Chumsri, S.

Abstract Background: The Breast Cancer Index (BCI) is clinically validated to stratify patients with hormone receptor-positive (HR+), early-stage BC based on the risk of overall (0-10 years [y]) and late (5-10 y) distant recurrence (DR) with N0/N1 lymph node involvement. Validation of BCI in patients with HR+ HER2+ BC has been limited, especially in patients treated with adjuvant trastuzumab-based chemotherapy. The HOXB13 and IL17BR genes constitute the predictive component of BCI and are known to exhibit an ER-dependent correlation pattern with HER2. We evaluated the prognostic performance of BCI in patients with HR+ HER2+ disease enrolled in the NCCTG N9831 (Alliance) trial treated with the combination of chemotherapy and trastuzumab. Methods: All available tumor specimens from patients with HR+ HER2+ N0/N1 BC in arms B (sequential trastuzumab) and C (concurrent trastuzumab) of the trial were included. BCI testing was performed blinded to clinical outcomes. The primary endpoint was distant recurrence-free interval (DRFI). BCI (N0) and BCIN+ (N1) risk groups were calculated based on prespecified cut points. Kaplan-Meier analysis and log-rank testing were used to assess the prognostic significance of BCI/BCIN+ risk groups based on DR. Cox proportional hazard models with and without clinical covariates were used to estimate hazard ratios (HRs), and the associated 95% confidence intervals (CIs). Results: A total of 454 HR+ patients (234 arm B, 220 arm C), including 69 N0 and 385 N1 patients, were analyzed (median age was 47 y, 85% N1, 55% T2, 63% G3). BCIN+ scores were available for 378 of the 385 N1 patients due to missing tumor grade for 7 patients. The median follow-up was 12.7 years. In the overall cohort, BCI/BCIN+ stratified 51 (11%), 24 (5%) and 372 (83%) patients as low-, intermediate- and high-risk with 10-y DRFI rates of 96% (95% CI: 84-99%), 100% (100-100%) and 89% (86-92%), respectively. Since the low and intermediate risk groups had few patients and a similar risk of 10-y DRFI, they are combined into a single low-risk group hereafter. Thus, for overall 10-y DR, 75 (17%) and 372 (83%) of patients were classified by BCI/BCIN+ as low and high risk with 10-y DRFI rates of 97% (95% CI: 89-99%) and 89% (95% CI: 86-92%), respectively (HR=4.50, 95% CI: 1.09-18.61; p = 0.038). BCI/BCIN+ remained statistically significant in the multivariable analysis, adjusting for age, treatment, and nodal status (HR=7.54, 95% CI: 1.34-42.64; p = 0.022). For 69 N0 patients, BCI stratified 54 (78%) and 15 (22%) patients as low and high risk with 10-y DRFI rates of 100% (95% CI, 100-100%) and 77% (95% CI, 44-92%), respectively (p = 0.012). For 378 N1 patients, BCIN+ stratified 21 (6%) and 357 (94%) of patients as low and high risk with 10-y DRFI of 90% (95% CI, 66-97%) and 90% (95% CI, 86-93%), respectively (p = 0.07). Of the 21 patients classified as BCIN+ low-risk, two had BCIN+ scores of 6.75 and 6.73 in close proximity to the cut point (6.93) and experienced DR after 5 years, resulting in the same 10-y DRFI for both BCIN+ low- and high-risk patients. Conclusions: BCI is significantly prognostic for the risk of overall DR (0-10 y) in patients with HR+ HER2+ disease treated with adjuvant trastuzumab-based chemotherapy. Optimization of BCI cut points may further improve prognostic performance in HR+ HER2+. Additional validation in other HR+ HER2+ cohorts treated in the modern setting with anti-HER2-based therapy is warranted, given the small sample size of N0 patients and relatively low DR rate in the late recurrence cohort. In summary, these results provide further evidence of BCI’s utility to support clinical decision-making for adjuvant endocrine therapies for HR+ HER2+ BC. Support: U10CA180821, U10CA180882, U24CA196171, Genentech; Clinicaltrials.gov Id: NCT00005970; https://acknowledgments.alliancefound.org. Citation Format: S. Chumsri, E. Liu, K. Edwards, Y. Wen, Y. Zhang, A. K. Anderson, A. H. Partridge, L. Carey, K. Ballman, E. Perez, K. Treuner, M. Goetz, E. Thompson. Prognostic performance of Breast Cancer Index in patients with early-stage HR+ HER2+ breast cancer (BC) treated with adjuvant trastuzumab: NCCTG N9831 (Alliance) [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 PS3-08-08.

Abstract Background: Tertiary lymphoid structures (TLS), which are organized ectopic lymphoid aggregates resembling secondary lymphoid organs, have been associated with improved outcomes across various cancer types. However, limited data exist on the prognostic relevance of TLS in HER2+ BC pts treated with trastuzumab. In our prior work, we demonstrated that the presence of functional TLS, defined by lymphoid aggregates with elevated expression of BCL6 or IL21R, correlated with improved outcomes in the NCCTG N9831 trial. In this study, we evaluated TLS-related gene and outcomes in the real-world database. Methods: 1344 HER2+ (HR-HER2+: 560; HR+HER2+: 784) BC samples were analyzed via NGS (592-gene panel, NextSeq; WES/WTS, NovaSeq; Caris Life Sciences). Above (high) or below (low) the 50th percentile expression levels of TLS-related genes were used to calculate overall survival (OS). IL21R-high (H) and -low (L) tumors were classified by RNA expression above or below the 50th percentile. Real-world OS was derived from insurance claims and calculated from date of biopsy to last contact using Kaplan-Meier. Statistical significance was assessed using chi-square and Mann-Whitney U with multiple comparison adjustments (q < 0.05). Results: Among pts with HR-HER2+ BC, high expression of IL21R, CXCL9, CD38, and CXCL10 were associated with improved OS (Table). In contrast, among pts with HR+HER2+ BC, high expression of CXCR3, PDCD1, TBX21, CD200 and IL21R, were associated with improved OS. In both HR-HER2+ and HR+HER2+, IL21R-H groups had 1) higher infiltration of B cells (HR-HER2+: 4.4% vs 2.9%; HR+HER2+: 5.6% vs 4.2%), M1 MΦ (HR-HER2+: 3.9% vs 2.6%; HR+HER2+: 3% vs 2.1%), M2 MΦ (HR-HER2+: 4% vs 3.2%; HR+HER2+: 5.7% vs 4.4%), NK cells (HR-HER2+: 2.8% vs 2.5%; HR+HER2+: 2.7% vs 2.4%), CD8+ T cell (HR-HER2+: 0.9% vs 0%; HR+HER2+: 0.7% vs 0%), Tregs (HR-HER2+: 2.9% vs 1.4%; HR+HER2+: 2.8% vs 1.5%), all q<.05; 2) higher T cell inflamed score (HR-HER2+: 87.5 vs -95; HR+HER2+: 71 vs -86), IFNy score (HR-HER2+: -0.19 vs -0.48; HR+HER2+: -0.27 vs -0.49), all q<.05; 3) higher expression levels of MHC class I genes (HLA-A, HLA-B, HLA-C, HLA-E, FC: 1.6-2.3; all q<.05), MHC class II genes (HLA-DQA1, HLA-DOA, HLA-DPA1, HLA-DMA, FC: 2.2-3.2; all q<.05) compared to IL21R-L group. Conclusion: Consistent with our prior findings, overexpression of TLS-related genes was associated with improved outcomes in HER2+ BC in real-world data. Notably, the prognostic impact of TLS-related genes differed by hormone receptor (HR) status. Among these, IL21R emerged as the only gene significantly associated with clinical outcomes across both datasets and HR subgroups. Higher IL21R expression is associated with more B cell, M1 MΦ, CD8 T cell and NK cells. These findings underscore the potential importance of the IL21/IL21R axis in HER2+ BC and warrant further investigation to validate its role and therapeutic relevance. Citation Format: S. Chumsri, S. Deshmukh, Y. Liu, S. Wu, J. Xiu, N. Norton, S. Shipra Gandhi, E. Torres, M. Lustberg, E. Perez, A. Nassar, G. Sledge, E. Thompson, K. Knutson. Tertiary lymphoid structure (TLS)-related gene expression and outcomes in HER2-Positive (HER2+) breast cancer (BC) in the Real-World Database [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 PS2-09-08.

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive yet immunogenic subtype of breast cancer (BC) and the only one with FDA-approved immune checkpoint inhibitor (ICI) - pembrolizumab. Currently, PD-L1 (22C3) is the companion biomarker for predicting pembrolizumab benefit, but its utility is limited by spatial/temporal heterogeneity, assay variability, and modest correlation with treatment response. Chemokines such as CXCL9, CXCL10, and CCL5 recruit CD8+ T cells into the tumor microenvironment and have been associated with improved response to ICI, but their predictive value alongside PD-L1 remains unclear. Here, we examined the relationship between these chemokines, PD-L1, and pembrolizumab efficacy in TNBC. Methods: We analyzed 3662 TNBC tumors profiled by NGS (592, NextSeq; WES/WTS, NovaSeq; Caris Life Sciences). Immune cells were estimated using WTS deconvolution (Quantiseq). PD-L1 positivity was defined as >10% CPS (PharmDx 22C3). CXCL9/CXCL10/CCL5-high (H) and -low (L) TNBC were classified by RNA expression above or below 50th percentile. Real-world median overall survival (mOS) was derived from insurance claims and calculated from start of pembrolizumab to last contact using Kaplan-Meier. Statistical significance was assessed by chi-square and Mann-Whitney U with multiple comparison adjustments (q<0.05). Results: TNBC expressed higher levels of CXCL9, CXCL10, and CCL5 (N = 3662, median TPM: 5.3, 14.8, and 15.1) compared to HER2+ (N = 1344; 4.8, 10.1, 10.6) and HR+HER2- BC (N = 6097; 2.7, 6.6, 9.4), all q<0.05. Moderate correlations were observed between PD-L1 CPS and each chemokine (r = 0.41, p<.01). PD-L1-positive tumors had better mOS (24.3 months (m) vs 18.6 m, HR 0.75, 95% CI 0.6-0.92, p=0.007) compared to PD-L1-negative TNBC post-pembrolizumab. Among PD-L1-negative tumors, those with CXCL9-H had better mOS (26.9 m vs 15.8 m for CXCL9-L, HR 0.61, 95% CI 0.45-0.82, p=0.001) post-pembrolizumab. Among PD-L1-positive tumors, CXCL10-H and CCL5-H was associated with improved mOS (CXCL10-H: 25.7 m vs. 20.7 m for CXC10-L, p = 0.03; CCL5-H: 26.7 vs. 18.5 m for CCL5-L, p = 0.03) post-pembrolizumab. PD-L1 negative CXCL9-H, PD-L1 positive CXCL10-H and PD-L1 positive CCL5-H groups showed higher infiltration of CD8+ T cell, B cells, M1/M2 macrophages, Tregs. They also exhibited higher T cell inflamed score, IFNy score and MAPK activation score vs. CXCL9-L, CXCL10-L and CCL5-L groups (Table). Conclusions: In TNBC, a chemokine signature comprising CXCL9, CXCL10 and CCL5 refines PD-L1 based prediction of pembrolizumab benefit. Specifically, high CXCL10 or CCL5 expression in PD-L1-positive tumors, and high CXCL9 expression in PD-L1-negative tumors, were associated with improved survival post pembrolizumab and immune-rich microenvironment. These findings support the use of chemokine profiling to complement PD-L1 and enhance patient selection for ICI therapy in TNBC. Citation Format: S. Gandhi, S. Deshmukh, S. Wu, J. Xiu, G. Sledge Jr., D. Trapani, J. Leone, S. Chumsri, M. Lustberg, C. Hong, S. Yao, K. Takabe, M. Ernstoff, M. Opyrchal, P. Chalasani, C. Paulos, G. Lesinski, A. Madabhushi, S. Badve, K. Kalinsky, P. Kalinski. Chemokine signatures improve PD-L1-based prediction of pembrolizumab response 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 PS2-08-14.

Abstract Background: Fatty Acid Synthase (FASN) is a key lipogenic enzyme co-regulated with HER2 and highly expressed in ER+ and HER2+ BC. Its targeting downregulates HER2 expression. Our preclinical studies revealed that FASN inhibition can sensitize or restore sensitivity to trastuzumab, paclitaxel and ET in HER2+ BC models. Denifanstat (deni, formerly TVB-2640) is a novel FASN inhibitor that demonstrated promising clinical activity and safety in a prior phase I trial as either monotherapy or in combination with paclitaxel. The primary objective of this study was to evaluate the objective tumor response rate (ORR) of deni plus trastuzumab (H) in combination with paclitaxel (T) or ET. Methods: Two independent, phase II clinical trials were initiated to assess the efficacy and safety of deni plus H in combination with T (Cohort A) or ET (Cohort B). Key inclusion criteria included HER2+ MBC resistant to HER2-directed therapy, measurable disease, unlimited ET, ≤6 prior chemotherapy or antibody drug conjugate regimens, and confirmation of HER2+ MBC on a pre-registration biopsy. Deni was administered 100 mg/m2 orally once daily. HP or H plus ET (fulvestrant or aromatase inhibitor) was administered at standard dose and schedule. Blood was collected on Cycle 1 Day 1 (C1D1), C1D8 and C2D1 for serum FASN analysis. An additional MBC tumor biopsy was collected on C2D1 for correlative studies. A 2-stage Simon design with a safety-run in was used to test the null hypothesis that the true ORR is at most 5% against the alternative it is at least 20%. With a sample size of 32 patients (pts) per cohort, if ≥4 responses are seen, this design has a 90% chance of rejecting the true ORR is <5% when the true ORR is ≥20% at a 0.10 level of significance. The decision to close both trials to enrollment early was multifactorial. Results: From August 2017 - February 2024, 33 pts with HER2+ MBC pre-registered, among which 17 were eligible and 16 were evaluable (15 pts in Cohort A and 1 pt in Cohort B). The most common reason for ineligibility was HER2-negative status on central review of the pre-registration biopsy. Median age was 57 years (range 35-80). All pts were non-Hispanic and 13 (81.3%) were White. Median prior lines of therapy was 5 (range 0 - 11). In Cohort A, there were 5 partial responses; thus, the primary endpoint was met with the ORR estimated to be 33.3% (90% CI: 14.2-57.7%). The median PFS time was 7.0 months (95 %CI: 3.7 - 11.3), and median OS was 25.7 months (95%CI: 10.2 - NE). No dose limiting toxicities were observed during the C1 safety run-in (6 pts). In Cohort A, 7 (46.7%) developed a grade 3 or 4 adverse event (AE) attributed to combination therapy. Grade ≥2 AEs regardless of attribution occurring in ≥20% of all pts included anemia, fatigue, neutropenia, palmar plantar erythrodysesthesia (PPE), and peripheral neuropathy. Treatment was discontinued due to progression (12; 80%) or AE (3; 20% - pneumonitis; neuropathy; PPE). In Cohort B, the pt maintained stable disease through 8 cycles with grade 3 ALT increase, and she remained alive 31.2 months post-registration. Median C1D1 FASN level was 32.7 ng/mL (IQR: 18.7 - 49.6 ng/mL). There was minimal association between C1D8 and C2D1 FASN levels and time to disease progression (C1D8 n=9, Spearman rank correlation coefficient (r s) = 0.040; C2D1 n=13, r s = -0.019). Analyses of changes in MBC tumor expression of FASN, phospho-AKT, and phospho-S6 are in progress. Conclusion: Deni combined with HP provided clinical antitumor activity in pts with HER2+ MBC resistant to trastuzumab. Expected paclitaxel-associated AEs were observed. Nearly half of pts experienced a grade 3 or 4 AE attributed to the combination therapy, and 20% stopped treatment due to AE. Further study of deni, H plus ET is necessary to draw safety and efficacy conclusions. Citation Format: T. C. Haddad, V. J. Suman, R. R. Roa, B. J. Ernst, R. Lupu, T. Vander Steen, M. H. Solanki, M. Keeney, D. W. Northfelt, K. S. Anderson, T. J. Hobday, S. Chumsri, J. E. Hoppenworth, J. L. Carroll, K. V. Giridhar, C. C. O’Sullivan, R. A. Leon-Ferre, M. P. Goetz. Phase II Trial of the FASN Inhibitor, Denifanstat (TVB-2640), Plus Trastuzumab in Combination with Paclitaxel or Endocrine Therapy (ET) in Patients with HER2+ Metastatic Breast Cancer (MBC) Resistant to Trastuzumab [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 PS5-01-16.

Abstract Backgroud. Breast cancer (BC) is the most common and deadly cancer affecting women globally. While traditional treatments are used, factors limit their effectiveness, such as tumor heterogeneity, drug resistance, and non-targeted actions on cancer cells. Emerging evidence highlights the role of gut microbiota in modulating breast cancer risk, treatment response, and recurrence through various mechanisms, including the production of biologically active compounds and metabolites. These non-viable microbial products and metabolic byproducts from probiotic fermentation, known as postbiotics, exert beneficial effects on the host without the need for live microorganisms. Postbiotics derived from Lacticaseibacillus species represent a novel class of therapeutics with potential anti-cancer properties. In this study, we investigated the therapeutic effects of an innovative postbiotic product (iPB), developed through sequential fermentation of L.paracasei NPB01 and L.rhamnosus GG, in BC experimental models. Methods. The following cell lines were used to represent main BC subtypes: MCF7 cells (HR+/HER2-), MDA-MB-231 (triple negative breast cancer), SKBR3 (HER2+). MCF-10A (normal breast epithelial cells) and Caco-2 (enterocytes) cell lines were used as control. Cell viability was assessed by colony formation assay. Apoptosis was evaluated using Annexin V and 7-AAD staining and HLA class I surface expression was quantified by flow cytometry. Wound healing assay was applied to investigate the inhibitory effect of iPB on cell migration. Results. The iPB exposure resulted in >50% colony formation reduction in MCF7, MDA-MB-231 and SKBR3 cells starting from 0.5 mg/ml for 72 h. Furthermore, the iPB significantly increased early apoptotic cell rate in MCF7 cells starting at 0.5 mg/ml for 96 h. A similar trend was observed in MDA-MB231 and SKBR3 cells exposed to 5 mg/ml for 96 h iPB, as well as significant increase in late apoptotic cells detected by 7-AAD. On the contrary, control cells (MCF-10A, Caco-2) viability remained unaffected by the iPB exposure up to 5 mg/ml for 96 h. Evaluation of HLA class I surface expression revealed up to 30% increase in HLA class I expression in MCF7 cells induced by iPB starting at 0.5 mg/ml for 96 h, MDA-MB-231 cells at 1 mg/ml for 72 h, and SKBR3 cells at 5 mg/ml for 72 h, but there was no significant changes in MHC class I expression in control cells. Finally, the results of wound healing analysis revealed that iPB induced an up to 30% reduction in cell migration in all BC cell models, compared to the untreated cells where it induced a full wound closure. Conclusions. The iPB product reduces cell viability and cell migration in all main BC subtypes. These effects parallel with HLA class I surface expression increase in all BC cell phenotypes, potentially enhancing immunogenicity. These findings suggest that this iPB product may represent a promising novel agent in BC treatment and prevention. Citation Format: R. Berni Canani, C. Luongo, L. Pisapia, R. Di Santillo, A. Gaeta, C. Scocco, M. Michelini, V. Mauriello, A. Cadavere, C. Messuri, S. Chumsri, D. Mussallem. An Innovative Postbiotic Product Based on Sequential Fermentation of L.paracasei NPB01 and L.rhamnosus GG Demonstrates Antitumor Activity against 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 PS2-12-02.

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: Hispanic/Latinx (HL) and African American (AA) breast cancer (BC) patients (pts) have increased prevalence of high-risk features such as incidence of BC at age <50 and estrogen receptor negativity. We previously showed that AA and HL had a lower fraction of neutrophils and M2 macrophages (Mɸ) despite higher percentage of PD-L1 positive cases. This study aims to further characterize differences in the tumor immune microenvironment between racial and ethnic groups, with a focus on how these differences vary by site of biopsy. Methods: We analyzed 19,459 BC samples tested by NGS (592, NextSeq; WES, NovaSeq) and WTS (NovaSeq; Caris Life Sciences). Race/ethnicity data were self-reported by pts. Immune cell proportions were estimated using WTS deconvolution (Quantiseq). Gene expression was analyzed for T-cell inflammed score (TIS), MAPK Activation Score (MPAS) and interferon-gamma (IFNy) score. Real-world overall survival (OS) data were derived from insurance claims, calculated from tissue collection or treatment initiation to last contact, and analyzed using Kaplan-Meier. Statistical significance was assessed by chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q<0.05). Results: There were 9674 Non-Hispanic Whites (NHW) (primary BC, defined as biopsy (bx) from breast) pBC: n=3,537, 36.6%; (metastatic BC, defined as biopsy outside of the breast) mBC: n=6,137 63.4%, 1800 AA (pBC: n=750, 42%, mBC: n=1,050, 58%), and 1795 HL (pBC: n=842, 47%; mBC n=953, 53%) samples. There were 2364 TNBC (66% NHW, 19% NHB, 15% HL), 4318 HR+/HER2- (76% NHW, 11.6% NHB, 12.4% HL), and 893 HER2+ (68% NHW, 15% NHB, 17% HL). In pBC overall, AA had lower median cell fraction (%) of B cells (4.8% vs 5.3%), M2 Mɸ (3.4% vs 4.4%), but higher median cell fraction of dendritic cells (DC) (2.8% vs 2.6%) compared to NHW while HL had lower median cell fraction of M2 Mɸ (3.9% vs 4.4%) and higher median cell fraction of CD8+ T cell (0.4% vs 0.2%), all q<0.05. In pBC, HL had higher IFNy score (-0.3 vs -0.34), while AA had lower MPAS (-1.2 vs -0.96), all q<0.05. In lymph node bx, AA had lower median % of M2 Mɸ (3.4% vs 4.4%, q<0.05). No significant differences were seen in the liver bx by race but notably there were lower median cell fractions of CD4+, CD8+, and regulatory T cells (0.0 across NHW, AA, HL) across all race/ethnicities vs. other biopsy sites. By subtype, TNBC primary bx yielded much of the significant changes with AA having lower median cell fraction of M2 Mɸ (2.49% vs 3.04%, q= 0.01) compared to NHW. In primary bx, AA (35.6 m vs 48.5 m, HR 1.4, 95% CI 1.2-1.5, p<0.001) and HL (42.4 m vs 48.5 m, HR 1.2, 95% CI 1.1-1.4, p<0.001) had worse median OS versus NHW. In lymph node bx, AA also had worse OS (29.2 m vs 37.2 m, HR 1.2, 95% CI 1-1.5, p=0.03) compared to NHW. In liver bx, there was worse mOS overall but no difference by race (NHW 18.65m, AA 18.79m, HL 20.6m, p =0.73). In HR+/HER2- liver bx AA had worse OS (14.2 m vs 20.9 m, HR 1.3, 95% CI 1-1.7, p=0.03) versus NHW. In HER2+ primary bx, HL had worse OS (43.2m vs 66.9m, HR 1.7, 95% CI 1.1-2.6, p=0.009). There was a trend towards worse mOS in TNBC AA patients receiving pembrolizumab (18.42m vs HL 23.69m vs NHW 35.30m; p=0.16) and HER2+ AA patients receiving trastuzumab deruxtecan (AA: 16.06m vs HL: 26.81m vs NHW: not reached; p = 0.05). Conclusions: We found race and biopsy site specific differences in the breast cancer immune microenvironment. AA pts had lower M2 Mɸ in primary and lymph node bx while NHW pts showed higher CD8+ T cell in primary tumors. Liver biopsies exhibited low T cell infiltration across all groups. AA pts had worse mOS in primary and lymph node bx compared to NHW but not in liver bx of which we saw worse mOS in all races. These results highlight the need to consider both race and bx site in treatment decisions. Citation Format: R. Hsu, S. Deshmukh, N. Gyabaah-Kessie, E. Rallos, B. Al-Zubeidy, E. Gonzalez, A. G. Baugh, S. Carrel, M. Li, D. Zavala, A. Martynova, P. Jayachandran, D. Stewart, D. Spicer, S. Wu, J. Xiu, G. W. Sledge Jr., R. L. Mahtani, A. C. Sandoval Leon, S. Gandhi, S. Chumsri, M. Lustberg, M. C. Stern, E. T. Roussos Torres. Immune microenvironment and survival differences among Hispanic and African American breast cancer cases by biopsy site [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 PS3-12-06.

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.