Sledge, G. W.

Abstract Background: MHC class II molecules are normally restricted to professional antigen-presenting cells. Prior TNBC studies have focused on immune-compartment MHC-II. Using spatial transcriptomics, we recently identified aberrant tumor-cell MHC-II expression. Here, we characterized the immune architecture surrounding HLA-DRA-expressing tumor cells. Methods: High-plex single-cell spatial transcriptomics (CosMxTM SMI) was performed on treatment-naïve TNBC (Mayo TMA, n=65) and two neoadjuvant pembrolizumab cohorts (Mayo n=8; Emory n=4). Spatial neighborhoods were mapped relative to HLA-DRA-high tumor cells. Differential expression and adaptive immune gene-set scores were evaluated. Clinical relevance was assessed using FinXX (n=114), I-SPY2 (n=364), and Caris CODEai (n=3,662). Results: HLA-DRA-high tumor regions showed significant enrichment of adaptive immune subsets within ≤50 μm, including B cells, CD8+ T cells, NK cells, macrophages, and plasmacytoid dendritic cells. In pembrolizumab-treated cohorts, responders exhibited higher proportions of HLA-DRA-high tumor cells and greater numbers of B cells, plasmablasts, CD4+ and CD8+ T cells, and macrophages near tumor cells. Responders’ tumors showed upregulation of antigen-presentation machinery, B-cell/plasma-cell programs, CXCL13, and NOTCH3/DLL1. Across I-SPY2 and FinXX, higher HLA-DRA correlated with pathologic complete response and improved survival. In CODEai TNBC, high HLA-DRA expression correlated with higher CXCL13 (median TPM 7.5 vs. 1.1, q<0.05) and longer overall survival (24.2 vs. 18.5 months, HR 0.77, 95% CI 0.71-0.83, p<0.0001). This association was TNBC-specific. High CXCL13 also predicted improved survival (26.6 vs. 16.5 months, HR 0.64, 95% CI 0.59-0.69, p<0.0001). Conclusions: Tumor-cell HLA-DRA expression defines a highly organized adaptive immune niche enriched with B cells and activated T-cell populations in close proximity. Adaptive immune programs—including enhanced antigen presentation and CXCL13-mediated B-cell/plasma-cell pathways—consistently associate with improved outcomes across TNBC cohorts treated with chemotherapy and immune checkpoint blockade. Aberrant tumor-cell MHC-II expression may contribute to antitumor immunity and warrants further investigation as a potential therapeutic target. Citation Format: Yi Liu, Sachin Kumar Deshmukh, Thiti Susiriwatananont, Panuch Eiamprapaporn, Bogang Wu, Heikki Joensuu, Roberto A. Leon-Ferre, David Zahrieh, Judy C. Boughey, James Newell Ingle, Fergus J. Couch, Sharon Wu, Shipra Gandhi, Maryam Lustberg, George W. Sledge, Matthew P. Goetz, Keith L. Knutson, E Aubrey Thompson, Jodi Carter, Saranya Chumsri. Spatially resolved tumor-cell MHC class II shapes adaptive immunity and therapeutic response in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1200.

Abstract Background: Survivor plots permit adjustment by baseline demographics and clinical characteristics; log-normal survival analysis may be more robust than a Cox model under departures from model assumptions. We depicted distant disease-free survival (DDFS) for statistically standardized quantitated ER, PgR, and HER2. Methods: We utilized CCTG MA.27 (NCT00066573), an adjuvant phase III trial of exemestane versus anastrozole in postmenopausal women with ER+ and/or PgR+ tumours. IHC ER, PgR, and HER2 HSCORE were centrally assessed by machine image quantitation, and statistically standardized to mean of 0, standard deviation (SD) of 1 following Box-Cox variance stabilization transformations of square for ER, natural logarithm (ln) and square root for PgR, and ln for HER2. For the ln transformation, 0.1 was added to 0 HSCOREs for a stable transformation. The primary endpoint was DDFS at the longest trial follow-up of 4.1 years. For log-normal survival analysis, ln(time) is a linear function of covariates and had a normal distribution; the Cox models were log-linear for covariates. Survival was depicted with log-normal and Cox survivor plots adjusted at the means of age, T and N stage, grade, lymphovascular invasion, treatment, and baseline patient demographics. We examined ER and PgR ASCO/CAP cut-points at (0; >0), HER2 cut-points at (IHC 0, 0 stain; IHC 0, (0,10%] stain; IHC 1+; IHC 2+; IHC 3+), and ER/PgR/HER2 standardized cut-points at standard deviations about mean of 0 (<-1; (-1,0]; (0,1]; >1). P was based on the likelihood ratio criterion (∼Χ2 (1)) for the addition of ER/PgR/HER2 to the model of baseline covariates, and had nominal significance if P<0.05. Log-normal and Cox multivariable regressions with unstandardized and standardized categorical, and continuous, ER, PgR, and HER2 were adjusted by baseline covariates; 2-sided Wald tests had nominal significance if P<0.05. Results: Of 7576 MA.27 patients, 2325 patients had quantitated image analysis assessments for ER, PgR, and HER2; 113 of 2325 (4.9%) patients experienced a DDFS event during follow-up. Multivariable log-normal survival analysis indicated DDFS was significantly longer for patients with T1 (p=0.01), N0 (p=.002), and grade 1/2 tumors (p=0.01); similar significance levels were found with a Cox model. Log-normal survivor plots were smooth, while Cox survivor plots were step-wise discontinuous at events. Adjusted log-normal survivor plots indicated similar 5-year DDFS for patients by standardized ER categories (p=0.43): 95.4% for those with ER <-1.0 (n=401); 95.8%, for (-1.0,0.0] (n=737); 96.1%, for (0.0,1.0] (n=737); and 96.4%, for >1.0 (n=450). The log-normal standardized PgR survivor plots indicated patients had significantly different 5-year DDFS (p<.001) by categories, with both ln and square root transformations of HSCORE: with a ln transformation, 93.7% for <-1.0 (n=607); 95.4%, for (-1.0,0.0] (n=390); 96.8%, for (0.0,1.0] (n=826); and 97.8%, for >1.0 (n=502). Patients had similar 5-year DDFS by standardized HER2 categories (p=0.71): 96.2% for <-1.0 (n=83); 96.0%, for (-1.0,0.0] (n=1201); 95.9%, for (0.0,1.0] (n=527); and 95.7%, for >1.0 (n=514). In full fit multivariable log-normal survival analyses, DDFS was not significantly different for ER (p=0.51-0.89) and HER2 (p=0.12-0.98); higher PgR was associated with significantly better DDFS (p=0.01 to <.001). Adjusted Cox survival analyses had similar results. Conclusions: Survivor plots adjusted for baseline demographic and clinical characteristics provided similar indications of significance for standardized ER, PgR, and HER2 as those seen in multivariable models with both log-normal and Cox survival analyses. Higher PgR was associated with significantly better DDFS. Citation Format: J. W. Chapman, J. Bayani, S. SenGupta, J. M. S. Bartlett, T. Piper, M. A. Quintayo, S. Virk, P. E. Goss, J. N. Ingle, M. J. Ellis, G. W. Sledge, G. T. Budd, M. Rabaglio, R. H. Ansari, R. Tozer, D. P. D'Souza, H. Chalchal, S. Spadafora, V. Stearns, E. A. Perez, K. A. Gelmon, T. J. Whelan, C. Elliott, L. E. Shepherd, B. E. Chen, K. J. Taylor. Survivor plots for quantitated adjunctive statistically standardized ER, PgR, and HER2 in adjuvant postmenopausal breast cancer: Canadian Cancer Trials Group MA.27 trial of exemestane versus anastrozole [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 PS3-07-23.

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: Male breast cancer (MaBC) is a rare malignancy, accounting for approximately 1% of all breast cancer cases. Our previous studies suggest that MaBC is biologically distinct from female breast cancer (FeBC), exhibiting a unique mutational profile and immune-genomic microenvironment. While the impact of these molecular alterations on site-specific metastases has been explored in FeBC, it remains poorly understood in MaBC. This gap limits accurate risk stratification and prediction of disease trajectory in male patients. In this study, we investigated site-specific genomic and immune alterations across metastatic sites in MaBC. Methods: 238 MaBC samples were tested by NGS (592, NextSeq; WES, NovaSeq) and WTS (NovaSeq; Caris Life Sciences, Phoenix, AZ). We performed analysis in hormone receptor-positive (HR+) and HER2-negative (HER2-) subtypes which are most common in male. HR+HER2- was defined as estrogen receptor (ER) or progesterone receptor (PR) positive and HER2 IHC 1+ or 2+ with negative chromogenic in situ hybridization (CISH) assay. Immune cells were estimated using WTS deconvolution (Quantiseq). Gene expression profiles were analyzed for T-cell inflamed score, interferon-gamma (IFN-gamma) score and MAPK Activation Score. We classified patients by site of biopsy/specimen site. Statistical significance was assessed with chi-square and Mann-Whitney U tests, p<0.05 is considered statistically significant. Results: Our cohort included 86 HR+HER2- MaBC samples biopsied from primary breast cancer (pBC) (n=39), bone (n=15), liver (n=8), lung (n=8), lymph nodes (n=7), and skin (n=9) mets. We report here only the data on statistically significant differences, but the overall landscape has been depicted across all defined-sites investigated. pBC showed a significantly lower prevalence of PIK3CA mutations (23.6% vs. 53.3%, p<0.05) compared to bone mets. ESR1 mutations were absent in pBC but present in 25% of liver and 25% of lung mets (all comparisons p<0.05). Immune profiling revealed reduced infiltration of M2 macrophages (4.3% vs. 6%, p<0.05) and neutrophil (2.2% vs. 6.9%, p<0.05) in pBC relative to bone Mets. Further, CD4+ T cells were lower in pBC compared to lymph nodes (0% vs. 2.4%, p<0.05). pBC had lower expression of immune checkpoint genes (PDCD1LG2, HAVCR2 and CD47) compared to other mets. pBC exhibited lower MAPK pathway activation score (-1.6 vs. 0.17, p=0.03) and a trend toward lower T cell-inflamed gene expression scores (-65 vs. 8, p=0.06) compared to bone mets. Bone mets had higher infiltration of M2 macrophage compared to lung (6% vs. 4.4%, p<0.05) and lymph node (6% vs. 4.5%, p<0.05). Neutrophil infiltration was elevated in bone versus liver (6.9% vs. 2%, p<0.05) but T regulatory cells were lower in bone compared to lymph nodes (1% vs. 2.4%, p<0.05). Bone showed reduced expression of CD47 (median TPM 50.6 vs. 109.9, p<0.05) compared to skin. Further bone had lower IFN-gamma scores (-0.41 vs. -0.17, p<0.05) compared to lymph node. Data on prior number and types of cancer treatment to give a context to the findings will be presented at the meeting. Conclusions: Our study highlights that each MaBC metastatic site harbors a unique genomic and immune landscape that can influence tumor behavior and potentially impact patient prognosis. Better understanding of metastatic site-specific molecular features in larger cohorts can help in the future research efforts and guiding treatment decisions. Citation Format: D. Trapani, S. Deshmukh, S. Wu, J. Xiu, N. Lin, G. Curigliano, P. Spanheimer, P. Advani, P. Barreto Coelho, M. Lustberg, G. Sledge Jr, S. Tolaney, J. Leone. Site specific Genomic and Immune Landscapes Underpinning in metastatic Male 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 PS2-08-04.