MULTI-OMICS DISSECTION OF EBV-DRIVEN HETEROGENEITY, METABOLIC REMODELING, AND TUMOR CELLULAR LANDSCAPE IN ENKTL PROGRESSION

Introduction: ENKTL is an aggressive lymphoma associated with EBV infection. While predominantly involving the upper aerodigestive tract (UAT) with better-prognosis early-stage disease (Model-I), advanced UAT-ENKTL (Model-II) and non-UAT (NUAT; Model-III) exhibit worse survival outcomes, validated in our 341-patient cohort. We utilized multi-omics approach integrating EBV virome analysis with tumor microenvironment and metabolic reprogramming to decipher the molecular drivers of clinical heterogeneity.

Methods: The study enrolled 65 ENKTL patients (pts) from our center. We profiled using DNA-target gene sequencing (N = 42), RNAseq (N = 35), metabolomic assay (N = 42) and scRNAseq with EBV tag (N = 23) (Figure A).

Results: We classified ENKTL into five groups: early-stage TN (GroupA), early-stage PD (GroupB), advanced UAT-ENKTL TN (GroupC), non-UAT TN (GroupD), and non-UAT PD (GroupE). Single-cell RNAseq (n = 280,939 cells) revealed T/NK cells, macrophages, and fibroblasts as predominant cell types (Figure B). By leveraging EBV tags at single-cell resolution, we characterized EBV infection patterns, showing predominant infection of NK cells (10%–67%, except NK_C13) and Teff cells (Figure C). Contrary to prior understanding, all EBV+ NK/T cells exhibited latency type I infection (Figure D). EBV+ NK cells were more prevalent in nasal versus non-nasal lesions and in PD versus TN lesions (GroupB > A, E > D). InferCNV analysis identified malignant NK cells, and they distributed in distinct groups with different functions (Figure E). Widely distributed NK_C1/C3 displayed functional trends aligned with the above. Bulk RNAseq validated the associations of NK_C4/C8 with advanced stages and poor prognosis by ssGSEA score (Figure F-G). EBV+ NK_C3/C8/C11/C12/C14 showed enhanced DNA modification and innate immunity, whereas EBV- NK cells mediated adaptive immunity, migration, and energy metabolism (Figure H). Gene module analysis uncovered two mutually exclusive modules: Module 4 featured immune activation (NK_C1/C2/C4), while Module 8 involved metabolic reprogramming (glycolysis/nucleotide metabolism) linked to poor prognosis (Figure I–L). Serum metabolomics revealed lipid metabolism enrichment in Model-III versus deficiency in Model-I. Monocle2 analysis demonstrated increasing blast NK cells during progression, with early-stage pts dominated by mature NK cells retaining cytotoxic functions (Figure M–O). CytoTRACE analysis confirmed higher stemness in EBV+ versus EBV- NK subpopulations, supporting EBV-driven proliferation (Figure P). Spatial heterogeneity was observed in fibroblasts: groupA/B/C contained Fibroblasts_MMP1 mediating ECM remodeling, while groupD/E featured Fibroblasts_ADH1B involved in antigen presentation and IFN signaling (Figure Q–S).

Conclusions: In this largest multi-omics study focused on ENKTL, different models display distinct clinical, metabolic, and cellular landscape features, comprehensively revealing the heterogeneity and potential mechanisms of disease progression.

Research funding declaration: No funding disclosure

Encore Abstract: EHA 2025

Keywords: tumor biology and heterogeneity; aggressive T-cell non-Hodgkin lymphoma; metabolism

No potential sources of conflict of interest.