191 is a clinical-stage ADC bearing a novel topoisomerase I inhibitor payload and targeted to the clinically-validated tumor antigen folate receptor alpha (FRα). Experimental Design: The nonclinical activity and tolerability profiles of ZW191 were assessed in vitro and in vivo, focusing on properties of its antibody and payload components. Results: ZW191 has a highly differentiated efficacy and tolerability profile, markedly improving on the FDA-approved FRα-targeted ADC mirvetuximab soravtansine in its activity in tumors with both high and low levels of FRα in patient-derived xenograft models of ovarian cancer, endometrial cancer, NSCLC, and TNBC. ZW191’s novel FRα-targeting antibody demonstrates unique binding properties and is superior to multiple other clinical-stage ADC antibodies in its internalization, payload delivery, and penetration through 3D tumor spheroids. ZW191’s novel payload drives strong anti-tumor activity in vitro, including bystander activity. Further, ZW191 is effective in vivo in combination with clinically relevant standard of care drugs olaparib, bevacizumab, and paclitaxel. ZW191’s best-in-class tolerability profile in GLP NHP toxicity studies is owing to its payload’s moderate potency – an ADC feature that we show from a clinical landscape analysis allows for higher dosing in humans. Conclusions: ZW191 demonstrates favourable preclinical efficacy and tolerability. This differentiated profile is supported by initial clinical data, potentially positioning ZW191 to meaningfully improve responses and substantially widen the targetable patient population over standard of care.

Abstract Background: Circulating tumor DNA (ctDNA) is a clinically validated biomarker in breast cancer for predicting the risk of recurrence, monitoring response to neoadjuvant treatment, and detecting molecular relapse after adjuvant therapy. Signatera™ is a tumor-informed, multiplex PCR-NGS ctDNA assay that may be developed from a backbone of whole-exome sequencing (WES) or whole-genome sequencing (WGS). Early clinical validation of Signatera Genome in a pan-cancer cohort demonstrated improved performance in certain clinical scenarios. In this study, we specifically assessed the performance of the Signatera Genome assay in the surveillance setting for breast cancer. Methods: A retrospective analysis was performed using clinically annotated residual patient samples from commercial testing with WES-based Signatera. Treatment decisions and cadence of ctDNA testing were at the provider’s discretion. Signatera Genome assays were designed, consisting of 64 high-quality tumor-specific variants, from matched tumor and normal whole genome sequencing data. These assays were then used to detect ctDNA in the corresponding patients’ plasma. ctDNA levels were quantified as mean tumor molecules per mL of plasma (MTM/mL). Longitudinal blood samples represented time points after surgery/definitive treatment until recurrence or the end of follow-up. The correlation between ctDNA status and distant recurrence-free survival (DRFS) was assessed using Cox regression analysis. Results: Clinical performance of the Signatera Genome assay was assessed in a real-world cohort of 228 patients with breast cancer [triple-negative breast cancer (TNBC), n=130; HR+/HER2-, n=85; and HER2+, n=13]. In a landmark analysis, patients with ctDNA-positivity within 3 months of surgery were significantly at a higher risk of distant disease and/or molecular recurrence (HR: 8.1, 95% CI: 3.5-8.6, P <0.001). With longitudinal testing, ctDNA-positivity at any time post-definitive treatment was associated with significantly worse DRFS (HR: 22.2, 95% CI: 11.0-44.7, P <0.0001). ctDNA detection in the surveillance setting preceded clinical recurrence as confirmed by imaging with a sensitivity and specificity of 100%. Upon analyzing ctDNA dynamics, patients with unfavorable ctDNA dynamics (persistently/converted to positive) were observed to have significantly shorter DRFS than those with favorable ctDNA dynamics (serially/converted to negative) (HR with Firth’s penalized likelihood hazard model: 97.8, 95% CI: 11.5-12768.9, P <0.001). Conclusions: These data indicate the robust clinical performance of the Signatera Genome in breast cancer for detecting recurrence after surgery or definitive treatment. Prospective clinical trials are warranted to establish the clinical utility of the assay for guiding treatment decisions for patients with molecular non-radiographic recurrence. Citation Format: W. McHayleh, C. Scalise, E. Kalashnikova, B. Sridhar, J. Feeney, W. Tan, A. Hsieh, A. Butskova, J. Ortega, S. Velichko, C. Palsuledesai, H. Sethi, G. Heilek, J. McKenzie, A. Rodriguez, M. Liu, M. George. Clinical performance of Signatera Genome assay for predicting recurrence in patients with 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 PS2-07-26.

Abstract Introduction: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) combined with endocrine therapy are the standard of care for hormone receptor-positive, human epidermal growth factor receptor 2-negative (HR+/HER2-) metastatic breast cancer (MBC). Although most patients eventually develop resistance, a subset achieves durable responses lasting at least 24 months with a single CDK4/6i. Identifying prognostic factors associated with this long-term benefit is essential for guiding clinical practice. Methods: We conducted a retrospective cross-sectional study of HR+/HER2- MBC patients treated with CDK4/6i plus endocrine therapy at Ho Chi Minh City Oncology Hospital between January 2021 and July 2023. Eligible patients were those who received continuous treatment with one CDK4/6i for ≥24 months. Progression-free survival (PFS) was estimated using Kaplan-Meier methods, and prognostic factors were evaluated by univariate and multivariate Cox proportional hazards regression models. Results: Of 236 patients treated with CDK4/6i, 80 met inclusion criteria by maintaining therapy with a single CDK4/6i for ≥24 months (30 with palbociclib [36.1%], 50 with ribociclib [63.9%]). Median follow-up was 31.4 months (range, 24.08-46.16). The median PFS for the cohort was not reached. Subgroup analysis demonstrated that patients achieving partial or complete response (PR/CR) did not reach median PFS, whereas those with stable disease (SD) had a median PFS of 37.65 months (95% CI, 34.11-41.19; p = 0.024). In multivariate analysis, both treatment response (PR/CR vs SD) and Ki-67 >30% were significantly associated with PFS. Conclusion: In HR+/HER2- MBC patients achieving long-term response (≥24 months) to a single CDK4/6i plus endocrine therapy, treatment response and high Ki-67 index (>30%) emerged as significant prognostic factors for PFS. These findings highlight the prognostic value of clinical response and proliferative activity in predicting durable benefit. Citation Format: T. N. Nguyen, D. T. H. Phan, T. B. Diep, H. D. Vo, P. H. M. Vo, Q. H. Nguyen, T. T. T. Nguyen, H. N. Vo. Analysis of Prognostic Factors in HR+/HER2- Metastatic Breast Cancer with Long-Term Response to CDK4/6 Inhibitors [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 PS1-09-01.

Abstract Background: Clinical trials are essential to improving cancer outcomes, yet racial and ethnic minorities remain underrepresented. Structural and social barriers such as limited access to care, geographic distance, financial burden, lack of information, and cultural mistrust contribute to this persistent underrepresentation. Despite longstanding initiatives to improve inclusivity, recent data show most clinical trial participants continue to be White (W). An understanding of the factors that influence trial enrollment, including both sociodemographic and geographic determinants, is essential to designing more equitable and representative research. In this study, we used the National Cancer Database (NCDB) to evaluate disparities in clinical trial enrollment among patients (pts) with breast cancer (BC), with a particular focus on the impact of race, income, education, and geographic access. Method: We conducted a retrospective cohort study using data from the NCDB including pts with complete data on race/ethnicity, clinical trial enrollment status, clinical stage, sociodemographic variables, and survival time. Race/ethnicity in the NCDB may be based on self-report, administrative data, or observation. Education and income quartiles were assigned based on ZIP code-level U.S. Census data, per NCDB methodology. Descriptive statistics were used to compare enrollment rates across racial and ethnic groups. Multivariable logistic regression was performed to estimate adjusted odds ratios (AORs) for clinical trial enrollment, with race/ethnicity as the primary predictor and adjustment for age, stage, grade, Charlson-Deyo comorbidity index, income, education level, facility type, and geographic location. A p-value of < 0.05 was considered statistically significant. All analyses were conducted using SAS software version 9.4. Results: A total of 1,798,690 BC pts were included in the analysis. The median age was 62 years. The cohort was 79.7% W, 10.9% Black, 4.9% Hispanic, and 4.6% other races/ethnicities. The majority of pts resided in metro areas (84.7%), followed by urban (13.3%) and rural (2.0%). Only 1,645 (<1%) were documented as enrolled in clinical trials. In multivariable logistic regression, rural residence was independently associated with lower clinical trial enrollment (AOR= 0.381; 95% CI: 0.24-0.60; p< 0.0001) as was travel distance > 50 miles to the treating facility (AOR= 0.44-0.52; p< 0.0001). Treatment at non-academic center was associated with a higher enrollment compared to academic centers (AOR 1.438, 95% CI 1.15-1.80; p<0.0012). After adjustment for clinical and sociodemographic variables, race/ethnicity, income and education level were not significant predictors of clinical trial participation. Conclusion: In this large national cohort of BC pts, clinical trial enrollment was strikingly low, with fewer than 1% of pts documented as participants. Rural residence and long travel distances to treatment facilities were significantly associated with lower enrollment, underscoring the critical role of geographic and structural barriers in accessing research opportunities. Unexpectedly, patients treated at non-academic centers had higher odds of trial enrollment, a finding that may reflect variation in reporting practices rather than true differences in access. Race, income, and education were not independent predictors after adjustment, suggesting access may be the primary driver of disparities. A key limitation is the NCDB’s underreporting of trial participation. The very low number of patients identified as enrolled highlights the urgent need for improved and standardized reporting mechanisms. Enhanced data accuracy is essential to guide equity-focused interventions and ensure representative participation in cancer research. Citation Format: A. Sandoval-Leon, Y. Chamorro, M. Roy, M. Rubens, N. Dempsey, L. Carcas, M. Ahluwalia, L. Dumeny, R. Mahtani. Geographic Barriers are Key Predictors of Clinical Trial Enrollment in Breast Cancer: A National Cancer Database Analysis [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 PS4-09-10.

Abstract R-loops, transcription-induced three-stranded nucleic acid structures, play essential roles in gene regulation but jeopardize genomic integrity when unresolved. BRCA2, a key tumor suppressor required for homologous recombination (HR) repair of DNA double-strand breaks (DSBs), also maintains R-loop homeostasis, though its mechanisms remain incompletely understood. To identify BRCA2-interacting factors involved in R-loop resolution during DNA repair, we employed biotin-based proximity labeling and mass spectrometry (BioID-MS), uncovering HELZ—a previously uncharacterized RNA-specific helicase—as a direct BRCA2 binding partner. BRCA2 enhances HELZ’s catalytic activity in vitro and promotes its recruitment to transcription-associated R-loops in vivo, particularly at DSB sites. Functionally, HELZ resolves co-transcriptional R-loops, facilitates DNA end resection, and promotes efficient HR. HELZ depletion results in R-loop accumulation, impaired HR capacity, and heightened sensitivity to genotoxic stress. Notably, HELZ activity is context-dependent and proves indispensable in hormone-driven malignancies such as estrogen receptor-positive (ER+) breast cancers, which exhibit transcriptional dysregulation and elevated R-loop burden. These findings establish HELZ as a critical mediator of BRCA2-dependent genome integrity in R-loop-rich chromatin environments. HELZ expression or function may serve as a biomarker for therapeutic response, and HELZ helicase activity offers a promising target to restore HR proficiency in transcriptionally dysregulated breast tumors. By integrating proximity proteomics with functional assays, our study identifies HELZ as a novel and therapeutically actionable regulator of R-loop resolution and BRCA2-mediated repair in breast cancer. Citation Format: W. Li, B. Wu, B. Gao, E. M. Irvin:, A. Arijit Ghosh, L. Eliaz, Y. Huang, Y. Kwon, C. M. Stiefel, T. T. Nguyen, D. Zhao, H. J. Suarez, T. Ni, S. Alejo, O. Fitzgerald, X. Song, E. V. Wasmuth, S. Zheng, J. Leung, X. Xue, H. Wang, J. Ji, L. Lan, W. Zhao. Helz-mediated r-loop resolution orchestrates brca2-dependent dna repair in hormone-driven breast cancers [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 PS2-12-29.

Abstract Introduction: The I-SPY2 neoadjuvant platform trial (NCT01042379) evaluates new therapies in high molecular risk, stage 2-3 breast cancer patients. Between 2010 and 2022, 24 therapies were tested in 2,117 patients. One-third of these patients had HER2-negative tumors with low expression of immune genes and no DNA repair deficiency (DRD) (HER2-/Immune-/DRD- subtype). These patients had very low pathologic complete response (pCR) rates (∼10% on average) across all treatment and control arms. We performed a high-throughput drug screen (n=386 compounds) in triple-negative (TN), immune-low, DRD-low breast cancer patient-derived organoids with confirmed in vitro resistance to platinum chemotherapy to identify effective treatments for this tumor type. HSP90 inhibitors, including ganetespib, were one of the most potent drug classes in the screen. HSP90 is an abundant chaperone protein involved in the folding of oncoproteins under basal conditions and during stress response. Neoadjuvant ganetespib was tested in I-SPY2 in combination with standard chemotherapy in HER2-negative breast cancer but did not meaningfully improve pCR rates (PMID 36456573). Here, we re-evaluate the ganetespib arm by Response Predictive Subtypes with 5-year median follow-up data, focused on patients with breast tumors of the HER2-/Immune-/DRD- subtype. Methods: HER2-/Immune-/DRD- patients (N=221; 79% HR+, 21% TN) and HER2-/Immune+ and/or DRD+ patients (N=219; 32% HR+, 68% TN) were randomized to neoadjuvant ganetespib (150 mg/m2 ganetespib every 3 weeks with paclitaxel, then doxorubicin plus cyclophosphamide (AC)) (n=38 HER2-/Immune-/DRD- and n=55 HER2-/Immune+ and/or DRD+), or standard neoadjuvant chemotherapy control (paclitaxel-AC) (n=183 HER2-/Immune-/DRD- and n=164 HER2-/Immune+ and/or DRD+). The primary endpoint, pCR, was defined as the absence of invasive disease in the breast and regional nodes at surgery. Distant relapse-free survival (DRFS) was assessed using Kaplan-Meier curves, with hazard ratios (HR) estimated by Cox regression. Residual cancer burden (RCB) and serial MRI-based functional tumor volume (FTV) were collected as investigational early measures of treatment efficacy in the neoadjuvant setting. Results: In HER2-/Immune-/DRD- patients, pCR rates were low in the ganetespib and control arms (13% vs. 9%), compared with 38% vs. 31%, respectively, in HER2-/Immune+ and/or DRD+ patients. In both subtypes, patients who achieved a pCR with either treatment had excellent DRFS (100% at 5 years). Interestingly, in HER2-/Immune-/DRD- patients treated with ganetespib with no pCR (n=32), we observed only 1 DRFS event, compared with 35 DRFS events in HER2-/Immune-/DRD- control-treated patients with no pCR (n=166); HR 0.12 (95% CI 0.02-0.90; p-value: 0.039). The respective DRFS at 5 years was 96% with ganetespib vs. 77% with control, showing a significantly improved DRFS with neoadjuvant ganetespib for HER2-/Immune-/DRD- patients, despite their non-pCR. This treatment effect remained significant when adjusted for hormone receptor status. Among non-pCR patients, RCB measure and FTV decrease explained a portion of ganetespib’s significant DRFS benefit. In contrast, ganetespib did not improve DRFS in HER2-/Immune+ and/or DRD+ patients compared with control. Conclusion: Our data suggest a promising role for ganetespib in improving long-term outcomes for patients with HER2-/Immune-/DRD- tumors, which account for 38% of all molecularly high-risk breast cancers, even in the absence of a pathologic complete response. Continuous measures of RCB and FTV may serve as early predictors of ganetespib’s long-term survival benefit in HER2-/Immune/-DRD- patients. Citation Format: T. B. V. Bui, D. M. Wolf, C. Yau, R. W. Sayaman, K. Chow, S. W. Choi, M. C. Bruck, K. Santos-Parker, A. M. Glas, J. E. Lang, A. Forero-Torres, L. Brown-Swigart, G. L. Hirst, E. F. Petricoin, J. Perlmutter, W. F. Symmans, P. R. Pohlmann, D. Yee, L. Pusztai, I-SPY investigators, N. M. Hylton, A. M. DeMichele, J. M. Rosenbluth, L. J. Esserman, L. J. Van 'T Veer. Neoadjuvant HSP90 inhibition with ganetespib significantly improves distant relapse-free survival in I-SPY2 patients with HER2-negative breast cancer with low expression of immune genes and no DNA repair deficiency [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 PS3-11-10.

Abstract Background: Predicting the anatomical patterns of relapse after an initial diagnosis of early-stage breast cancer (BC) and understanding their association with circulating tumor DNA (ctDNA)-based molecular residual disease (MRD) detection by tumor subtype and treatment history may inform surveillance strategies and enable earlier therapeutic intervention. Methods: This retrospective study explored the association between sites of relapse and ctDNA detection rates and dynamics in a real-world cohort (2019-2024) of patients (pts) with early-stage BC. We utilized Natera’s proprietary real-world database linked to Komodo's Healthcare Map® claims database and results of tumor-informed ctDNA testing (SignateraTM, Natera, Inc.). Hormone receptor (HR), HER2 status, and the date of relapse were inferred from the claims codes associated with interventions and secondary neoplasms (i.e., metastases). Pts with a diagnosis code of secondary neoplasms were categorized by the anatomic site(s) of recurrence. ctDNA detection rate (required time points: ctDNA positivity within 6 months prior to relapse; for ctDNA-negative pts, three or more time points were included), and dynamics were analyzed in relation to subtype and relapse site. Results: Evaluation of Natera's real-world database identified 532 pts with early-stage breast cancer who developed subsequent distant metastatic disease. The distribution of patients by subtype was: HR+/HER2-: 52% (276/532), triple-negative (TNBC): 34% (182/532), and HER2+: 14% (74/532). Among pts with isolated relapses (N=259), the most common sites were bone ([HR+HER2-: 59%; 82/138], [HER2+: 36%; 14/39] [TNBC: 30%; 25/82]) and lung ([HR+HER2-:16%; 22/138, [HER2+: 31%; 12/39], [TNBC: 34%; 28/82]), concordant with common sites of metastasis by subtype. Relapses occurred within a median of 1.84 years (yrs) after surgery, varying by subtype (TNBC: 1.33 yrs, HER2+: 1.92 yrs, HR+HER2-: 2.41 yrs). Among pts with required timepoints (77%, 411/532), ctDNA was detectable prior to/at the time of recurrence in 91% (373/411) of cases; 90% (130/145) for TNBC, 90% (187/208) for HR+HER2- and, 97% (56/58) for HER2+ disease. For the subset of patients with an isolated relapse, the rate of ctDNA positivity at the time of recurrence was the highest for liver (100%; 23/23), followed by bone (93%; 95/102) metastases. In terms of ctDNA quantification, median ctDNA levels differed by subtypes, with TNBC showing the highest levels (16.4 MTM/mL, range: 0.03-17053) compared to HR+/HER2- (5.58 MTM/mL, range: 0.018-8343) and HER2+ (5.44 MTM/mL, range: 0.03-2879). ctDNA levels among secondary neoplasms prior to/at the time of recurrence were highest for multi-site relapses (median = 15 MTM/mL, IQR:1.5-143), followed by lung (median: 19.9, IQR: 1.5 - 82), bone (median: 5.6, IQR: 0.9-29), and liver (3.4, IQR: 0.52-76.8) only metastases. Serial ctDNA measurements preceding the date of secondary neoplasms showed rising ctDNA levels over time in >90% of cases, with the most rapid increase observed in TNBC. The cases that exhibited declining ctDNA levels without achieving clearance were predominantly HR+HER2- pts with bone metastases. Conclusions: Our findings highlight the potential utility of serial ctDNA monitoring in clinical practice to identify pts at high risk of relapse across multiple organ sites, including isolated progression in individual organs. These findings could potentially inform future ctDNA-guided intervention strategies, including increased surveillance by imaging for pts with ctDNA positivity. Citation Format: D. Stover, B. Dwivedi, P. Dutta, S. Beyer, H. LeFebvre, E. Kalashnikova, J. McKenzie, A. Rodriguez, M. Liu. Circulating Tumor DNA Dynamics and Anatomical Patterns of Relapse Following Curative Therapy in Early-Stage 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 PS2-09-20.

Abstract Background: Recent studies have demonstrated the utility of longitudinal circulating tumor DNA (ctDNA) testing for monitoring treatment response and predicting long-term outcomes in patients (pts) with breast cancer (BC). In this study, we evaluated ctDNA detection rates post-surgery in pts with HER2+ BC in the adjuvant treatment and surveillance settings. Methods: This retrospective study included 59 patients with stage I-IV HER2+ BC. Commercial CTDNA testing was performed postoperatively at the physician's discretion. A clinically validated, tumor-informed ctDNA assay (SignateraTM, Natera, Inc.) was used to detect and quantify ctDNA in blood samples collected after primary resection and serially during surveillance. ctDNA status and dynamics were correlated with clinicopathological features and surgical outcomes. Results: The cohort included 59 patients with HER2+ BC; the majority of pts had clinical T1-3 stage disease at diagnosis (T1, 25%, 15/59; T2, 20%, 12/59; and T3, 24%, 14/59), the remaining pts were T4 (7%; 4/59) or had unknown stage (24%; 14/59). Lymph node involvement was reported in 46% of pts (27/59; stage N1, 22/59; N2, 3/59; N3, 2/59); 47% had grade 3 (28/59) and 46% had grade 2 (27/59) disease. Sixty-four percent of cases (38/59) were estrogen receptor (ER)-positive; 36% (21/59) were ER-negative. Neoadjuvant treatment was received by 71% (42/59) of pts, this information was unavailable for 4% (2/59); the rest of the pts (25%; 15/59) did not receive neoadjuvant treatment. Among pts receiving neoadjuvant therapy (n=42), residual cancer burden (RCB) class was available for 27 (RCB0: 22%, 6/27; RCB1: 19%, 5/27; RCB2: 40%, 11/27; RCB3: 19%, 5/27). Among pts with longitudinal ctDNA results and complete clinical information available (n=42), ctDNA-positivity any time after surgery was observed in 14% (6/42) with a median time to ctDNA-positivity of 34 months (range: 14-792) from surgery. Of these six ctDNA-positive patients, one experienced recurrence 13 months after the first positive ctDNA test, one experienced ctDNA clearance after adjuvant therapy and remained event-free for 4.6 mos, and four remained recurrence-free with a median follow-up since ctDNA-positivity of 6.7 months and ongoing monitoring. Among patients with a favorable response to neoadjuvant treatment (RCB 0/1; n=11), no ctDNA-positivity was observed after surgery. Among pts with residual disease at surgery (RCB 2/3; n=10), ctDNA-positivity was observed in 20% (2/10). Among pts with persistent ctDNA-negativity during surveillance (n=30, median follow-up of 15.4 months, range: 5.9-50.0 months), no clinical recurrences were reported. Conclusions: In addition to clinicopathological features and surgical outcomes, longitudinal ctDNA monitoring helps identify pts with HER2+ BC who may be at high risk of recurrence. Further research is needed to assess the true utility of a ctDNA-guided treatment approach in this patient population. Citation Format: Y. Gao, M. Zaidi, A. Naqvi, F. Azim, S. Satta, C. Brewer, C. Palsuledesai, E. Kalashnikova, K. Sun, P. Niravath, H. Mai, J. McKenzie, A. Rodriguez, M. Liu, A. Puri. Single Institution experience of longitudinal post-surgical circulating tumor DNA monitoring in patients with HER2+ 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 PS4-02-25.

Abstract Background: Triple-negative breast cancer (TNBC) is a highly heterogeneous and aggressive subtype of breast cancer, with the features of tumor microenvironment and the underlying molecular mechanisms of immune-regulation remaining largely elusive. This study aimed to explore the immune landscape of TNBC by assessing tumor-infiltrating lymphocytes (TILs) level, programmed cell death ligand 1 (PD-L1) expression, and the degree of TIL infiltration within the tumor. Methods: Utilizing the GeoMX Digital Spatial Profiler platform, we also performed spatially resolved transcriptomic profiling of tumor, immune, and stromal cells, focusing on the distinctions between immune-activated and immune-suppressed phenotypes. Results: Immune-activated phenotypes, characterized by high TILs, PD-L1 positivity, and close proximity between tumor cells and TILs, were found to correlate with the basal-like immune-activated subtype based on transcriptomic alterations derived solely from tumor cells. These phenotypes were also associated with the upregulation of known immune-related pathways. In contrast, immune-suppressed phenotypes were linked to pathways involved in tumor progression, such as epithelial-mesenchymal transition, TGF-beta signaling, and angiogenesis. Notably, our transcriptomic analysis suggests that tumor cells play a more dominant role than immune cells in shaping the immune phenotype, as indicated by the greater number of differentially expressed genes in tumor cells rather than immune cells. Stratifying patients based on TILs and PD-L1 status revealed that the TIL+PD-L1+ subtype exhibited the most favorable prognosis, a finding further supported by TIL+PD-L1+ signature with excellent survival outcomes in public gene datasets. Conversely, the TIL-PD-L1- subtype displayed characteristics of an "cold" tumor, potentially driven by desmoplastic changes within the tumor microenvironment. Interestingly, the TIL+PD-L1- subtype, despite a high TILs level, was associated with poorer prognosis, likely due to a higher proportion of myeloid cells, decreased activity of immune cells, and increased activation of the adipogenesis pathway across cell types. This finding underscores that in the TIL+PD-L1- subtype, immune cell activity remains low even when TILs infiltrate the intra-tumoral region, highlighting PD-L1 as a critical marker reflecting the immune system status in patients with high TILs. Conclusion: In summary, this study emphasizes the clinical significance of integrating TILs and PD-L1 expression as prognostic biomarkers in TNBC and underscores the central role of tumor cells in dictating immune phenotypes. Furthermore, our findings suggest potential underlying mechanisms and target genes regarding the immune system regulation in TNBC. Based on these insights, future research is warranted to develop targeted therapies, especially for immune-suppressed subtypes, aiming to improve clinical outcomes for patients with TNBC. Citation Format: Y. Cha, S. Bae, J. Kim, K. Kim, Y. Kook, A. Kim, S. Ahn, J. Jeon. Deciphering the tumor microenvironment related to immune regulation in triple-negative 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 PS2-11-29.