Circulating extracellular Vesicle–Derived mirnas identify a high-risk subgroup in extranodal NK/T-cell lymphoma with caebv-like features

Abstract Background Extranodal natural killer (NK)/T-cell lymphoma (ENKTL) is a distinct lymphoma subtype associated with Epstein-Barr virus (EBV) infection and characterized by unique clinical features. Most patients present with localized disease (stage I/II) involving the nasal cavity or nasopharynx. In this group, outcomes have improved significantly with concurrent chemoradiotherapy using non-anthracycline, L-asparaginase–based regimens. However, a subset of patients presents with stage IV involving extranasal sites such as the skin or gastrointestinal tract and often exhibits clinical features of hemophagocytic lymphohistiocytosis (HLH). These presentations overlap with those of chronic active EBV infection (CAEBV) and aggressive NK-cell leukemia (ANKL), and are frequently associated with rapidly progressive, fatal courses. Moreover, some patients initially diagnosed with localized ENKTL may later experience systemic progression and outcomes resembling CAEBV or ANKL. We hypothesized that these patients may harbor distinct biological features linked to CAEBV-like biology, which are not currently identifiable through standard staging. To date, no reliable molecular tools exist to distinguish stage I/II ENKTL patients at high risk of systemic progression with occult CAEBV-like features. Methods We conducted comprehensive small RNA sequencing of intravesicular contents from serum-derived extracellular vesicles (EVs) in ENKTL patients (n = 50) and ENKTL cell lines. EVs were isolated using the Total Exosome Isolation reagent, and small RNA libraries were prepared using the SMARTer smRNA-Seq Kit for Illumina and sequenced on the NovaSeqX platform. Data processing was performed using miRDeep2 for alignment and identification of known and novel miRNAs. Reads were sequentially aligned to the human reference genome (GRCh38), miRBase v22.1, and the RNAcentral non-coding RNA database (release 14.0). Raw counts were normalized using the Trimmed Mean of M-values (TMM) method in edgeR. miRNAs absent in over 51% of samples were excluded, leaving 735 mature miRNAs for downstream analysis. Differentially expressed miRNAs were identified using edgeR's exact test, with significance defined as |fold change| ≥ 2 and p-value < 0.05. Results Archived serum samples from 50 ENKTL patients enrolled in a prospective cohort study were used for EV isolation. Six ENKTL or CAEBV-derived cell lines (SNK-1, -6, -8, -10, -15, and -16) served as reference controls. Among the patients, 30 had early-stage disease (stage I, n = 20; stage II, n = 10), while 20 presented with advanced disease (stage IV). At diagnosis, five patients fulfilled the diagnostic criteria for HLH, and an additional 19 exhibited HLH-like clinical and laboratory features. EBV DNA was markedly elevated (>10,000 IU/mL) in 14 patients, moderately elevated (<10,000 IU/mL) in 22, and undetectable in the remaining 14. Following first-line treatment, 15 patients achieved and maintained complete remission without relapse. However, 13 patients initially diagnosed with stage I/II disease experienced relapse or progression, while 22 patients—predominantly those with stage IV disease—developed systemic progression or relapse, resulting in death. Analysis of EBV-derived microRNAs revealed that six patients with stage IV disease and HLH-like features exhibited EBV microRNA expression patterns, including BART3-3p, BART10-3p, and BART17-5p, that closely resembled those seen in ENKTL/CAEBV cell lines. Further analysis of host-derived microRNAs showed that hsa-miR-1285-3p, hsa-miR-5684, and hsa-miR-8485 were significantly upregulated in patients with HLH-like features and high EBV DNA titers (>10,000 IU/mL). In particular, hsa-miR-1285-3p was elevated in patients with stage IV disease and those stage I/II patients who later developed systemic relapse. This microRNA, previously implicated in cancer progression, also demonstrated significantly higher expression in patients with high EBV DNA compared to those with undetectable EBV DNA (P < 0.05). Conclusions Our findings suggest that a subset of ENKTL patients—particularly those with advanced-stage disease or HLH-like features—exhibits distinct circulating EBV and host microRNA profiles, resembling those seen in CAEBV and ENKTL-derived cell lines. Notably, elevated levels of hsa-miR-1285-3p were associated with systemic relapse and high EBV DNA titers, indicating its potential as a biomarker for identifying patients at high risk of aggressive disease progression.