Background Low response rates have been observed with anti-PD1 (pembrolizumab) immunotherapy in triple-negative breast cancer (TNBC). However, combining pembrolizumab with radiation therapy (RT) has shown improved response rates, although the underlying mechanisms remain unclear. Therefore, in this study, we aimed to investigate immune cell profiles and T cell clonal dynamics in tumor and peripheral blood mononuclear cells (PBMCs) of patients receiving anti-PD1 and anti-PD1+RT, in order to identify potential biomarkers associated with treatment response.

Methods In this trial, a total of 66 patients with operable breast cancer were enrolled to investigate the efficacy of the Preoperative Combination of Pembrolizumab and Radiation Therapy. Patients received intravenous administration of pembrolizumab at a dose of 200 mg on Day 1 and 21, along with three daily fractions of 8 Gray (Gy) stereotactic radiation specifically targeting the breast tumor between Day 21 and 28. PBMC and Tumor biopsies were obtained at baseline and after each treatment cycle from 9 patients (6 responders(R) and 3 non-responders (NR). CD45+ positive stained cells were sorted for subsequent single-cell RNA and T-cell receptor sequencing analysis. T cell clones were identified and labelled using Scanpy and Scirpy packages.

Results Single-cell analysis of CD8+ TILs identified 12 transcriptionally defined clusters, including clusters associated with exhaustion and pre-exhaustion, which were more clonally expanded in responders. Pre-exhausted CD8 Tcells were predominantly present in Blood at pre-treatment (non-emergent clones), and in tumor with each treatment cycle we identified increased emergent clones in responders compared to NR’s. In addition, we observed an increased proportion of mucosa-associated invariant T (MAIT) cells in PBMC’s after anti-PD1+RT treatment compared to baseline levels, particularly in non-responders. Furthermore, responders showed an elevated proportion of B cells in their PBMCs at baseline, similar to tumor microenvironment proportions and indicated a better treatment response.

Conclusions These findings suggest that PBMC’s reflect ongoing tumor response to anti-PD1 and anti-PD1+RT therapy and blood-based biopsy could serve as a potential non-invasive method for monitoring treatment response in TNBC patients.