Anti–PD-1 Efficacy in Patients with Metastatic Urothelial Cancer Associates with Intratumoral Juxtaposition of T Helper-Type 1 and CD8+ T cells

Abstract

Purpose: PD-1 inhibition results in durable antitumor responses in a proportion of patients with metastatic urothelial cancer (mUC). The majority of patients, however, do not experience clinical benefit. In this study, we aimed to identify early changes in T-cell subsets that underlie anti–PD-1 efficacy in patients with mUC. Experimental Design: Paired samples were collected from peripheral blood, plasma, and metastatic lesions of 56 patients with mUC at baseline and weeks 6 and 12 after initiating pembrolizumab treatment (200 mg intravenously, every 3 weeks). Samples were analyzed using multiplex flow cytometry, ELISA, and in situ stainings, including cellular network analysis. Treatment response was evaluated as best overall response according to RECIST v1.1, and patients were classified as responder (complete or partial response) or nonresponder (progressive disease). Results: In responders, baseline fractions of CD4+ T cells expressing cosignaling receptors were higher compared with nonresponders. The fraction of circulating PD-1+ CD4+ T cells decreased at weeks 6 and 12, whereas the fraction of 4-1BB+ CD28+ CD4+ T cells increased at week 12. In metastatic lesions of responders, the baseline density of T helper-type 1 (Th1) cells, defined as T-bet+ CD4+ T cells, was higher as compared to non-responders. Upon treatment, Th1 cells became localized in close proximity to CD8+ T cells, CD11b+ myeloid cells, and tumor cells. Conclusions: A decrease in the fraction of circulating PD-1+ CD4+ T cells, and juxtaposition of Th1, CD8+, and myeloid cells was associated with response to anti–PD-1 treatment in patients with mUC.

A blood-based immune marker for resistance to pembrolizumab in patients with metastatic urothelial cancer

“…The presented data extend our previous study on the frequency of T-cell subsets in blood samples of 56 out of these 71 patients with mUC (18). In this earlier study we demonstrated that responders harbor higher frequencies of CD4+ T-cells that express PD1 and 4-1BB when compared to non-responders at baseline, and responders showed changes in frequencies of these subsets during treatment.…”

“…In other words, we cannot support a direct relationship between a high MNTR in blood with tumor cell-entrapment by neutrophils in tumor tissue. Previously, we showed that a lack of CD4+ T helper type 1 (Th1) cells in the tumor at baseline, and their inability to cluster with CD8+ T-cells and myeloid cells upon treatment, were associated with resistance to pembrolizumab (18). Also, in case of the MNTR, it appeared that lack of CD4+ rather than CD8+ T-cells was predominantly associated with nonresponse and limited survival.…”

“…Patients with locally advanced or mUC of the bladder or upper urinary tract with an indication for pembrolizumab were included in a phase II prospective biomarker discovery study (NCT03263039), and treated as described previously (pembrolizumab, 200 mg intravenously, 3-weekly (18)). Patients were classified as responder (ongoing complete or partial response, or stable disease) or non-responder (progressive disease) at 6 months after treatment initiation according to response evaluation criteria in solid tumors (RECIST) v1.1.…”

“…Multiplex immunofluorescence was performed using OPAL reagents (Akoya Biosciences, Marlborough, MA, USA) on 4-μm sections of FFPE tumor biopsies as described previously (18). The sequence of antibody stainings was as follows: 1.…”

“…DAPI. Digital image analysis was also performed as described previously (18). Cellular densities were calculated by dividing the number of cells with a certain phenotype by the total area of that region and were averaged per patient across all regions of interest.…”