FOXP3+ T-cell infiltration is associated with improved outcomes in metastatic urothelial carcinoma (mUC) treated with immune-checkpoint inhibitors (ICI).
Authors: Elio Adib, Talal El Zarif, Dory Freeman, Muhieddine Labban, Catherine Curran, Bijaya Sharma, Jane Song, Kristen Felt, Amin Nassar, Matthew Davidsohn, Scott J. Rodig, Toni K. Choueiri, David J. Kwiatkowski, Praful Ravi, Guru P. Sonpavde
Journal: Journal of Clinical Oncology (2022)

549 Background: Given that ICIs benefit a minority of mUC patients and are associated with significant costs, biomarkers are necessary to optimally utilize them in the clinic. Although FOXP3+ T-cells have been associated with an immune-cold environment in many cancers, studies in urothelial carcinoma have shown an opposite trend. Methods: Formalin-fixed paraffin-embedded slides from tumor specimens were collected for patients with mUC treated with ICI at Dana-Farber Cancer Institute. A novel multiplex immunofluorescence (mIF) panel, ImmunoProfile, was performed for PD-L1, PD-1, FOXP3, CD8 and DAPI, then scanned by a Vectra Polaris platform. Regions of interest were defined and used for quantitative analysis using PerkinElmer/Akoya. Intratumoral (IT) and tumor-stroma interface (TSI) density (cells/mm 2 ) of each cell type was calculated. Clinical data was collected through chart review, and associations between cell density and response were assessed. Wilcoxon Rank-Sum test between responders (CR/PR) and non-responders (SD/PD) was used to generate p-values, followed by Benjamini-Hochberg correction. Receiver-operating curve (ROC) and area-under-curve (AUC) calculations were performed to determine the optimal cutoff (OC) differentiating responders from non-responders. Cox proportional hazards models were used to estimate OS and PFS, accounting for type of therapy (single vs. combination ICI), baseline neutrophil-to-lymphocyte ratio (NLR), PD-L1 CPS, prior therapy, non-urothelial component, ECOG-PS and liver metastases (mets). Results: Of 35 patients assessed by ImmunoProfile, 32 were evaluable for response. Most patients (88%) were male and the median age at ICI start was 73 years. Median number of prior lines of therapy was 1 (range 0 – 3), and the majority (72%) was treated with single-agent ICI. Eight patients (25%) had CR/PR, eight had SD and 16 had PD as best response. Of all IF stains assessed, IT-TSI FOXP3 was the strongest predictor of objective response (q-value = 0.006), followed by IT-CD8 (q = 0.014). ROC analysis yielded an AUC of 0.812 (0.656 – 0.969) and the optimal cutpoint was set at 75 IT-TSI-FOXP3 cells/mm 2 . ORR was 46% in FOXP3-high ( &gt; 75/mm 2 ) vs 14% in FOXP3-low mUCs. A combined model using IT-TSI-FOXP3 and clinical covariates (NLR, ECOG-PS, line of therapy, histology, and liver mets) had an AUC of 0.929. PFS was significantly longer in the FOXP3-high group (7.9 [5.7 – NR] months) compared to the FOXP3-low group (2.3 [2.1 – 6.1] months) on multivariable analysis (p = 0.007). OS also showed the same trend (p = 0.1). Conclusions: In this pilot study of ImmunoProfile, a novel mIF panel, higher FOXP3+ infiltration in tumors was associated with better outcomes and a composite clinico-IHC panel exhibited robust prognostic impact in mUC pts treated with ICI. Further study of this mIF panel is warranted to implement it in routine practice.