T-cell receptor (TCR) repertoire dynamics of response to neoadjuvant chemo-immunotherapy in triple- negative breast cancer (TNBC).

Abstract

e12660 Background: Neoadjuvant pembrolizumab plus chemotherapy is the current standard for cT2N0-3 TNBC, yet ~40% of patients (pts) do not achieve a pathological complete response (pCR). This increased efficacy of the addition of pembrolizumab comes at the expense of significant immune related and financial toxicities. To better understand drivers of pCR, we analyzed longitudinal TCR dynamics in pCR patients versus those with residual disease (non-pCR). Methods: Blood samples from TNBC pts were collected at baseline (BL), on-treatment (DT), and post-surgery (PS). Peripheral blood mononuclear cells were isolated by density gradient centrifugation. Single-cell RNA sequencing was performed (Parse Biosciences platform). Paired α/β TCR repertoires were analyzed for diversity, overlap, and homeostasis. Longitudinal tracking identified persistent clonotypes across timepoints, with expansion quantified as log₂ fold changes. Transcriptional states of persistent and convergent clones were characterized using GSEA (MSigDB Hallmark and a targeted panel of T-cell signaling pathways [C2/GO:BP]) and differential expression analyses, comparing pCR vs. non-pCR pts and longitudinal clonal dynamics. Results: Among 18 pts, 11 achieved pCR. A total of 21,622 TCR clones were analyzed. pCR pts had 2,682 unique clones at BL, 3,834 at DT, and 5,274 at PS; 78 clones were present at both BL and DT, and 68 were detected across all timepoints. Non pCR pts had 1,864 clones at BL, 2,997 at DT, and 4,538 at PS; 27 clones were present at both BL and DT, and 49 persisted across all timepoints. Most clones were not shared between pCR and non pCR pts. While non pCR CD8+ T cells showed greater numeric expansion (137% vs 76%), their repertoires exhibited a marked loss of large clonotypes (20.2% to 7.2%) compared with pCR pts (24.5% to 17.6%). Persistent pCR clones demonstrated stronger V gene correlations, and their corresponding T cells showed upregulation of NFκB pathways. None of the clonotypes persisting in either pCR or non pCR pts had been previously associated with breast cancer in the literature. Convergent clones were most frequent in CD8+ T cells (6.1%) and exhibited higher degeneracy, particularly at the D2 level, which was increased in pCR patients. These clones showed upregulation of TCR signaling pathways, MMP14 , and AGMAT (markers of tissue homing and proliferation), whereas non pCR pts showed downregulation of NFκB signaling. Conclusions: pCR may be driven by the stability and functional quality of the TCR repertoire rather than by expansion alone. Non pCR was characterized by repertoire instability and loss of inflammatory signaling. In contrast, pCR was associated with the maintenance of robust and persistent clonotypes exhibiting invasive and proliferative phenotypes. Peripheral clonal stability may represent a more informative biomarker of treatment efficacy than simple diversity metrics.