Abstract 6253: Single-cell RNA sequencing reveals metastatic site-specific biomarkers for predicting response to CDK4/6 inhibition in advanced HR+/HER2- breast cancer

Abstract Background: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), in combination with endocrine therapy, are established as standard treatment for advanced HR+/HER2- breast cancer (BC). However, response heterogeneity is observed, with approximately one-third of patients exhibiting intrinsic resistance. This study investigates the mechanisms of variable responses to CDK4/6i through single-cell RNA sequencing of metastatic site biopsies, revealing distinct biomarkers for predicting CDK4/6i response. Methods: Single-cell RNA sequencing analyzed metastatic tumors from advanced HR+/HER2- BC patients pre-CDK4/6i treatment at baseline (BL) and/or at disease progression. BL samples were from CDK4/6i responders (n = 8, median progression-free survival mPFS = 17 months), while progressors were categorized as early-progressors (EP, n = 3, mPFS = 3 months) and late-progressors (LP, n = 8, mPFS = 10 months). Metastatic sites included liver (10), pleural effusions (6), ascites (2), and bone (1). InferCNV distinguished tumor cells, and functional analysis utilized the Molecular Signatures Database. Results: Both EP and LP tumor cells exhibited reduced estrogen response and increased IFN-γ pathways compared to BL tumors. LP tumors displayed enhanced Myc targets, EMT, TNF-α, and inflammatory pathways compared to EP tumors, revealing differential mechanisms of early versus late progression. Predictive signatures, including TFF3 and MGP, were significantly upregulated in BL tumor cells across different metastatic sites. Metastasis-specific markers included APOA and SERPINA1 (liver), and AGR2 (pleural effusion). BL and LP responders showed increased tumor infiltrating CD8+ T cell and NK cells compared to EP non-responders. Notably, despite a high CD8+ T frequency in responder tumors, functional analysis revealed a significant upregulation of genes associated with stress/apoptosis and exhaustion in proliferative CD4+ and CD8+ T cells from BL compared to LP tumors, including HSP90 and HSPA8, associated with poor response to immune checkpoint inhibitors. These findings suggest potential benefits of immunotherapy for CDK4/6i late progressors. Longitudinal biopsies further revealed dynamic NK cell expansion and enhanced cytotoxic T cell activity alongside the downregulation of stress/apoptosis gene expression at progression compared to BL tumors. Conclusion: This study underscores the significance of metastatic site-specific biomarkers in predicting outcomes to CDK4/6i. Tumor-infiltration lymphocytes may also serve as baseline predictors, while stress/apoptosis gene signatures suggest checkpoint inhibitor usage in CDK4/6i late progressors. These potentially actionable findings emphasize the need to optimize therapeutic approaches based on microenvironment-specific changes following disease progression. Citation Format: Linjie Luo, Peng Yang, Sofia Mastoraki, Yan Wang, Nicole M. Kettner, Akshara Singareeka Raghavendra, Debasish Tripathy, Senthil Damodaran, Kelly K. Hunt, Ziyi Li, Khandan Keyomarsi. Single-cell RNA sequencing reveals metastatic site-specific biomarkers for predicting response to CDK4/6 inhibition in advanced HR+/HER2- breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6253.