Biological pathways of response to neoadjuvant chemo-immunotherapy in triple-negative breast cancer (TNBC)

Background: KEYNOTE-522 established neoadjuvant pembrolizumab plus chemotherapy as standard care for early-stage TNBC, improving pathological complete response (pCR) rates. However, up to 45% of patients (pts) do not achieve pCR, and mechanisms driving differential responses are unclear. This study aimed to identify biological pathways distinguishing pCR from non-pCR pts at baseline (BL) and to characterize how these pathways evolve during treatment (DT) and post-surgery (PS). Methods: Blood samples from TNBC pts were collected at BL, DT, and PS. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Single-cell RNA sequencing was performed using the Parse Biosciences platform. Gene Set Enrichment Analysis identified enriched pathways using the Molecular Signatures Database (MSigDB) hallmark (50 gene sets) and Reactome (1,787 gene sets) collections. Comparisons were made between pCR and non-pCR patients at each timepoint and within groups (DT vs. BL). Results: Among 18 sequenced pts, 11 achieved pCR. A total of 883,183 cells across 17 cell types were analyzed. At BL, responders showed elevated interferon (IFN) and cytokine signaling, mainly from lymphocytes, along with chemokine upregulation in classical monocytes (CM) and increased DAP12 expression in CD8+ T cells. During treatment, responders demonstrated a myeloid-driven TNFalpha/NFκB inflammatory response and upregulated GPCR signaling in CM. In contrast, non-responders exhibited ineffective myeloid IFN signaling and downregulated TNFalpha/NFκB activity across all cell types DT. Conclusions: pCR is distinguished by a coordinated lymphocyte and myeloid immune activation. Non-responders exhibit ineffective signaling and suppressed inflammatory pathways. These divergent mechanisms highlight potential biomarkers to guide patient stratification and therapeutic optimization in TNBC. Table 1. Most prominent pathways differing between responders and non-responders Pathway MSigDB Gene set Cell Type Baseline (responders vs non-responders) On treatment (responders vs non-responders) Post-Surgery (responders vs non-responders) Antigen Presentation & Immune Activation ANTIGEN_PROCESSING_CROSS_PRESENTATION Reactome Classical Monocytes, Non-classical Monocytes Downregulated Downregulated - BINDING/UPTAKE BY SCAVENGER RECEPTORS Reactome CD8+ T-cells - Upregulated Upregulated REGULATION OF TLR BY ENDOGENOUS LIGAND Reactome Plasma cells - Downregulated Upregulated T-Cell & Cytokine Signaling INTERFERON_GAMMA SIGNALING Reactome CD8+ T-cells Upregulated - Upregulated INTERFERON_ALPHA_RESPONSE and INTERFERON_GAMMA_RESPONSE Hallmark CD8+ T-cells, CD4+ T-cells, B cells, Plasma cells, regulatory T-cells, NK-cells Upregulated Upregulated Upregulated INTERFERON_ALPHA_RESPONSE and INTERFERON_GAMMA_RESPONSE Hallmark Classical Moncoytes, Non-classical monocytes, Dendritic cells Upregulated Downregulated Upregulated INTERFERON_GAMMA SIGNALING Reactome CD8+ T-cells Upregulated - Upregulated TNFA_SIGNALING_VIA_NFKB Hallmark CD8+ T-cells, CD4+ T-cells, B cells, Plasma cells, regulatory T-cells, NK-cells Upregulated Upregulated Upregulated CYTOKINE SIGNALING IN IMMUNE SYSTEM Reactome CD8+ T-cells, CD4+ T-cells Upregulated - Upregulated INTERFERON_ALPHA_BETA_SIGNALING Reactome Classical Monocytes, CD8+ T-cells Upregulated - Upregulated INTERFERON_SIGNALING Reactome CD8+ T-cells, CD4+ T-cells Upregulated - Upregulated INTERFERON_SIGNALING Reactome Classical Monocytes - Downregulated Upregulated DAP12_INTERACTIONS Reactome CD8+ T-cells Upregulated - Upregulated Cellular Signaling & Trafficking SIGNALING_BY_GPCR Reactome Classical Monocytes Upregulated Upregulated - GPCR_LIGAND_BINDING Reactome Classical Monocytes Upregulated Upregulated Upregulated GPCR_LIGAND_BINDING Reactome Plasma cells - Upregulated Upregulated CHEMOKINE_RECEPTOR_BIND_CHEMOKINES Reactome Classical Monocytes Upregulated Upregulated Upregulated FORMATION OF BETA-CATENIN TCF COMPLEX Reactome Classical Monocytes Upregulated Upregulated - FORMATION OF BETA-CATENIN TCF COMPLEX Reactome Non-classical Monocytes Upregulated Upregulated Downregulated Cell Cycle & DNA Replication DNA_METHYLATION Reactome Classical Monocytes Upregulated Upregulated - DNA_METHYLATION Reactome Non-classical Monocytes Upregulated Upregulated Downregulated DNA_METHYLATION Reactome Plasma cells Downregulated - Downregulated CELL_CYCLE_MITOTIC Reactome Plasma cells Downregulated - Downregulated CELL_CYCLE_CHECKPOINTS Reactome Plasma cells Downregulated - Downregulated DNA_REPLICATION Reactome Plasma cells Downregulated - Downregulated Metabolism & Biosynthesis RESPIRATORY_ELECTRON_TRANSPORT Reactome Non-classical Monocytes Downregulated Downregulated - HEME_SIGNALING Reactome Immature T-cells Upregulated Upregulated - HEPARAN_SULFATE/HEPARIN METABOLISM Reactome Classical Monocytes Downregulated - Downregulated