Comprehensive analysis of nasopharyngeal carcinoma using sequencing

Abstract

Nasopharyngeal carcinoma (NPC), endemic in Southern China, is the distinct type of head and neck cancer that originates from the nasopharynx and frequently metastasises to neck lymph nodes and distal organs such as bone, lung and liver. Genetic predisposition, environmental factors and Epstein-Barr virus (EBV) have been associated with NPC tumourigenesis.

Recently, tumour microenvironment (TME) studies showed expression of programmed cell death protein 1 (PD-1) inhibitory receptor on cytotoxic tumour-killing T cells and its ligand programmed death-ligand 1 (PD-L1) in tumour cells of NPC. Immunotherapy targeting PD-1/PD-L1 on NPC has gained some insights into novel treatment possibilities. However, responsive rate still remains limited.

In this study, we recruited 18 patients, collected 41 samples including 16 normal, 17 primary tumour and 8 lymph node samples, and launched single-cell sequencing of NPC with largest scale to comprehensively characterize the cellular components in NPC TME and tumour cells. First we found that T cells, B cells, tumour-associated macrophages (TAMs), cancer-associated fibroblasts and dendritic cells were major TME cells in NPC. Next, we confirmed that most druggable inhibitory receptors were mainly found on T cells and TAMs. Interestingly, in particular, we found that CD4 Treg expressing especially high level of previously overlooked inhibitory receptors CTLA4 and TIGIT rather than PD-1 were highly correlated with locoregional spread to neck lymph node, an indicator of poor prognosis in NPC. Novel immune-suppressive TAMs expressing HAVCR2 was also identified to be highly correlated with CD4 Treg in NPC. Our results suggested that we should target CTLA4+TIGIT+ CD4 Treg and HAVCR2+ TAMs in immunotherapy.

Distinct from other cancers, most common cancer-driving gene mutations were found to be infrequently mutated in NPC. Usually patients are diagnosed of NPC at advanced stage and quite a number of patients treated with standard chemo-radiotherapy eventually still relapse. It still remains largely elusive if rare but aggressive subpopulation of NPC tumour cells may pre-exist and contribute to progression of NPC to advanced stage. Identification of previously overlooked tumour subpopulation is important to develop combination therapy to limit progression.

In this regard, we first confirmed with aberrant copy number variations (CNVs) that our extracted tumour cells were of high purity. Then we characterized genetic heterogeneity and evolution of NPC tumour cells based on copy number variations (CNVs). It was found that subclone accumulation and some CNV aberrations were highly associated with locoregional neck lymph node spread. Next, comprehensive transcriptomic characterisation in combination of trajectory inference of heterogenous tumour cell subpopulations in NPC showed more advanced NPC may be associated with ability to induce immunosuppressive or exhausted T cells and tumour cells with cell adhesion molecules (CAMs) acquired at later tumourigenic stage. Interestingly, we found that early stage patients may also have the potential to develop to advanced stage based on existence of more aggressive tumour subpopulations. Finally, we had also identified a number of novel immune check point pathways that may contribute to tumour development.

In short, our results supported that immunosuppressive and/or exhausted TME, and heterogeneity of NPC tumour cells were associated with more advanced NPC and CTLA4/TIGIT/LAG3/HAVCR2 pathways may be new directions for NPC immunotherapy.