Single-cell and spatial profiling identify three response trajectories to pembrolizumab and radiation therapy in triple negative breast cancer

Cancer immunotherapy trials have produced encouraging results, but resistance remains a problem necessitating strategies to identify patients that benefit from immunotherapy alone or who require additional combinations like chemotherapy or radiotherapy. Here we employ single-cell transcriptomics and spatial proteomics to profile triple negative breast cancer biopsies taken before and after one cycle of pembrolizumab and after a second cycle of pembrolizumab given with radiotherapy. Non-responders lack immune infiltrate before and after therapy and exhibit minimal therapy-induced immune changes. Responding tumors form two groups that are distinguishable by a classifier prior to therapy, with one showing high MHC expression, evidence of tertiary lymphoid structures and displaying anti-tumor immunity before treatment. The other responder group resembles non-responders at baseline and mounts a maximal immune response after the combination therapy, which is characterized by cytotoxic T cell and antigen presenting myeloid cell interactions and is mirrored in murine breast tumors only after radiation. From November 2017 through June 2021, we enrolled 50 patients in our phase Ib/II trial entitled “Preoperative Combination of Pembrolizumab and Radiation Therapy in Patients with Operable Breast Cancer”03366844, clinicaltrials.gov). Patients with tumors greater than 2 cm with any nodal status as determined by imaging without metastatic disease were eligible for the trial. Patients received pembrolizumab at a dose of 200 mg, administered intravenously, on Day 1 and 21 and received three daily fractions of 8 Gray (Gy) stereotactic radiation to the breast tumor only between Day 21 and 28. Biopsies were taken prior to each treatment and patients were consented to analysis of the biopsies which included, but were not limited to, sequencing and immunohistochemistry. We utilized the E0771 orthotopic murine model of TNBC and performed a four-arm study consisting of control IgG treatment, anti-PD1 treatment, control IgG + radiation therapy, or anti-PD1 + radiation therapy. We then performed single-cell RNA-sequencing on CD45-positive cells isolated from tumors (n=2, with 2 mice pooled per replicate) from each of the arms.