Using longitudinal spatial-omics to demonstrate in-situ Epstein-Barr virus reduction in responders to immunotherapy treated nasopharyngeal cancer.

6025 Background: Epstein-Barr virus (EBV) related nasopharyngeal cancer (NPC) is endemic in Southern China and Southeast Asia. Limited studies of tumor-immune microenvironment (TIME) modulated by dual checkpoint inhibitors (CPI) exist in relation to intratumoral EBV viral load. Methods: Spatial-omic analysis on longitudinally collected fresh frozen tissue from a phase 2 study of nivolumab/ipilimumab in NPC (NCT03097939) was done. Serial sections (10 μm) were cut for both H&E staining, and Stereo-seq assay (BGI, USA) that uses DNA nanoballs to capture mRNA which is amplified & reverse transcribed into cDNA, and then sequenced to a depth of 1 billion reads. Sequencing data was mapped to the human genome GRCh38.p14 and EBV-1 genome to identify Unique Molecular Identifiers (UMI), summarized into BIN100 niches (50μm by 50μm), processed into counts per million (CPM) and normalized for analysis. Cell type composition was estimated for each BIN100 using EBV+ NPC scRNA-seq datasets and cell2location . EBV viral load and the proportions of different cell types were associated with pre/post CPI in responders (R) and non-responders (NR) using Generalized Estimation Equation to account for repeated measures in the same slide. Colocalizations of different cell types within each BIN100 were also evaluated, adjusting for cell type proportions across all samples. P-value < 0.05 on two sided testing was considered statistically significant. Results: Samples were collected at baseline pre-treatment, and 2 weeks into treatment, and associated with clinical response. Although 21/40 patients were biopsied, only 7 pairs of pre- and on-treatment samples (3R vs 4NR) had sufficient tissue quality for analysis. The most abundant EBV genes were RPMS1, EBNA1.1, LMP2B, LMP2A, and LMP1 (using mean expression). In pre/post treatment comparisons, the EBV viral load significantly reduced in R, as reflected by EBNA1.1 (p=4.9E-11) and LMP2A (p=7.4E-23), controlling for the proportion of epithelial cells (EC), but it did not change in NR. In R, a significant reduction of B cells (p=8.2E-71) and increase of myeloid (p=1.3E-3) and natural killer cells (p=3.1E-3) was observed, while no changes in cell composition were seen in NR. Colocalization analysis of EC with two immune cell (IC) types, CD4+ T cell and CD8+ T cell, identified significantly increased colocalization only in NR (CD4+ T cell: p=4.1E-7; CD8+ T cell: p=4.6E-4), suggesting IC infiltration is induced by CPI in NR but insufficient for cell kill. Conclusions: This is the first study to suggest intratumoral viral transcriptional activity reflected by reduction in EBNA1.1 and LMP2A on CPI treated NPC correlates with response and changes in immune cell composition and colocalization. Validation of these findings at protein level using multiplex IHC/IF and spatial analysis of the virus and epithelial/immune cell neighborhood is being completed. Clinical trial information: NCT03097939 .