Spatial and Temporal Heterogeneity of Tumor-Infiltrating Lymphocytes in Advanced Urothelial Cancer

Checkpoint inhibitors targeting PD-(L)1 induce objective responses in 20% of patients with metastatic urothelial cancer (UC). CD8(+) T cell infiltration has been proposed as a putative biomarker for response to checkpoint inhibitors. Nevertheless, data on spatial and temporal heterogeneity of tumor-infiltrating lymphocytes in advanced UC are lacking. The major aims of this study were to explore spatial heterogeneity for lymphocyte infiltration and to investigate how the immune landscape changes during the disease course. We performed multiplex immunohistochemistry to assess the density of intratumoral and stromal CD3(+), CD8(+), FoxP3(+) and CD20(+) immune cells in longitudinally collected samples of 49 UC patients. Within these samples, spatial heterogeneity for lymphocyte infiltration was observed. Regions the size of a 0.6 tissue microarray core (0.28 mm(2)) provided a representative sample in 60.6 to 71.6% of cases, depending on the cell type of interest. Regions of 3.30 mm(2), the median tumor surface area in our biopsies, were representative in 58.8 to 73.8% of cases. Immune cell densities did not significantly differ between untreated primary tumors and metachronous distant metastases. Interestingly, CD3(+), CD8(+) and FoxP3(+) T cell densities decreased during chemotherapy in two small cohorts of patients treated with neoadjuvant or palliative platinum-based chemotherapy. In conclusion, spatial heterogeneity in advanced UC challenges the use of immune cell infiltration in biopsies as biomarker for response prediction. Our data also suggests a decrease in tumor-infiltrating T cells during platinum-based chemotherapy.