Abstract 723: Tumor microenvironment and epithelial subtypes in nasopharyngeal carcinoma for precision oncology

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated epithelial malignancy endemic to Southeast Asia, Southern China, and the Middle East. Profiling the gene expression of NPC has been challenging due to its dense inflammatory microenvironment admixed with tumor epithelial cells, as well as limited tissue availability from small 4mm transnasal endoscopic biopsies. We performed laser-capture microdissection on a cohort of formalin-fixed paraffin-embedded (FFPE) biopsy samples obtained from NPC patients (n = 69) treated at the National University Health System Singapore and nasopharyngeal biopsies from healthy controls (n = 7). Primary, pretreatment tumors from patients who developed recurrent or metastatic disease (Group A) were compared with tumors from patients who remained healthy (Group B), matched for stage of disease. Tumor epithelial (TUM) and microenvironment (TME) regions were separately isolated. Gene expression libraries were prepared using a specialized mRNA-Seq technique optimized for tiny amounts of FFPE material, followed by next generation sequencing. Post-filtering, 330 gene expression libraries were analyzed, including 165 TUM, 134 TME, 4 dysplasia, 18 normal-adjacent and 9 normal libraries. Principal component analysis showed distinct clustering of epithelial and microenvironment gene expression libraries, confirming the purity of microdissection. Unsupervised k-means clustering revealed three distinct TME clusters (C1, C2, C3), which were also confirmed by silhouette analysis. Gene set enrichment analysis demonstrated significant enrichment of immune processes in C1, proliferative pathways in C2 and stromal processes in C3. Deconvolution of cell-type signatures with ESTIMATE showed that C1 had the highest immune score (1057, 129 and 733 respectively, p < 2e-16) while C2 had the highest tumor epithelial purity score (0.666, 0.852 and 0.715 respectively, p = 3e-16). C2 and C3 tumors had a higher recurrence rate compared to C1 (42.85%, 73.07% and 71.43% respectively, p = 0.004). Within the tumor epithelial compartment, Group A tumors were enriched for vascular endothelial growth factor signaling (NES = 1.55) and fibroblast growth factor receptor signaling (NES = 1.53), when compared to Group B. Consistent with our previous work interrogating receptor kinase signaling in NPC, FGF2 was significantly upregulated in Group A tumors (log2FC = 3.612). Microdissected gene expression profiling of FFPE patient biopsies identifies biomarkers and microenvironment subtypes important for prognosis and targeted treatment. VEGF and FGF pathway enrichment in high-risk tumors forms the basis of ongoing anti-VEGF clinical trials for NPC at our institution, with the potential of using a gene signature approach for patient selection. Citation Format: Wei Keat Teo, Yi Ren, Chee Yit Lim, Luvita Suryani, Joseph W. Foley, Bing Cheng Wu, Kwok Seng Loh, Raymond KY Tsang, Joshua K. Tay. Tumor microenvironment and epithelial subtypes in nasopharyngeal carcinoma for precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 723.