Nasopharyngeal carcinoma at the virology precision oncology nexus: decoding molecular alterations for early intervention and therapeutic innovation

Nasopharyngeal carcinoma at the virology precision oncology nexus: decoding molecular alterations for early intervention and therapeutic innovation | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 7 May 2025 V1 Latest version Share on Nasopharyngeal carcinoma at the virology precision oncology nexus: decoding molecular alterations for early intervention and therapeutic innovation Authors : Na Liu , Bin Meng , Yueshuo Li , Min Tang , Y Cao 0000-0002-3558-3336 , Li Shang , and Feng Shi 0009-0007-9118-7622 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174661396.69523146/v1 Published Biomolecules Version of record Peer review timeline 392 views 195 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Nasopharyngeal carcinoma (NPC) represents an Epstein-Barr virus (EBV)-associated malignancy showing elevated incidence in East and Southeast Asia. Early detection remains vital, as molecular abnormalities precede visible histological changes during tumor development. This review summarizes recent progress in decoding NPC’s molecular profile, including genetic mutations, epigenetic alterations, non-coding RNA networks, and proteomic alterations. Importantly, these molecular discoveries are increasingly informing clinical approaches to disease management. Modern diagnostic strategies combine histopathological evaluation, EBV DNA/antibodies detection, and imaging technologies. However, locoregional recurrence and distant metastases continue to dominate as primary causes of NPC-related deaths. Immunotherapy has demonstrated growing potential for treating recurrent/metastatic NPC, showing encouraging clinical translation prospects. Nasopharyngeal carcinoma at the virology precision oncology nexus: decoding molecular alterations for early intervention and therapeutic innovation Na Liu 1,3 , Bin Meng 1,3 , Yueshuo Li 1,3 , Min Tang 1,3,4 , Ya Cao 1,3,4 , Li Shang 1,2,3 *, Feng Shi 1,2,3 * 1 Key Laboratory of Carcinogenesis and Cancer Invasion of Chinese Ministry of Education, XiangYa Hospital, Central South University, Changsha 410078, China 2 Department of Pathology, National Clinical Research Center for Geriatric Disorders/ XiangYa Hospital, Central South University, Changsha 410078, China 3 Key Laboratory of Carcinogenesis of National Health Commission, Cancer Research Institute and Xiangya School of Basic Medical Sciences, Central South University, Changsha 410078, China 4 Molecular Imaging Research Center of Central South University, Changsha 410008, Hunan, China * Corresponding author: Feng Shi, Key Laboratory of Carcinogenesis and Cancer Invasion of Chinese Ministry of Education, Department of Pathology, XiangYa Hospital, Central South University, Changsha 410078, China. E-mail address: [email protected] . Li Shang, Department of Pathology, National Clinical Research Center for Geriatric Disorders/ XiangYa Hospital, Central South University, Changsha 410078, China. E-mail address: [email protected] . Abstract : Nasopharyngeal carcinoma (NPC) represents an Epstein-Barr virus (EBV)-associated malignancy showing elevated incidence in East and Southeast Asia. Early detection remains vital, as molecular abnormalities precede visible histological changes during tumor development. This review summarizes recent progress in decoding NPC’s molecular profile, including genetic mutations, epigenetic alterations, non-coding RNA networks, and proteomic alterations. Importantly, these molecular discoveries are increasingly informing clinical approaches to disease management. Modern diagnostic strategies combine histopathological evaluation, EBV DNA/antibodies detection, and imaging technologies. However, locoregional recurrence and distant metastases continue to dominate as primary causes of NPC-related deaths. Immunotherapy has demonstrated growing potential for treating recurrent/metastatic NPC, showing encouraging clinical translation prospects. Keywords : Nasopharyngeal carcinoma, Viral infection, Molecular alteration, Immunotherapy Introduction NPC tumorigenesis involves a multistep interplay of EBV oncogenesis, genetic susceptibility, environmental carcinogens, and epigenetic dysregulation 1-5 . Notably, over 70% of global NPC cases occur in East and Southeast Asia, with crude incidence and mortality rates in China reported as 3.09 and 1.57 per 100,000, respectively 1,6,7 . Locoregional recurrence and distant metastasis remain leading causes of mortality, contributing to a five-year survival rate of 50–60% for advanced-stage (III/IV) patients 8,9 . In contrast, early-stage (I/II) patients exhibit survival rates exceeding 90%, underscoring the importance of early detection 10,11 . Molecular alterations, including genetic mutations, epigenetic dysregulation, and viral oncogenic mechanisms, precede histopathological changes, offering promising avenues for biomarker discovery and therapeutic innovation. Traditional treatment strategies such as radiotherapy, chemotherapy, and surgery are often associated with severe adverse effects and limited efficacy. In recent years, immunotherapy has emerged as a promising treatment approach for patients with NPC. A growing body of clinical research has validated the safety and effectiveness of immunotherapy and demonstrates its considerable potential in this field. 1 EBV infection mechanisms and molecular pathogenesis NPC is an infection-associated malignancy primarily driven by EBV. EBV, the first identified human tumor virus, is strongly implicated in the etiology of multiple lymphoid and epithelial cancers 12 . B Lymphocytes and epithelial cells are the main target cells of EBV. During EBV infection of epithelial cells, the viral BMRF2 protein first anchors to integrin. Subsequently, the gH/gL heterodimer cooperatively binds to both integrin and ephrin receptor A2, triggering conformational activation of the fusion protein gB, which ultimately mediates the fusion of the viral envelope with the host cell plasma membrane 13,14 . Thus, the EBV envelope glycoproteins gH/gL and gB are critical mediators of EBV infection in epithelial cells. Following initial infection, EBV establishes lifelong latency in the host. EBV exhibits three latency types: type I, type II, and type III. NPC is associated with type II latency, during which the virus expresses latent proteins such as EBNA1, LMP1, LMP2, along with non-coding RNAs including BARTs and EBERs 14 . These latent molecules disrupt intracellular signaling pathways and drive NPC pathogenesis through multiple mechanisms 15 . Histologically, NPC is classified into keratinizing squamous cell carcinoma, differentiated non-keratinizing carcinoma, undifferentiated non-keratinizing carcinoma, and basaloid squamous cell carcinoma. Notably, EBV is detected in nearly all cases of undifferentiated NPC, particularly in endemic areas 1,16 . Given its strong epidemiological association with NPC, EBV-derived biomarkers have become cornerstone tools for screening and diagnosis. Detections targeting EBV DNA and EBV antigens (anti-VCA IgA, anti-EBNA1-lgA and anti-EA IgG) are widely used for NPC screening in high-risk populations 17,18 . 2 Current diagnostic The diagnostic paradigm for NPC integrates multimodal approaches, with histopathological confirmation remaining the diagnostic gold standard (Figure 1). This involves endoscopic-guided biopsy of suspicious nasopharyngeal lesions, the procedure’s invasiveness, patient discomfort, and risk of complications have driven exploration of advanced non-invasive diagnostic modalities. Emerging deep learning technologies now augment traditional imaging: a Siamese deep convolutional neural network (S-DCNN) leveraging both white light imaging (WLI) and narrow-band imaging (NBI) achieves 94.9% diagnostic accuracy, significantly outperforming single-modality models, offering a robust computer-aided tool for early screening 19 . High-resolution magnetic resonance imaging (MRI) and NBI, are used as first-line investigations for detecting subtle mucosal abnormalities and guiding targeted biopsies in early-stage lesions that evade conventional endoscopic visualization 20-22 . MRI encounters diagnostic challenges in distinguishing T1-stage NPCs from benign hyperplasia and identifying diffuse symmetrical tumors lacking focal masses, requiring endoscopic correlation despite demonstrating higher sensitivity than endoscopic examination 23 . Positron emission tomography/computed tomography (PET/CT) has demonstrated established feasibility and efficacy in tumor diagnosis, treatment planning, prognostic evaluation, and disease surveillance. Fluciclovine F-18, gallium Ga-68 DOTATATE, and lutetium Lu-177 DOTATATE are FDA-approved for PET imaging 24 . A multicenter cohort study demonstrates the potential of artificial intelligence (AI) in enhancing MRI-based surveillance for NPC recurrence. While AI-matched MRI performed in sensitivity (74.3% vs. 74.7%, P=0.89), AI-aided MRI significantly improved specificity (92.5% vs. 85.0%, P=0.034) and approached positron emission tomography/computed tomography (PET/CT) equivalence in select cohorts 25 . Contrast-enhanced CT demonstrates superior prognostic stratification over traditional tumor-node-metastasis (TNM) staging, with a model combining radiomics score (flatness, mean, gray level non-uniformity in gray level dependence matrix) and N stage effectively identifying locoregionally advanced NPC candidates for deintensified therapy (5-year survival: 90.7%) 26 . At the tissue level, in situ hybridization for EBERs remains the gold standard for confirming EBV association, demonstrating near-universal positivity in NPC specimens 27,28 . Immunohistochemical detection of latent membrane protein 1 (LMP1) points to a role for this viral oncoprotein as a key effector molecule in NPC pathogenesis 29 . Whole slide imaging has revolutionized pathology diagnosis by enabling AI-driven digital image analysis, demonstrating potential to reduce diagnostic time burdens on pathologists 30 . Recent advances in weakly supervised computational pathology, notably the Tokens-to-Token Vision Transformer framework (WS-T2T-ViT), achieved an AUC of 0.989 for NPC classification on whole slide images without manual annotation 31 . WS-T2T-ViT addressed labor-intensive workflows while demonstrating robustness across both NPC and CAMELYON16 datasets 31 . Molecular alterations in premalignant lesions often precede morphological changes, highlighting the potential of molecular biomarkers for early detection and precision classification. Given the strong association between EBV infection and NPC, EBV-related molecular markers are frequently used to assist in diagnosis 32 . Clinically validated markers include plasma EBV DNA loads and serological profiles of EBV-specific antibodies (e.g., anti-VCA IgA, anti-EBNA1-lgA, anti-EA IgG), which exhibit high sensitivity for NPC detection 1,33,34 . A landmark prospective trial demonstrated that combining VCA-IgA and EBNA1-IgA serology improved early NPC diagnosis rates by 21–79% and reduced NPC-related mortality by 88% 35,36 . A meta-analysis involving 5729 NPC patients demonstrated EA-IgA levels significantly predicted overall survival (HR = 1.63, 95% CI 1.07–2.48) 37 . Plasma EBV DNA load exhibits superior diagnostic specificity in resolving false-positive cases, while also serving as a robust prognostic indicator 8,38-40 . Post-treatment EBV DNA persistence correlates with poor clinical outcomes, including diminished 3-year overall survival, distant metastasis-free survival, and disease-free survival 41 . Meta-analyses validated plasma EBV DNA’s utility in detecting locoregional recurrence (sensitivity: 68.8%; specificity: 80.0%; accuracy: 85.9%,), regional recurrence (sensitivity: 80.2%; specificity: 80.0%; accuracy: 78.2%) and distant metastasis (sensitivity: 91.1%; specificity: 80.0%; accuracy: 92.8%) 42 . Notably, plasma EBV DNA outperforms peripheral blood mononuclear cell (PBMC)-based assays in specificity and sensitivity for EBV-associated diseases 43 . These findings collectively establish EBV DNA and specific antibodies as a pivotal biomarker for early diagnosis, recurrence monitoring, and survival prediction in NPC. Nasopharyngeal brushing is an effective sampling method for NPC diagnosis 44 . The quantified EBV DNA loads in nasopharyngeal brushing samples demonstrated comparable levels to parallel biopsy results, establishing it as a valid diagnostic biomarker 45 . It directly reflects EBV genomic content at the primary tumor site surface in NPC, with its elevation attributed to tumor-derived viral DNA rather than newly replicated virus 45,46 . Currently, nasopharyngeal brush sampling remains predominantly dependent on endoscopic guidance. Notably, EBV DNA methylation analysis (Cp-promoter region) in nasopharyngeal brushing samples without endoscopy shows high accuracy (AUC=0.902-0.928), surpassing EBV DNA load quantification (AUC=0.865) and offering a practical method for population screening without the need for specialized endoscopic procedures 47 . These virological markers are increasingly integrated into diagnostic algorithms, with emerging multi-analyte models combining EBV DNA quantification and circulating tumor biomarkers showing promise for risk stratification and treatment monitoring. 3 Molecular regulatory networks of nasopharyngeal cancer NPC pathogenesis arises from multifaceted molecular changes encompassing genetic mutations, epigenetic modifications, and non-coding RNA/protein dysregulation. Comprehensive elucidation of these mechanisms enables precise identification of diagnostic biomarkers and therapeutic targets. 3.1 Genetic aberrations The pathogenesis of NPC involves a multistep process with cumulative genetic changes across multiple pathways (Figure 2). This malignancy exhibits a relatively low mutation rate and frequent copy number alterations. Analysis of 40 NPC patients through targeted next-generation sequencing identified KMT2D and TP53 as the most frequently mutated genes, consistent with findings from the COSMIC and cBioPortal databases 48 . In addition, mutations in the retinoblastoma-related gene RB2/p130 (exons 19-22), identified in 30% of North African NPC cases, demonstrate its tumor suppressor role and therapeutic targeting potential 49 . Whole-genome sequencing (WGS) of NPC tissues and cell lines identified recurrent chromosomal imbalances, including frequent gains at 1q, 3q, 8q, 11q, 12p, and 12q, with critical amplified regions at 3q27.3-28, 8q21-24, and 11q13.1-13.3 50-54 . A 5.3-Mb amplicon at 11q13.1-13.3 is particularly prominent, driving amplification and overexpression of the cyclin D1 ( CCND1 ) in NPC primary tumors 55 . Functional studies demonstrated that siRNA-mediated knockdown of CCND1 significantly suppressed NPC cell proliferation 55 . Notably, amplification of the 11q13 locus (encompassing CCND1 , FGF14 , FGF3 , and FGF4 genes) correlates with reduced therapeutic response to toripalimab (anti-PD-1 therapy) 52 . This association was evidenced in a phase II trial (NCT02915432) of 190 NPC patients, where none of the 12 subjects harboring this genomic alteration achieved objective response (objective response rate: 0%), highlighting its clinical significance in immunotherapy resistance 52 . PIK3CA gene amplification is found in 21.6% of NPC cases and was strongly associated with distant metastasis, lymph node involvement, and advanced tumor stage 56 . Another oncogene related to cell proliferation and cellular differentiation, MYC , located in 8q24, is amplified in 62% of NPC tumors 53 . Conversely, high-frequency allelic losses are observed at 3p, 9p, 9q, 11q, 13q, 14q and 16q, with minimally deleted regions mapped to 3p14.1-22, 11q13.3-24, 13q14.3-22, 14q24.3-32.1 and 16q22-23 50,51 . The deletion of key oncogenes located within these chromosomal regions exerts critical functional roles in driving nasopharyngeal carcinogenesis. Notably, homozygous deletions of CDKN2A / CDKN2B and MTAP at 9p21.3, along with recurrent TGFBR2 (3p24.1), TRAF3 (14q32.3), and CYLD (16q12.1) losses, drive constitutive NF-κB activation, immune evasion, and EBV persistence in tumorigenesis 57 . CHL1 (3p26.3) is downregulated in 4/6 NPC cell lines and 74.7% of primary tumor specimens 58 . Re-expression of CHL1 suppresses clonogenic potential and attenuates migratory capacity 58 . Collectively, these findings demonstrate that NPC progression is driven by cumulative genetic alterations disrupting tumor suppression, activating oncogenic signaling and enhancing immune evasion, thereby delineating molecular vulnerabilities for precision therapeutic intervention. 3.2 Epigenetic dysregulation DNA methylation plays pivotal roles in NPC carcinogenesis 59 . DNA methylation is catalyzed by a family of DNA methyltransferases that transfer methyl groups from S-adenosylmethionine to the 5th carbon position of cytosine residues, forming 5-methylcytosine 60 . To elucidate the functional roles and clinical relevance of DNA methylation in NPC, this review systematically analyzes methylation patterns and their associations with disease screening, survival outcomes, and prognostic stratification. Aberrant promoter methylation of tumor suppressor genes and proto-oncogenes drives malignant phenotypes. Specifically, hypermethylation-mediated silencing of RASSF1A , NFAT1 , ACAT1 , USP44 , HOPX , and 14-3-3 sigma coupled with hypomethylation-driven overexpression of FGF5 , ELF3 and S100A4 collectively promote NPC progression by enhancing proliferation, migration, immune evasion, and therapy resistance 61-70 . Methylation-driven oncogenic pathways hold clinical utility as diagnostic biomarkers. Multi-gene methylation panels analyzed in tissue specimens, including AIM1 , APC , CALCA , DCC , DLEC , DLC1 , ESR , KIF1A and PGP9.5 , exhibit high diagnostic specificity (>92%) albeit with variable sensitivity (26–66%) 71 . FHIT methylation demonstrates lower specificity in comparison 71 . Hypermethylation of RERG and ZNF671 in circulating cell-free DNA (ccfDNA) exhibits high diagnostic accuracy for NPC screening, outperforming conventional tissue-based methylation markers in a cohort of 79 NPC patients and 29 controls 72 . Methylation-sensitive high-resolution melting analysis demonstrated that a four-gene methylation panel ( RASSF1A , WIF1 , DAPK1 , RARβ2 ) detected in noninvasive plasma and nasopharyngeal brushing samples significantly improved both sensitivity and specificity for early-stage and recurrent NPC detection when combined with EBV DNA testing 73 . Blind nasopharyngeal brushing combining EBV methylation marker BILF2 and host-derived IMPA2 methylation achieves 84.62% sensitivity and 98.44% specificity for noninvasive NPC detection 74 . In addition, SEPT9 methylation detected in nasopharyngeal swabs exhibits promise as a minimally invasive biomarker for early NPC detection 75 . Methylation profiling reveals prognostic biomarkers in NPC, where hypermethylation of WIF1 (84% frequency), UCHL1 (61% frequency), RASSF1A (49% frequency), CCNA1 (52% frequency), TP73 (95% frequency) and SFRP1 (85% frequency) demonstrates significant correlations with both shorter disease-free survival (HR= 2.26, 95% CI 1.28-4.01) and worse overall survival (HR= 2.47, 95% CI 1.30-4.71) 76 . High methylation levels of HOPX in NPC tissues predict advanced tumor stage and inferior clinical outcomes 77 . These findings collectively establish DNA methylation as a dual-purpose tool in NPC management—facilitating early diagnosis through non-invasive sampling and enabling risk stratification via prognostic signatures. 3.3 MicroRNA dysregulation in nasopharyngeal carcinoma: mechanisms, biomarkers, and therapeutic frontiers MicroRNAs (miRNAs) are endogenous non-coding single-stranded RNAs approximately 22 nucleotides in length that regulate gene expression by binding to the 3′-UTR, 5′-UTR and coding regions of target mRNAs 78 . They mediate mRNA destabilization or translational repression to modulate critical signaling pathways and cellular processes 79-81 . In NPC, the host’s intrinsic regulatory network is disrupted by synergistic interactions between EBV-encoded miRNAs and host-derived dysregulated miRNAs, promoting tumor initiation, metastasis, immune evasion, and therapeutic resistance (Figure 3). For instance, EBV-miR-BART11 and EBV-miR-BART17-3p promote immune evasion, while EBV-miR-BART22 and host-derived miR-106A-5p drive malignant transformation and therapy resistance 82-84 . Circulating miRNAs exhibit remarkable stability in biofluids such as plasma, serum, and saliva, positioning them as transformative biomarkers for non-invasive cancer diagnosis and prognosis 85 . A 5-miRNA panel (let-7b-5p, miR-140-3p, miR-192-5p, miR-223-3p, miR-24-3p) achieves AUCs of 0.910-0.968 across multi-stage validation cohorts, while a 4-miRNA prognostic classifier (miR-22, miR-572, miR-638, miR-1234) independently predicts overall survival and distant metastasis risk 86,87 . Elevated pretreatment and post-treatment plasma EBV-miR-BART8-3p levels demonstrate significant associations with reduced overall survival, distant metastasis-free survival, and locoregional relapse-free survival in NPC 88 . The diagnostic landscape is further enriched by the concept of ’tumor-educated platelet (TEP)’, where platelet-absorbed miR-34c-3p and miR-18a-5p demonstrate exceptional diagnostic accuracy (combined AUC=0.954) 89 . Salivary miRNA profiling has emerged as a novel diagnostic strategy for NPC, with a recently identified 12-miRNA signature (miR-30b-3p, miR-575, miR-650, miR-937-5p, miR-1202, miR-1203, miR-1321, miR-3612, miR-3714, miR-4259, miR-4478, and miR-4730) that demonstrates exceptional diagnostic accuracy in differentiating NPC patients from healthy controls (AUC=0.999, sensitivity: 100.00%, specificity: 96.00%) 90 . Therapeutic exploitation of miRNA networks advances through dual strategies: suppression of oncogenic miRNAs (e.g., targeting the miR-28-3p/BIN1 axis or EBV-miR-BART22/MOSPD2 pathway to reverse protumorigenic signaling) and restoration of tumor-suppressive miRNAs (e.g., miR-205 mediated EMT inhibition or miR-873/ZIC2-driven stemness regulation to overcome chemoresistance) 84,91-93 . Given the multifaceted roles of miRNAs in nasopharyngeal carcinogenesis, future investigations should prioritize: (1) systematic identification of optimal miRNA biomarkers for NPC diagnosis and therapeutic monitoring; (2) development of miRNA-based precision therapeutics targeting critical pathological pathways to enhance treatment efficacy. 3.4 Long non-coding RNAs in nasopharyngeal carcinoma: oncogenic drivers, prognostic signatures, and therapeutic frontiers Long non-coding RNAs (lncRNAs) represent a structurally and functionally heterogeneous subgroup of non-coding RNAs defined as transcripts exceeding 200 nucleotides in length 94 . lncRNAs have emerged as central orchestrators of NPC pathogenesis, driving tumorigenesis, metastasis, and therapeutic resistance through multifaceted regulatory mechanisms (Figure 4). lncRNAs are categorized into competing endogenous RNAs (ceRNAs) and activating non-coding RNAs 95 . For example, lncRNA FAM225A accelerates tumor progression by sponging miR-590-3p and miR-1275 to hyperactivate the FAK/PI3K/AKT signaling axis, a mechanism tightly correlated with advanced TNM staging and dismal survival outcomes 96 . BC200, an EBV-upregulated lncRNA that promotes NPC proliferation and migration through miR-6834-5p sequestration, which relieves the miRNA’s repression on TYMS, thereby driving TYMS-mediated metabolic reprogramming 97 . RRFERV stabilizes TEAD1 by competitively sequestering miR-615-5p/miR-1293, promoting radiotherapy resistance while transcriptionally upregulating ACSL4/TFRC-dependent lipid peroxidation to sensitize nasopharyngeal carcinoma to ferroptosis 98 . Activating ncRNAs positively regulate nearby genes 95 . PVT1 fosters radioresistance by activating DNA repair pathways, and HOTAIRM1 reinforces this resistance through FTO-mediated m6A demethylation that promotes CD44 alternative splicing and suppresses irradiation-induced ferroptosis via the HOTAIRM1-FTO-YTHDC1-CD44V axis 99,100 . The METTL3-stabilized lncRNA SUCLG2-AS1 mediates long-range chromatin looping between SOX2 enhancers and promoters to drive NPC metastasis and radiosensitivity 101 . Meanwhile, circulating POU3F3 independently drives metastasis via TGF-β1 activation, establishing its prognostic value in NPC progression 102 . In addition, circulating lncRNAs demonstrate remarkable clinical potential as dynamic biomarkers. MALAT1, AFAP1-AS1, and AL359062 exhibit stage-dependent elevation and EBV co-expression, with serial monitoring revealing significant post-treatment declines, underscoring their utility in liquid biopsy applications 103 . 3.5 Proteomic alterations Proteomic analysis serves as a pivotal approach for investigating cancer biomarkers. In NPC, proteomics has been extensively utilized to systematically identify potential biomarkers and perform comparative analyses of differentially expressed proteins. Laser capture microdissection combined with 2D electrophoresis and mass spectrometry identified 36 differentially expressed proteins between NPC and normal tissues, with stathmin upregulation and 14-3-3sigma/annexin I downregulation strongly correlating with poor histologic differentiation, advanced clinical stage and recurrence 104 . Secretome profiling identified plasma CCL5 as a promising biomarker, demonstrating significant efficacy in distinguishing NPC patients from healthy controls (AUC = 0.801) 105 . The synergistic combination of serum exosomal cyclophilin A and EBV-VCA-IgA achieved enhanced diagnostic specificity for NPC screening 106 . Using 2D electrophoresis and mass spectrometry to analyze serum proteins from 42 healthy individuals, 27 non-lymph node metastasis NPC patients, and 37 lymph node metastasis NPC patients, three proteins (HSP70, sICAM-1, SAA) were identified as potential metastasis-specific biomarkers for NPC through ELISA and immunohistochemistry 107 . Prognostically, integrating plasma EBV DNA load with serum high-sensitivity C-reactive protein levels significantly improved risk stratification in advanced cases, showing heightened predictive value for disease-free survival and distant metastasis 108 . The profound impact of EBV infection on NPC pathogenesis has been further elucidated through proteomic investigations. The systematic mapping of virus-host interactomes through iTRAQ-coupled two-dimensional liquid chromatography–tandem mass spectrometry revealed 12 significantly upregulated proteins (VDAC1, S100-A2, Hip-70, Ubiquitin, TPT1-like protein, 4F2hc, Keratin-75, TB8, Dynein light chain 1, LDH-B, TIM and HMG-1) in EBV-infected NPC cells 109 . The tumor microenvironment’s proteomic complexity further amplifies disease progression, as evidenced by comparative stromal protein analyses between NPC and normal nasopharyngeal mucosa. Overexpression of the extracellular matrix protein periostin demonstrates significant associations with advanced clinical stage, lymph node metastasis, and reduced overall survival 110 . These proteomic discoveries not only enhance our understanding of NPC pathogenesis but also provide tangible candidates for clinical biomarker development across different disease stages. 4 Immunotherapy for Nasopharyngeal Carcinoma More than 70% of NPC patients present with stage III/IV disease at initial diagnosis due to the nasopharynx’s concealed anatomy and nonspecific early symptoms 111 . And the standard treatment for locoregionally advanced NPC involves induction chemotherapy combined with concurrent chemoradiotherapy, where gemcitabine and cisplatin constitute a first-line induction option 112 . Despite this intensified approach, 20-30% of patients experience disease recurrence 112,113 . Immunotherapy has emerged as a promising therapeutic approach for recurrent/metastatic NPC, with primary strategies encompassing immune checkpoint blockade, EBV-directed vaccination, and adoptive T-cell therapy (Table 1). NPC exhibits elevated PD-L1 expression, with 83-92% of patients showing PD-L1 positivity in tumor cells or tumor-associated immune cells, making them potential candidates for anti-PD-1 immunotherapy 112,114 . Four landmark phase 3 trials (NCT03581786, NCT03707509, NCT03924986 and NCT03700476]) established the efficacy of PD-1 inhibitors (toripalimab, camrelizumab, tislelizumab and sintilimab) in recurrent or metastatic NPC 112,114-116 . Despite clinical success, anti-PD-1 monotherapy demonstrates only modest overall response rates in advanced cancers 117 . Thus, combination therapies targeting PD-1 with other immune checkpoints are urgently needed. CTLA-4 acts as a negative regulator of T cell activation. And CTLA-4 overexpression in tumor tissue predicts poor survival in NPC patients 118 . While single-agent checkpoint inhibitors exhibit limited activity in EBV-related NPC, combined nivolumab (anti-PD-1) and ipilimumab (anti-CTLA-4) therapy demonstrates enhanced efficacy, as evidenced by a phase II trial (NCT03097939) reporting a best overall response rate of 38% and median progression-free survival and overall survival of 5.3 and 19.5 months, respectively, in recurrent or metastatic EBV-positive NPC 119 . And a single-arm, open-label, phase 2 trial (ChiCTR2200067057) also confirmed the clinical efficacy of cadonilimab (PD-1/CTLA-4 bispecific) combined with chemotherapy in anti-PD-1-resistant recurrent or metastatic NPC 120 . QL1706 (PSB205) comprises two engineered monoclonal antibodies: anti-PD-1 IgG4 and anti-CTLA-4 IgG1 121 . In the phase I/Ib clinical trial (NCT04296994 and NCT05171790), QL1706 at the recommended phase 2 dose (RP2D) demonstrated an objective response rate of 24.5% (27/110) with a median duration of response of 11.7 months in patients with NPC 121 . LAG-3, an inhibitory receptor, is highly expressed on exhausted T cells 122 . It suppresses T cell function by interacting with major histocompatibility complex class II molecules 122 . An open-label, multicenter, Ib/II trial (NCT05102006) demonstrated manageable safety and promising antitumor activity of LBL-007 (anti-LAG-3 antibody) combined with toripalimab in advanced solid tumors, including NPC 117 . So, these findings warrant further validation in future studies. Given the etiological role of EBV in nasopharyngeal carcinogenesis, EBV prophylactic vaccines hold considerable promise for preventing EBV-associated malignancies by blocking primary infection or suppressing viral latency establishment. EBV envelope glycoproteins, including gH/gL and gB, play critical roles in EBV entry into and infection of epithelial cells, with neutralizing antibodies triggered by these glycoproteins demonstrated to block viral infection 13,123,124 . A self-assembling nanoparticle vaccine displaying the EBV gH/gL elicited potent neutralizing antibodies and conferred robust protection against lethal EBV infection in humanized mouse models 123 . Nanoparticle-based EBV gB constructs, gB-I53-50 NPs, exhibited enhanced structural stability and protected against viral challenge in murine and non-human primate preclinical models 125 . EBV therapeutic vaccine targets focus on EBNA1, LMP2. EBNA1 serves as the primary target for CD4⁺ T cells, whereas LMP2 represents the predominant target for CD8⁺ T cells 126,127 . In a phase I trial (NCT01256853) of recombinant modified vaccinia ankara (MVA-EL) encoding an EBNA1/LMP2 fusion protein, sustained remission (>12 weeks) was maintained post-initial treatment 128 . And MVA-EL efficiently expanded the EBNA1- and LMP2-specific CD4+ and CD8+ T cells from the peripheral blood lymphocytes of seropositive healthy donors in vitro 128 . A second UK phase I trial (NCT01147991) of the MVA-EL vaccine demonstrated enhanced polyfunctional differentiation of EBNA1/LMP2-specific CD4+/CD8+ T-cell subsets 129 . Although EBV vaccines have demonstrated promising clinical benefits, further investigation is warranted. The persistent presence of EBV in NPC cells enables targeting through T-cell recognition of expressed viral antigens, including EBNA1, LMP1, and LMP2. Building on this approach, adoptive T-cell therapy employing in vitro-expanded cytotoxic T lymphocytes (CTLs) targeting type II latency antigens (EBNA1, LMP1, LMP2) has been successfully developed for clinical application 13,130-132 . A clinical trial of autologous EBV-specific CTLs achieved disease control in 60% (6/10) of refractory stage IV NPC patients 130 . Therefore, immune cell therapy targeting NPC has emerged as another highly promising therapeutic approach. Conclusions and future directions NPC exemplifies a malignancy shaped by intricate interactions between EBV oncogenesis, host molecular vulnerabilities, and environmental cofactors. Its distinct molecular landscape, characterized by epigenetic reprogramming, genetic alterations, non-coding RNA dysregulation, and proteomic rewiring, provides critical insights into tumor initiation, progression, and therapeutic resistance. Early detection remains paramount, as molecular aberrations in premalignant lesions precede morphological changes, offering a window for intervention. Current diagnostic paradigms integrate histopathological validation, EBV biomarker profiling and advanced imaging modalities. Therapeutic innovation is increasingly guided by molecular stratification. Immunotherapy has emerged as a cornerstone for recurrent/metastatic NPC, with PD-1 inhibitors (toripalimab, camrelizumab, tislelizumab, sintilimab) demonstrating durable responses in phase III trials, albeit limited by genomic resistance mechanisms (e.g., 11q13 amplification). And EBV-targeted strategies, including EBV-directed vaccination and adoptive T-cell therapy, show preclinical promise in neutralizing viral entry and suppressing oncoprotein signaling, while adoptive T-cell therapies achieve disease control in refractory cases. The key focuses of NPC research will involve exploring the biological mechanisms of pathogenesis, improving screening and staging strategies, and identifying biomarkers linked to prognosis risk stratification and treatment benefits. These efforts will be complemented by ongoing work to optimize treatment strategies for different patient subgroups and develop novel therapies. Looking ahead, future research must prioritize the systematic integration of multi-omics data to refine biomarker panels for enhanced early detection, precise risk stratification, and dynamic therapeutic monitoring. Acknowledgments This work was supported by the National Natural Science Foundation of China (82073030, 81602402 and 81502109); the Hunan Provincial Natural Science Foundation of China (2022JJ20083); the Program of Education and Teaching Reform in Central South University (2023ALK032 and 2024JY035); and Independent Exploration and Innovation Project for Postgraduate Students at Central South University (2025ZZTS0234). Conflict of interests The authors declare no competing interests. Authors’ contributions Na Liu: Conceptualization, Visualization, Writing–original draft. Bin Meng: Review and editing, Visualization. Yueshuo Li: Review and editing, Visualization. Min Tang: Review and editing, Visualization. Ya Cao: Supervision, Funding acquisition. 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Table 1 Progress in immunotherapy strategies Immune checkpoint blockade NCT03581786 (JUPITER-02) Phase III Toripalimab + chemotherapy Recurrent or metastatic NPC patients 289 Toripalimab vs Placebo (median OS: NE vs 33.7 months; median PFS: 21.4 vs 8.2 months; ORR: 78.8% vs 67.1%; DCR: 88.4% vs 80.4%; median DOR: 18.0 vs 6.0 months) 114 NCT03707509 (CAPTAIN-1st) Phase III Camrelizumab + chemotherapy Recurrent or metastatic NPC patients 263 Camrelizumab vs Placebo (ORR: 87·3% vs 80·6%; DCR: 96·3% vs 94·6%; median DOR: 8·5 vs 5·6 months) 115 NCT03924986 (RATIONALE-309) Phase III Tislelizumab + chemotherapy Recurrent or metastatic NPC patients 263 Tislelizumab vs Placebo (ORR: 69.5% vs 55.3%; DCR: 89.3% vs 84.8%; median DOR: 8.5 vs 6.1 months) 116 NCT03700476 (CONTINUUM) Phase III Sintilimab + chemotherapy High-risk non-metastatic stage III–IVa locoregionally advanced NPC patients 425 Sintilimab vs Standard (EFS rate at 36 months: 86% vs 76%; DMFS rate at 36 months: 90% vs 83%; LRFS rate at 36 months: 93% vs 86%; OS rate at 36 months: 92% vs 92%) 112 NCT03097939 Phase II Nivolumab + ipilimumab Recurrent or metastatic EBV-associated NPC patients 40 PR: 37.5%; SD: 17.5%; PD: 42.5%; DCR: 55% 119 ChiCTR2200067057 Phase II Cadonilimab (PD-1/CTLA-4 bispecific) + chemotherapy Anti-PD-1-resistant recurrent or metastatic NPC patients 25 ORR: 68%; DCR: 92%; CR: 12%; PR: 56%; SD: 24%; PD: 4% 120 NCT04296994 and NCT05171790 Phase I/Ib QL1706 (PSB205) NPC patients 110 CR: 0%; PR: 24.5%; SD: 24.5%; PD: 47.3%; ORR: 24.5%; DCR: 49.1% 121 NCT05102006 Phase Ib/II LBL-007 + toripalimab Advanced NPC patients 30 Immunotherapy-naive vs immunotherapy-treated (PR: 33.3% vs 11.8%; SD: 41.7% vs 52.9%; PD: 25.0% vs 35.3%; ORR: 33.3% vs 11.8%; DCR: 75.0% vs 64.7%) 117 EBV-directed vaccination Preclinical studies gH/gL nanoparticle vaccine Humanized mice Induce effective neutralizing antibody responses and prevent lethal EBV challenge 123 Preclinical studies gB nanoparticle vaccine Mice and non-human primate Induce effective neutralizing antibody responses and prevent lethal EBV challenge 125 NCT01256853 Phase I MVA-EL (encode an EBNA1/LMP2 fusion protein) EBV-positive NPC patients 18 T cells in 15/18 patients exhibit enhanced responses to vaccine antigens 128 NCT01147991 Phase I MVA-EL (encode an EBNA1/LMP2 fusion protein) EBV-positive NPC patients 14 T cells in 8/14 patients exhibit enhanced responses to vaccine antigens 129 Adoptive T-cell therapy Clinical trial Autologous virus-specific cytotoxic T lymphocytes Stage IV NPC patients 10 Induces LMP-2 specific immunologic responses 130 ORR: objective response rate; DCR: disease control rates; DOR: duration of response; PFS: progression-free survival; OS: Overall survival; PFS: Progression-free survival; Chemotherapy: gemcitabine-cisplatin; NE: not evaluable; EFS: event free survival; DMFS: distant metastasis free survival; LRFS: locoregional recurrence free survival; PR: partial response; SD: stable disease; PD: progressive disease; CR: complete response; PR: partial response; SD: stable disease Figure 1 Integrated Framework for Multimodal Diagnosis and Digital Pathology in NPC. Nasopharyngoscopy is the most effective method for diagnosing nasopharyngeal lesions, enabling biopsy of abnormal mucosa for pathological confirmation. White light imaging and narrow-band imaging enhance traditional endoscopic visualization. High-resolution magnetic resonance imaging, positron emission tomography/computed tomography and contrast-enhanced CT provide critical adjunctive diagnostic value. EBV infection is strongly linked to nasopharyngeal carcinogenesis. EBV DNA and EBV-specific antibodies serve as biomarkers for early screening. At the tissue level, in situ hybridization for EBERs remains the gold standard for confirming EBV association. Immunohistochemical detection of LMP1 aids in clinical diagnosis. Tokens-to-Token Vision Transformer framework facilitates AI-driven digital pathology, reducing diagnostic time burdens for pathologists. Figure 2 Genetic and Molecular Drivers of NPC Pathogenesis. NPC exhibits a relatively low mutation rate with frequent copy number alterations. The disease progression is driven by cumulative genetic alterations that disrupt tumor suppression mechanisms, activate oncogenic signaling pathways, and enhance immune evasion capabilities. KMT2D, TP53, and RB2/p130 are the most frequently mutated genes. Whole-genome sequencing identified recurrent chromosomal imbalances, including frequent gains at 1q, 3q, 8q, 11q, 12p and 12q, with key amplified oncogenes (CCND1, FGF14, FGF3, FGF4, PIK3CA and MYC). High-frequency allelic losses occur at 3p, 9p, 9q, 11q, 13q, 14q, and 16q, involving critical tumor suppressor genes (CDKN2A/CDKN2B, MTAP, TGFBR2, TRAF3, CYLD and CHL1). Figure 3 Regulatory networks of viral and host miRNAs in NPC progression. Viral miRNAs: EBV-miR-BART11 and EBV-miR-BART17-3p target FOXP1 and PBRM1, respectively, relieving their suppression of PD-L1 transcription and enabling immune evasion via PD-1/PD-L1 interaction; EBV-miR-BART-22 promotes EMT and metastasis by targeting MOSPD2, activating Wnt/β-catenin signaling. Host miRNAs: MIR106A-5p, transactivated by EGR1/SOX9, suppresses autophagy via BTG3 targeting and MAPK signaling activation, driving malignant transformation; miR-28-3p inhibits apoptosis and reduces BIN1 protein levels; miR-205 suppresses HER3, while miR-873 downregulation enhances ZIC2 expression, activating AKT signaling and cancer stemness. Figure 4 Long noncoding RNAs and associated molecules in NPC tumorigenesis, metastasis, and radioresistance. Competing endogenous RNAs involves: (1) LncRNA FAM225A acting as a ceRNA to sponge miR-590-3p/miR-1275, upregulating ITGB3 and activating pro-metastatic signaling; (2) EBV-associated BC200 binding miR-6834-5p to derepress TYMS, fueling metabolic reprogramming; (3) RRFERV stabilizing TEAD1 via miR-615-5p/miR-1293 sequestration, while transcriptionally upregulating ACSL4/TFRC to modulate lipid peroxidation. Activating non-coding RNAs involves: (1) PVT1 and c-Myc interaction activating ATM phosphorylation; (2) HOTAIRM1 enhancing FTO stability through acetylation, which drives m6A demethylation of CD44 transcripts, favoring CD44V splicing and ferroptosis suppression; (3) METTL3-mediated m6A modification stabilizing SUCLG2-AS1, facilitating chromatin looping to amplify SOX2 expression; (4) POU3F3 promoting TGF-β1-mediated migration and invasion. Information & Authors Information Version history V1 Version 1 07 May 2025 Peer review timeline Published Biomolecules Version of Record 5 Feb 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords antiviral agents epstein-barr virus pathogenesis virus classification Authors Affiliations Na Liu Central South University View all articles by this author Bin Meng Central South University View all articles by this author Yueshuo Li Central South University View all articles by this author Min Tang Central South University View all articles by this author Y Cao 0000-0002-3558-3336 Central South University View all articles by this author Li Shang Central South University View all articles by this author Feng Shi 0009-0007-9118-7622 [email protected] Central South University View all articles by this author Metrics & Citations Metrics Article Usage 392 views 195 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Na Liu, Bin Meng, Yueshuo Li, et al. Nasopharyngeal carcinoma at the virology precision oncology nexus: decoding molecular alterations for early intervention and therapeutic innovation. Authorea . 07 May 2025. 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