Single-cell profiling of natural killer T-cell lymphoma reveals stratified immune features and therapeutic strategies

Abstract Background :Natural killer T-cell lymphoma (NKTCL) is a rare subtype of non-Hodgkin lymphoma (NHL) characterized by rapid progression and poor prognosis. The cornerstone of treatment for NKTCL patients is L-asparaginase-based chemotherapy, which exploit the metabolic vulnerability of NKTCL resulting from its dependence on extracellular asparagine for tumor growth. Similar to many other hematologic malignancies, immune checkpoint inhibitors (ICIs) are increasingly used to treat advanced NKTCL in the first-line setting. However, approximately 40% of patients are resistant to ICIs. Given the molecular heterogeneity among patients, stratified targeted therapies are essential to improve treatment efficacy and clinical outcomes. In the current study, we performed the scRNA-seq analysis on 63 samples and identified four subgroups in NKTCL, each associated with distinct immune-related TME characteristics, e.g., immune exhaustion, immune desert, immune exclusion, and tertiary lymphoid structures (TLSs) enrichment. Methods:For the scRNA-seq analysis, we included a total of 63 samples from 53 individuals, comprising five normal nasal mucosa samples from healthy controls and 58 NKTCL patient samples. Among the 58 NKTCL samples, 10 were obtained from publicly available datasets, while 48 were self-collected from our institution. The remaining two datasets were obtained from the GEO and GSA database under accession number GSE203663 and HRA000772, respectively. Xenium in situ expression analysis was conducted in 14 treatment-naive samples from 14 patients. Results Four distinct subgroups (MP1-MP4) were identified, each linked to specific molecular and immune features with varying clinical outcomes. MP1 was characterized by an immune-exhausted TME and high PD-L1 expression, suggesting a potential response to immune checkpoint blockade. MP2 was characterized by an immune-desert phenotype with elevated HDAC2 and MKI67 expression, indicating a role for epigenetic regulation in tumor proliferation. MP3 was characterized by a myeloid-dominant TME and upregulated inflammatory pathways, association with JAK/STAT pathway activation, hemophagocytic lymphohistiocytosis (HLH), and an aggressive clinical course. MP4 was characterized by tertiary lymphoid structures (TLS) and enhanced amino acid metabolism, and favorable prognosis and high sensitivity to asparaginase-based regimens. Based on these findings, We propose tailored therapeutic strategies for each MP, including combination therapies targeting immune checkpoints, epigenetic regulators, or inflammatory pathways, currently under clinical investigation. We are currently conducting a series of clinical trials investigating these proposed therapeutic strategies. These trials, including PD-1 blockade combined with anti-LAG3-antibody (NCT06649656), HDAC inhibitor (NCT04994210) or JAK1 blockade (NCT06733051), and asparaginase-based regimens (NCT06583083), aim to translate our molecular findings into tailored treatment approaches for NKTCL. Conclusion In this study, we performed a comprehensive analysis of the molecular and immune landscapes of NKTCL at the single-cell level. We identified four MP subgroups distinguished by specific tumor-intrinsic and immune features, with varying clinical outcomes and associations with potential biomarkers and therapeutic strategies. These findings were further validated by spatial transcriptomics, bulk RNA sequencing, metabolomics, proteomics, as well as by in vitro and in vivo functional experiments. Our classification reveals tumor heterogeneity in NKTCL and lays the groundwork for precision treatment strategies.