Nasopharyngeal carcinoma (NPC) is a distinct EBV-associated head and neck cancer with insidious spread patterns, often presenting as locoregionally advanced (LA) disease, requiring combinatorial chemoradiotherapy. LA-NPC presents in four subtypes: limited (L), ascending (A), descending (D), and ascending-descending (AD).

We analyzed 994 LA-NPC patients using germline and somatic whole exome sequencing (WES), transcriptomics, multiplex immunohistochemistry (mIHC), and spatial histopathology. AD-subtypes had the most germline variants, while D-subtypes showed the most immune-dense tumors. A- and AD-subtypes exhibited higher extracellular matrix gene expression, while D- and AD-subtypes had enriched immune gene expression. N0/N+ L-subtypes resembled A/D-subtypes, suggesting distinct evolutionary paths. Tumour immune microenvironment (TIME) composition correlated with disease-free survival in AD-subtypes, underscoring its role in NPC progression and treatment strategies.

The mIHC analysis showed more B cells in D-subtypes, while A-subtypes had a higher Treg/CD8+ T cell ratio, suggesting a more immunosuppressive TIME. L-subtypes (early-stage NPC) had distinct N0/N+ TIME profiles: N+ L-subtypes were immune-rich, resembling D-subtypes, whereas N0 L-subtypes had more M2 macrophages, CLDN1+ tumor cells, and lower immune infiltration. These findings suggest N+ L-subtypes may evolve into D-subtypes, while N0 L-subtypes may transition to A-subtypes. AD-subtypes, with a mixed TIME, have an unclear lineage.

mIHC images of tumor and immune-related markers from 31 patients with various NPC subtypes.

• A-subtype
• D-subtype
• N+ L-subtype – Panel 1 ; N+ L-subtype – Panel 2
• N0 L-subtype – Panel 1 ; N0 L-subtype – Panel 2
• AD-subtype – Panel 1 ; AD-subtype – Panel 2

Panel 1: CD4, CD8, CLDN1 and CK/EpCAM Panel 2: CD15, CD20, CD68, CD163 and FOXP3