Differences in arm-level copy number gains and losses by source data set (CBCS vs TCGA)

Oncogenic pathway level alterations and associations with subtype

566 Background: Endocrine therapy (ET) resistance (ETR) remains a primary challenge in ER+ breast cancer. Analyzing pretreatment tumor transcriptomes across trials with early response endpoints can reveal shared and specific ETR signatures. This study utilizes baseline RNA data from the Phase III ALTERNATE trial (Alliance A011106, NCT01953588; Anastrozole [A], Fulvestrant [F], or AF) and the ACOSOG Z1031B trial (NCT00824941) to identify predictors of early Ki67 response in postmenopausal ER+/HER2– patients. Methods: ETR was defined as week-4 on-treatment Ki67 >10%. Baseline gene expression from ALTERNATE was analyzed to identify differentially expressed (DE) genes (Wilcoxon test, P<0.05) and Hallmark pathways associated with ETR, both across and within individual treatment arms. Feature selection was performed using mixOmics. A Pan-Endocrine Therapy Signature (PETS) was developed by uniting DE genes identified across all three ALTERNATE arms and Z1031B. All statistical analyses were conducted in R (P<0.05). Results: Overall ETR rate in the ALTERNATE RNA-seq cohort (n=733) was 26%. ETR was associated with high Risk of Recurrence (ROR), Oncotype RS, and Mammaprint scores (calculated from RNA-seq data in research setting). In luminal tumors (n=649), ETR was linked to chr 3q13.33, 8q24.13, and 20q13.12 cytoband upregulation and 17q21, 18q23, 3p21.1, and 10q24.32 cytoband downregulation. ETR tumors showed T-cell, E2F target, and interferon-γ enrichment; sensitive tumors favored early estrogen response and muscle differentiation. At individual gene level, high MYBL2 , PIF1 , TROAP and with low HJURP predicted ETR across all samples (AUC>0.70). A deep learning model using all protein-coding genes achieved AUC 0.82 (training) and 0.79 (test) in predicting ETR. Cross-trial integration identified ETR-associated PETS, enriched for genomic instability. PETS performed comparably to established signatures and strongly correlated with MYBL2 signature (r=0.93). Top ETR predictors were MYBL2 , AURKB , and EME1 for Arm A and IL4I1 , TNFAIP6 , and ANLN for Arm AF. AF-resistant tumors were enriched for systemic lupus and RIG-I–like receptor signaling; sensitive tumors favored PI3K–AKT, EGFR TKI resistance, AMPK, and insulin signaling. Conclusions: Baseline transcriptomics identify shared and therapy-specific ETR markers. The 15-gene PETS defines a convergent resistance signature, performing similar to established signatures in predicting ETR and correlating with MYBL2. Enrichment of cell-cycle and immune pathways in resistant tumors may suggest patient stratification approach for alternative or combinatorial strategies to overcome early ETR in ER+ breast cancer. Acknowledgement: https://acknowledgments.alliancefound.org. Support: U10CA180821, U10CA180882, U24CA1. Clinical trial information: NCT01953588 .

Complete tabulation of mutational frequency and proportion by self-reported race [either Black vs non-Black or Non-Hispanic white vs Non-Hispanic Black only] and age at diagnosis.

While cancer immunotherapies have primarily focused on activation of cytotoxic CD8 cells, CD4 T cell activity is also associated with survival and immunotherapeutic response in numerous cancers. We applied integrated single-cell RNA sequencing and multiplexed protein epitope profiling to breast cancer samples to resolve the complexity of immune cell states within the tumor microenvironment. This approach enhanced phenotypic resolution, identifying three distinct states within the CD4 T follicular helper-like (Tfh) cell cluster. A CXCR4high progenitor state gave rise to two differentiated states: an IGFL2high subset resembling conventional Tfh cells and localised to B cell-rich lymphoid aggregates, and a CD103+ subset, exhibiting features of tissue residency, exhaustion, and cytotoxicity, which co-localised with tumor foci. CD103+ Tfh-like cells were found to interact with CXCL10+ macrophages through production of CCL chemokines and CSF1. A higher CD103+ Tfh to IGFL2high Tfh ratio, together with the selective clonal expansion of the CD103+ subset, was strongly associated with improved tumour immunity and superior responses to anti-PD-1 checkpoint blockade, surpassing the predictive value of exhausted CD8 T cells. These findings integrate Tfh and CD4 with cytotoxic potential in breast cancer, offering new insight into anti-tumor immunity and response to checkpoint blockade.

Flow diagram of sample and patient selection for study

Abstract In PREDIX LumB patients with estrogen receptor positive and human epidermal growth factor receptor negative (ER + /HER2-) breast cancer > 20 mm and/or with lymph node metastasis were randomized 1:1 to receive either paclitaxel weekly for 12 weeks followed by palbociclib and endocrine therapy for 12 weeks (arm A), or the reverse sequence (arm B). Primary endpoint is objective radiologic response at 12 weeks (ORR12), and key secondary endpoints are ORR24, pathologic complete response, event-free survival, safety and correlative studies of tissue and circulating biomarkers. Whole exome sequencing and RNA sequencing were performed on baseline fresh frozen tissue samples. In total, 179 patients comprise the intention-to-treat population. There is no statistically significant difference between the two arms in ORR12 (59% vs 45%, p = 0.058). An exploratory gene expression analysis identified differentially expressed genes and gene sets between responders and non-responders at 12 weeks. A predictive signature, CDKPredX, comprising 31 genes related to proliferation, ER signaling and immune activity was developed to identify patients resistant to chemotherapy but responding to palbociclib plus endocrine therapy (pinteraction=0.03). The predictive signature was independently validated in the CORALLEEN trial (pinteraction=0.048). Clinicaltrials.gov identifier: NCT02603679

BACKGROUND:Treatment approaches differ for isolated in-breast tumor recurrence (representing treatment failure) and new primary breast tumors (representing high etiologic risk). However, methods for distinguishing recurrences from second primaries (based on radiographic and histologic criteria) are subject to error. Gold-standard genomic datasets for assessing classification accuracy have been lacking. METHODS:To identify the scope of misclassification, we performed DNA sequencing of 1,200 genes in 108 participants with synchronous or metachronous second breast cancer in the Carolina Breast Cancer Study (CBCS3). DNA sequencing data included 87 second tumors and 42 paired first and second breast cancers in the same patient (14 contralateral and 28 ipsilateral). Recurrence status was classified based on mutations and copy number, accounting for site-specific mutation probabilities. DNA-based recurrence versus second primary classifications were compared with clinical and Surveillance, Epidemiology and End Results (SEER) classifications based on laterality, histology, quadrant, and latency. RESULTS:Twenty-two of 28 ipsilateral tumor pairs (79%) shared at least one mutation and were classified as recurrences. Pathologist classifications of ipsilateral second tumors were 79% accurate [95% confidence interval (CI), 60%-90%) with higher sensitivity (95%, 95% CI = CI, 78%-99%) and lower specificity (17%, 95% CI, 3%-56%). SEER classifications were 82% accurate (95% CI, 64%-92%) with lower sensitivity (77%, 95% CI, 57%-90%) and higher specificity (100%, 95% CI, 61%-100%). CONCLUSIONS:Although most genomically defined recurrences are captured by clinical recurrence definitions, new primaries are frequently misclassified. IMPACT:Given overtreatment harms, genomic methods may support treatment de-escalation for second breast cancers.

Association between RNA-based TP53 pathway dysfunction and DNA-based TP53 defects

Boxplot showing differences in predicted probability of recurrence by weighting schema

Differences in proportions of samples with detected mutations by stage at diagnosis (Stage I/II vs. Stage III/IV)

Enrichments for arm-level alterations by self reported race

Arm level amplification and deletion prevalence differences by age at diagnosis

Association between socioeconomic variables in CBCS and mutations or copy number alterations. All estimates are stratified by self reported race

Summary or different schema to classify IBTR vs second primary tumors

Accuracy and test metrics of genomic vs clinical schema stratified by prior treatment.

Abstract Chromatin remodeling complexes are critical regulators of transcription and therapeutic resistance in cancer, yet the mechanisms governing their recruitment and activity remain poorly understood. Here, we identify BRD8, a largely uncharacterized bromodomain protein within the EP400 chromatin remodeling complex, as a master regulator of treatment-induced transcriptional reprogramming in hormone receptor-positive/HER2-positive (HR+/HER2+) breast cancer. Using integrative multi-omics approaches—including paired single-cell RNA-seq and ATAC-seq, comprehensive ChIP-seq profiling, and functional genomics—we uncover a novel epigenetic mechanism whereby BRD8 orchestrates chromatin accessibility and transcription factor cooperation to drive therapeutic resistance. BRD8 depletion reduces H2A.Z acetylation and disrupts interactions between the EP400 complex and key transcription factors. Through both ER-dependent and ER-independent pathways, BRD8 cooperates with H2A.Z and acetylated H2A.Z (H2A.Zac) to facilitate recruitment of the EP400 complex to transcription factors including ER, FOXA1, and ETS family members, thereby promoting H2A.Z acetylation, chromatin accessibility, and expression of growth-promoting genes. BRD8 expression is markedly elevated in anti-HER2-resistant models, and its depletion sensitizes resistant cells to anti-HER2 therapy. To translate these findings therapeutically, we developed a potent and selective BRD8 PROTAC degrader with a DC50 of ∼100 nM. Combined treatment with the BRD8 degrader and anti-HER2 agents synergistically inhibits HR+/HER2+ breast cancer cell growth with superior efficacy and no detectable toxicity. Notably, the BRD8 degrader demonstrates fivefold greater potency than the BRD8 inhibitor DN02 across multiple HR+/HER2+ breast cancer cell lines. Finally, we developed a 3-gene BRD8 signature that predicts anti-HER2 therapy response in two independent clinical trials, establishing BRD8 as both a predictive biomarker and a central epigenetic regulator of therapeutic resistance. Our findings identify dual BRD8 and HER2 targeting as a promising precision medicine strategy for overcoming resistance in HR+/HER2+ breast cancer. Citation Format: Wei Xu, Ang Gao, Parth Khatri, Gui Ma, Huy Dinh, Charles Perou. BRD8 chromatin regulator mediates anti-HER2 response in HR+/HER2+ 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 375.

Differences in recurrent copy number alterations of mutations by self-reported race or age after stratifying on estrogen receptor (ER) status

Women diagnosed with metastatic triple negative breast cancer (mTNBC) have limited treatment options, are more prone to develop resistance and are associated with high mortality. A cold tumor immune microenvironment (TIME) characterized by low T cells and high tumor associated macrophages (TAMs) in mTNBC is associated with the failure of standard-of-care chemotherapy and immune checkpoint blockade (ICB) treatment. We demonstrate that the combination of immunomodulatory low-dose Cyclophosphamide (CTX) coupled with anti-CSF-1R antibody targeted therapy (SNDX-ms6352) and anti-PD-1 (ICB), was highly effective against aggressive metastatic Trp53 null TNBC transplantable syngeneic models that present with high macrophage infiltration. Mechanistically, CSF-1R inhibition along with CTX disrupted the M-CSF/CSF-1R axis which upregulated IL-17, IL-5 and type II interferon resulting in elevated B- and T cell infiltration. Addition of an anti-PD-1 maintenance dose helped overcome de novo PD-L1 intra-tumoral heterogeneity (ITH) associated recurrence in lung and liver mTNBC.

Oncogenic pathway dysfunction differences by race and age at diagnosis