Epstein-Barr Virus (EBV) is a ubiquitous oncovirus that affects more than 90% of adults worldwide, but is especially endemic in East Asia, South East Asia, North Africa and Polynesia (1). The oncogenic potential of EBV is well established and yet evolving still, and its role has been demonstrated in the pathogenesis of epithelial malignancies such as Epstein-Barr virus- associated gastric cancers (EBVaGCs), nasopharyngeal carcinoma (NPC), lymphoepithelial like carcinoma (LELC), as well as in hematological malignancies such as Burkitt’s lymphoma, Diffuse Large B Cell Lymphoma (DLBCL), Hodgkin’s disease, and natural killer T-cell lymphomas (2).

While EBV-associated epithelial cancers vary in presentation and cell origin, these malignancies have in common a viral-mediated immune-suppressed tumor immune microenvironment, mutational signatures and epigenetic hallmarks (3). Among these epithelial cancers, the pathogenesis of NPC has been the most well described, in particular the role of EBV in transforming nasopharyngeal epithelium in a possible multi-step process from dysplasia to carcinoma (4). However, this process has not been described for other epithelial cancers, especially in lymphoepithelial like carcinoma (LELC).

We aim to discuss the pathogenesis of NPC and EBVaGC, and hypothesize the pathogenesis of LELC based on their common epigenetic, genomic and somatic mutational signatures. In particular, we examine primary pulmonary LELC (PPLELC) and consider the hypothesis of the EBV transformation of dysplastic epithelial cells in the lungs.

EBV is known to be one of the etiological agents of NPC pathogenesis (46). While lytic replication typically predominates during the acute phase of EBV infection and early dysplasia, persistent latent EBV infection, clonal expansion of infected epithelial and lymphoid cells are thought to be drivers of NPC (47), and the lytic-latent switch is an important step in malignant transformation.

EBV is potentially acquired in early childhood when the immune system is not fully developed and evolved (48). On the contrary, if acquired during teenage years, EBV potentially presents as infectious mononucleosis (49, 50). One hypothesis proposes that in early life, persistent EBV infection arises from inadequate immune clearance due to the developing immune system resulting in an infection that is likely to persist for the remainder of the carrier’s lifespan. Because of this, early infection is more likely to result in latency, which when exposed to genetic and environmental factors, is particularly susceptible to malignant transformation (51). In the case of NPC, EBV expresses a type II latency program expressing specific gene products that enhances its ability to survive and spread. This will be further elaborated in the later sections.

One hypothesis of EBV pathogenesis in EBVaGC suggests that direct ingestion of the EBV virus could result in the infection of gastric epithelial cells (52, 53). It has also been postulated that during the reactivation of the lytic phase, resident B lymphocytes present in gastric mucosa release EBV, which subsequently infects more epithelial cells, a process thought to be aided by ephrin receptor A, integrins, and non-muscle myosin heavy chain IIA (NMHCIIA) (54). This process is further facilitated by upregulation of adhesion molecule-1 through integrin β1/β2 mediated contact between B lymphocytes and gastric epithelial cells and clathrin-mediated endocytosis (3). Congruent with this hypothesis, EBV infection is found in a small number of non-neoplastic gastric mucosa, suggesting that EBV infection precedes the monoclonal expansion of EBV-infected cells in tumorigenesis (55, 56). EBV anti-VCA and anti-EBNA antibody titres are also elevated in patients with gastric dysplasia on biopsy, indicating that EBV reactivation may be related to early tumorigenesis of EBVaGC.

After establishing infection in epithelial cells or B lymphocytes, EBV maintains a type I or II latency programme. This includes the expression of latent genes like EBERs, EBNA-1, Bam-HI A rightward transcripts (BARTs), miR-BARTs, and latent membrane protein 2A (LMP2A) (57, 58). EBER1 upregulates insulin growth factor 1, promoting the growth of EBVaGC (59). It also triggers chemoresistance and cell migration through the IL-6-STAT3 pathway (60), and facilitates tumorigenesis by blunting cellular responses to DNA damage through disruption of promyelocytic leukemia nuclear bodies (61). Furthermore, EBNA1 sequesters reactive oxygen species via miR-34a and NOX2, increasing cell viability (62). LMP2A was found to activate the nuclear factor kappa-light-chain-enhancer of B cells (NF-κb) pathway to upregulate miR-155-5p, resulting in the inhibition of p-Smad2 and Smad 2, inducing epigenetic alterations in the host genome (63).

PPLELC is a poorly differentiated carcinoma with a rich lymphoid infiltration in its stroma, which is histologically indistinguishable from metastatic NPC. This raises the possibility of a “converging” role of EBV transformation in these epithelial cells, leading to a poorly differentiated subtype. Whilst the majority of LELC existing in case series are of PPLELC, gastric LELC (64), intrahepatic cholangiocarcinoma (ICC)-LELC (65) and parotid LELC (29), which suggest an aerodigestive epithelial nidus of pathogenesis, not all LELCs described originate from epithelial cells directly in contact with environmental carcinogens or EBV infection.

At present, our understanding of how EBV contributes to PPLELC pathogenesis is limited, primarily relying on inferences drawn from our knowledge of EBV in the context of NPC, and similarities in the gene signatures and EBV latency in PPLELC. We propose that PPLELC adopts a similar model whereby a type II latency program in dysplastic tissue, accompanied by other predisposing factors, leads to the development of cancer ( Figure 1 ).

Current literature has also established the presence of EBV viral load in PPLELC and its correlation to treatment response and prognostication (66–68). Here, we also posit the idea that early childhood latent infection of EBV in patients who develop PPLELC in later years possess underlying genetic predispositions such as variations in HLA-haplotypes and TLR subtypes, similar to the existing multistep model proposed in NPC (4). Subsequently, EBV oncoproteins and its downstream signaling pathways drive airway epithelium into malignant epithelial cancers ( Figure 1 ). However, due to the rarity of this disease and its sparse literature, the genetic and environmental predispositions remain to be proven by genome-wide association studies and epidemiology studies.

With the nasopharynx being located in the upper airway, and the lungs in the lower airway, both are exposed to similar viral infections and carcinogens; similarly, the nasopharynx and the upper digestive tract also share other carcinogens through inhalation and ingestion.

Several environmental risk factors have been found to increase NPC and EBVaGC risks. Dietary consumption of salt-preserved fish and preserved foods such as eggs, meat, and vegetables (12, 17, 69, 70) are highly associated with NPC. Exposure to wood dust (71–75), formaldehyde and solvents such as phenoxy acid and chlorophenol (76, 77) have also been linked to NPC and EBVaGC (23–25). Additionally, steam, flammable products, cotton dust, and smoking also increased NPC and EBVaGC risks (22, 78). A case-control study conducted among male patients in the Guangdong province demonstrated that a smoking pack-year of 40 or more predisposed an individual to a significant odds ratio of 1.76 to contract NPC. Similarly, 20-40 smoking pack-years was associated with a significantly increased risk of NPC, with an odds ratio of 1.52.

Histological and geographical similarities between NPC and PPLELC have prompted increasing comparisons between the two. Even though epidemiological studies on PPLELC are sparse, a recent study in Singapore revealed racial similarities, in which the Chinese race is more frequently affected (79). While still unexplored, a Chinese predominance may suggest that risk factors common in the traditional diet or environment of endemic regions could also be associated with PPLELC. While race is a similarity between PPLELC and NPC, there are some differences. For example, PPLELC tends to occur in non-smokers (43) and females (79). More studies are warranted to better describe the environmental risk factors associated with PPLELC.

Apart from environmental and epidemiological risk factors, certain genomic variants increase susceptibility to NPC. Several HLA alleles have been associated with increased risks of NPC, such as HLA-A1, HLA-A2, HLA-A*0207, HLA-A*33:03, HLA-B14, HLA-B*38:02, and HLA-B46 (80, 81). On the other hand, HLA-A11, HLA-A*11:01, HLA-A23, HLA-A*31:01, HLA-B*13:01, HLA-B14, HLA-B22, HLA-B27, HLA-B55, HLA-B*55:02, and HLA-DR4 were found to be protective factors against NPC carcinogenesis (80, 81). Variants in expressions and polymorphisms in toll-like receptors (TLR), a mediator of the immune system, were also found to increase the risk of NPC. Sequence variants in TLR10 and a functional variant in the 3’UTR of TLR4 were associated with increased NPC risk (82, 83). Genetic polymorphisms of TLR3 (84), TLR9 (85), and TLR4 (86, 87) were also linked to increased risks of NPC.

Compared to NPC, little is known about HLA and TLR variants in EBVaGC and PPLELC. While one study with 52 EBVaGC patients found no interactions between EBV and TLR polymorphisms (88), other recent studies have shown a reduced level of TLR9 in EBVaGC, suggesting its potential involvement in EBV reactivation (89). In PPLELC, TLR variants and HLA haplotypes conferring increased susceptibility have yet to be investigated, and is an avenue for future research.

In NPC, early events such as genetic susceptibility, environmental carcinogens and epigenetic alterations drive normal epithelium to dysplasia through chromosomal loss and EBV-related malignant transformation (4). Subsequently, further somatic mutations and/or epigenetic inactivation events (cell cycle checkpoint, TP53 and other TSG inactivation) lead to tumor progression ( Figure 2 ). This process is accompanied by angiogenesis, metabolic dysregulation and increased invasiveness, resulting in metastasis and immune evasion.

Early studies suggest chromosomal aberrations that are detected in the dysplastic nasopharynx, including the loss of chromosomes 3p, 9p, 11q, and 14q (90–93), with the most widely known being 3p (95%) and 9p21 (85%) (4). Interestingly, these genetic deletions occurred even in the epithelium of people with no NPC in endemic regions, suggesting that these epigenetic changes occur early in disease, and may predispose individuals to EBV infection (91, 93, 94). Significant hypermethylation at chromosome 6p21.3 were found in both NPC and EBVaGC (95). This region contains numerous genes encoding for tumor suppressors and HLA genes that are known to be important risk factors in NPC (95, 96). Apart from the epigenetic alterations in tumor suppressor genes (TSGs), amplification of several oncogenes were also found in 8-20% of NPC. These include phosphatidylinositol-4,5-bisphosphonate 3-kinase catalytic subunit α (PIK3CA) and CCND1 on 3q26.3 and 11q13, respectively. The lymphotoxin-β receptor, a NF-κB signaller found on 12p13, was also amplified. Augmentations in PI3K-Akt and NF-κB signaling contribute to the tumorigenesis of NPC (97–99). Similar to NPC, mutations in PIK3CA are widely seen in EBVaGCs.

Genome sequencing and array-based methylomes have revealed a viral-mediated CpG Island hypermethylation Phenotype (CIMP) in NPC (95, 100, 101). EBV is known to induce a distinctive hypermethylated epigenotype in EBVaGC, NPC and LELC (63, 102–106), where important TSGs are inactivated by extensive methylation at the promoter region (107), in line with oncogenic behavior. Hypermethylation alters the balance between DNA methyltransferases and demethylases, maintaining type II latency. LMP1 and LMP2 are key players in promoting CIMP hypermethylation, which subsequently downregulates several TSGs such as Ras association domain-containing protein 2 (RASSF2A) (108), follistatin-like 1 (109), cyclin-dependent kinase inhibitor 2A (CDKN2A) (110), p16 (111), phosphatase and tensin homolog (PTEN) (112) and cadherin-1 (CDH1) (113), facilitating tumorigenesis and subsequent metastasis (94, 114). Although the pathogenesis of EBVaGC is not yet well defined, some have suggested that when EBV enters and establishes latency in the gastric epithelium, it undergoes genome wide methylation, a process similar to that in NPC (22, 115). The methylation of TSG (APC, PTEN and RASSF1A), cell adhesion molecules (THBS1 and E-cadherin), and CDKN2A are also widely found in EBVaGC (20, 116–119). Other methylated genes include MLH1, CXXC4, TIMP2 and PLXND1 (20).

Several molecular commonalities underlie PPLELC and NPC. Molecular profiling revealed similar LMP1 (120) and BART miRNA profiles between EBV positive PPLELC and NPC (121). Due to the expression of similar latent genes, it has been postulated that PPLELC and NPC share a similar pathogenesis involving a type II or III EBV latency programme, activating similar downstream pathways (122). Whole exome sequencing data from 30 patients with PPLELC showed that the mutational landscape of PPLELC closely resembled NPC, instead of other lung cancers or NKT cell lymphoma (123). NF-κB pathway genes, such as silencing mutations of negative regulators of NF-κB including TRAF3, were found in PPLELC, suggesting a similar oncogenesis pathway to NPC (122, 123). Likewise, mutations in TP53, JAK/STAT, and cell cycle genes such as CDKN2A and CCND1 were found in both NPC and PPLELC (116, 123, 124). High activation of AID/APOBEC genes (type 2 mutation signature), indicating a secondary immune response to EBV, is associated with the carcinogenesis of PPLELC (123), again pointing to the importance of EBV in shaping the somatic mutational landscape. While the tumor mutation load of PPLELC is low, large amounts of gene copy number changes, such as 11q13.3 amplification and 9p21.3 deletion were found, similar to the copy number variation in NPC (123).

EBV positive NPC has several unique traits which are distinct from other cancers, and these characteristics are reflected in PPLELC. Although a loss of function mutation of TSG p53 is present in many cancers, EBV associated NPC is known to lack this aberrance and usually have a normal profile of p53. Similarly, p53 levels were also found to be normal in EBV positive PPLELC tissues and with low levels of mutated p53 in PPLELC (122), suggesting a similar mechanism could be driving both NPC and PPLELC. This is similar to EBVaGC, where p53 mutations are rarely seen (125, 126).. EGFR activating mutations, ALK gene arrangement and ROS1 gene arrangement were also rarely found in PPLELC (127). Microsatellite instability is exceedingly rare in NPC (128), and this has been reflected in PPLELC (129). Also, the loss of heterozygosity at D5S346 (5q23) is common, suggesting that the pathogenesis of PPLELC is associated with a TSG in that region (129).

Existing models in NPC postulate a multistep pathogenesis process where early environmental exposures as well as distinct genetic profiles predispose an individual to EBV, and acquired epigenetic changes may result in susceptibility to EBV infection ( Figure 2 ). Upon infection by EBV, latent genes expressed upregulate certain gene products which facilitate cell survival and malignant transformation, resulting in the further development of NPC (91). In view of its similarities to NPC as well as a similar model described for EBV related lung cancers (130), we suggest that PPLELC may adopt a similar multistep pathogenesis pathway ( Figure 1 ). We hypothesize that EBV infection is an early transforming event, and that along with predisposing environmental risk factors, normal airway epithelial cells are transformed into poorly differentiated carcinoma.

EBV (human herpes virus type 4) is a double-stranded DNA (dsDNA) of approximately 170 KB. EBV is usually transmitted through the saliva (131) and infects both lymphoid and epithelial cells. Primary infection of EBV occurs as the virus enters the mucosal epithelium from the oropharynx and spreads to lymphoid tissues where it gains entry into the nasopharyngeal epithelial cells, facilitated by Ephrin A2 receptor and the vitronectin receptors αvβ5, αvβ6 and αvβ8 (132–134). Unlike in the latent phase where the EBV genome is only replicated once throughout the cycle, in the lytic phase, the EBV genome can undergo large numbers of amplification to generate numerous viral genomes which can infect other cells (81). While recent studies of the lytic phase and its involvement in tumorigenesis (135, 136) suggest that both phases might be involved in oncogenesis, the role of the latent phase in driving both lymphoid and epithelial tumors has been well established (91).

EBV enters a latent phase where the virus remains dormant in the nucleus and the carrier remains asymptomatic. EBV establishes latency, ranging from type I to III. In type I latency, latent genes such as EBNA1, EBER, and BART RNAs are expressed (137). In type III latency, nine proteins, namely (EBNA)-1, -2, -3a, -3b, -3c, and -LP, and (LMP)-1, -2a, and -2b are expressed. Of note, latency II gene expression is known to play a primary role in the pathogenesis of NPC (138), resulting in the expression of latent genes such as EBV nuclear antigen 1 (EBNA1), LMP1, LMP2, EBV-encoded small RNAs (EBER), Bam-HI A right frame (BARF) proteins, BARTs, and BART microRNAs (miR-BARTs). EBV effectively transforms and immortalizes B cells through various latent gene expressions such as LMP1, LMP2A (139) EBNA, BART and mir-BARTs (140), and a similar type II latency programme is suggested to exist in PPLELC (122). LMP1, LMP2a, and the NF-κB pathways were found to be involved in the carcinogenesis of PPLELC (122), suggesting that the pathway of tumorigenesis is likely to be highly similar ( Figure 1 ).

Stage 0 and I NPC are usually treated with radiotherapy to the site of disease, with prophylactic radiation to cervical lymph nodes (242). Patients with stage II, III, and IV NPC are treated with concurrent chemoradiation (chemoRT), with stage III and IV NPC requiring either induction or adjuvant chemotherapy on top of chemoRT, usually cisplatin or cisplatin with fluorouracil (5-FU) or adjuvant oral capecitabine (243). Recurrent or metastatic NPC can be treated with palliative radiotherapy, chemotherapy, targeted therapy and/or immunotherapy. On the other hand, no clear treatment guidelines for PPLELC have been outlined at present (34). In general, surgical treatment is the mainstay of treatment for early PPLELC, and treatment options like chemotherapy, radiotherapy are usually reserved for more advanced stages of PPLELC (45). Because PPLELC is uncommon, the adoption of standardized treatments has been challenging. Existing treatment regimes are adapted from those used for stage IV NPC, which involves gemcitabine-based chemotherapy (45), taxanes (244), anti-angiogenic therapy (245), and anti-PD1/PD-L1 therapy (246). The favorable response of PPLELC to NPC treatments is evident of the therapeutic implications of molecular similarities between the two cancer types, and the substantial overlap in their molecular characteristics provides an explanation for the effective response of PPLELC to chemotherapies typically used in NPC. For EBVaGCs, early stage cancers are treated surgically via endoscopic mucosal resection or gastrectomy. The treatment options for locally advanced EBVaGC is similar to that of other gastric cancers. This includes perioperative chemotherapy with primary resection. Advanced EBVaGCs are treated with palliative chemotherapy (taxanes, irinotecan, platinums, and fluoropyrimidines) and VEGF-R antagonist ramucirumab (247).

The role of immunotherapy in the treatment of NPC has been increasingly explored and this is especially so because the tumor microenvironment of EBV-driven NPC creates conditions favorable for immunotherapy. Much attention has been placed on immune-checkpoint inhibitors (248). In 44 patients with recurrent or pretreated metastatic NPC, patients receiving nivolumab achieved a 1-year overall survival rate of 95% and 1-year-progression free survival of 19.3% (249). In the KEYNOTE-028 study (250), 27 patients with PD-L1 positive unresectable or metastatic NPC achieved a 25.9% objective response rate of partial response and stable disease. The more recent JUPITER-02 study demonstrated that the addition of toripalimib to the standard first line chemotherapy treatment of gemcitabine and cisplatin yielded a significant increase in progression-free survival (HR = 0.52, 11.7 v.s 8.0 months) and overall response rate (77.4 v.s 66.4%) (251). The addition of camrelizumab to the standard gemcitabine and cisplatin chemotherapeutic agents in the CAPTAIN-1st trial also demonstrated a significantly increased progression-free survival (HR = 0.54, 9.7 v.s 6.9 months) (252). In RATIONALE-309, the addition of tislelizumab to standard chemotherapy resulted in a significantly improved progression-free survival (HR = 0.50, 9.6 v.s 7.4 months) (253). However, the efficacy of immunotherapy does not extend when it is used alone as KEYNOTE-122 showed no improvement in overall survival when pembrolizumab was used as a monotherapy as compared to the standard chemotherapy agents used for recurrent or metastatic NPC (254).

Similar to NPC, PD-1/PD-L1 inhibitors are potential therapeutic options in PPLELC (255).. A recent study demonstrated that the use of sintilimab, pembrolizumab, and nivolumab in patients with advanced PPLELC helped achieve stable disease in 60% of the patients (255). The use of nivolumab resulted in partial remission in 36.4% of the patients, and stable disease in 54.5% of the patients. While the predictive value of PD-L1 as a biomarker of immune checkpoint inhibitor remains debatable (256, 257), a recent study by Zhong et al. demonstrated that higher PD-1/PD-L1 and LAG3 levels were indicative of a significantly better progression-free survival in a retrospective study (19 months v.s 3.9 months) (257).

Immunotherapy such as pembrolizumab have also demonstrated efficacy in EBVaGC as one study demonstrated a 100% (6/6) response to pembrolizumab in patients with EBVaGC (258). However, given the small sample size of that study, it is necessary to validate this finding with larger studies. The positive response of immunotherapy could suggest a potentially synergistic effect when administered alongside chemotherapy in EBVaGC. Though not shown yet, this is currently seen in advanced gastric cancer patients where the combination of chemotherapy and immunotherapy such as pembrolizumab and nivolumab was superior to using chemotherapy as a single agent (259), as well as in NPC as seen in the JUPITER-02 study.

Apart from immunotherapy, another treatment option is to employ cytotoxic T lymphocytes (CTLs). EBV-CTL is an autologous T-cell therapy generated through irradiated EBV-transformed lymphoblastoid cell lines (LCL). It was found that infusion of autologous EBV-CTL post lymphodepletion had efficacy as an anti-tumor agent for patients with locoregional NPC. In a recent phase III trial (VANCE trial, ESMO 2022 Oral presentation) in patients with recurrent or metastatic NPC, EBV-CTL administration following gemcitabine and carboplatin (GC) treatment also did not reveal superior efficacy over first line standard of care GC. Still, in the VANCE trial, improved survival trends were seen in a subset country specific analysis of overall survival and progression free survival for Taiwan, United States, and Singapore (unpublished). EBV-CTL may still be a viable treatment option as CTLs specific for LMP2 were shown to be associated with a significantly higher overall survival in a phase II study (260). EBV-CTL also demonstrated a very favorable safety profile (261). Like NPC, EBV-CTL also displayed efficacy in the treatment of PPLELC. In an unpublished study, our group was able to demonstrate complete response to EBV-CTL in a patient with heavily pre-treated advanced PPLELC in combination with an immune checkpoint inhibitor after previous progression on immune checkpoint inhibitor after just a few months.

A phase II trial in patients with locally recurrent NPC found that patients with Endostatin, an inhibitor of angiogenesis, administered in addition to cisplatin and paclitaxel had longer progression free survival with increased disease control rates (262). It was also found that anti-angiogenic therapy on top of radiotherapy improved clinical outcomes (263). Similarly, angiogenesis inhibition was also efficacious in PPLELC. In one study, small molecule multi-targeted TKI, inhibiting VEGFR, platelet derived growth factor (PDGFR), FGFR, and c-KIT was used to treat a patient with FGFR3-TACC3 fusion, and response was favorable. While studies are limited, this suggests that anti-angiogenic therapy can potentially be employed in the treatment of PPLELC (245).

Targeting EBV latent proteins has also shown potential as a viable therapeutic option. Not only is EBNA1 the only viral protein expressed in all EBV infected cells regardless of latency type, its vital role in maintaining EBV episomal genome and its consistent expression of EBNA1 in all tumor cells makes it a promising therapeutic target (47). Administration of conditionally replicating adenovirus expressed in an EBNA-1-dependent manner (adv.oriP.E1A) in EBV-positive NPC cells demonstrated cytotoxic effects and tumor regression in xenografts models, albeit limited clinical potential due to liver sequestration (264, 265). Preventing DNA binding of EBNA1 has also been an area of study. In a high-throughput virtual screen, several small molecule inhibitors, such as LB2, LB3, LB7 and LC7, SC7, SC11, SC19, SC27) (266) have been identified to selectively inhibit EBNA1 DNA binding (267). Others have also developed molecules that could inhibit EBNA1 DNA binding in xenografts, such as 2,3-disubstituted benzoic acid (268), of which one of its inhibitors is in a Phase/I/IIa clinical trial (269) (NCT03682055) in EBV positive NPC (47). Apart from targeting EBNA1, inhibition of LMP1 targeting DNAzyme found lower short-term tumor regression rate in a clinical trial of 40 patients with NPC (270).

Lytic inducers have the benefit of sensitizing tumor cells to anti-virals via the expression of viral kinases (271) during EBV reactivation. During reactivation, highly immunogenic viral replication proteins and virions are expressed which results in a strong immune response against EBV reactivated tumor cells (272). Because of this, oncogenesis is largely dependent on latency, and therapeutic strategies have been devised to inhibit latent genes or activate lytic genes with the aim of inducing oncolysis. For instance, it was found that transfection of EBNA-driven CMV BZLF1 plasmid induced BZLF1 reactivating EBV, resulting in cell death (273). Other compounds, such as histone deacetylase inhibitors (HDACis) or protein kinase C (PKC) activators have also been used to activate EBV lytic genes, albeit with varying effectiveness (274–277). This variability is likely attributed to the highly cell-dependent sensitivity towards lytic inducers, which also explains the diverse responses observed in oncolysis following the administration of gemcitabine and valproic acid in Phase I/II trials (278). Somatic mutations in TP53 and TGFBR2 were also found to maintain EBV latency in NPC (122, 279, 280), and may affect a patient’s response to lytic-inducer treatment.

The goal of understanding the intricacies of cancer pathogenesis is to intervene as upstream as possible, thereby thwarting the progression into dysplastic changes. Mitigating dysplasia and minimizing exposure to risk factors play pivotal roles in substantially lowering cancer risk. Consequently, there has been increasing focus on advancing vaccine development, implementing nuanced screening methodologies, and cancer biomarker development as part of a comprehensive preventative approach. Patients with NPC were found to have persistently elevated plasma EBV DNA (281) and plasma EBV is increasingly used as a screening tool for NPC (9) and a surveillance biomarker for patients in remission (282). Posttreatment plasma EBV DNA is the strongest independent predictor for cancer recurrence and long term survival, with high EBV DNA levels strongly correlating with disease progression, and low plasma EBV DNA posttreatment correlating with high progression-free survival rate at 2 years (283). Because EBV insidiously results in disease, therapeutic EBV vaccines can be highly efficacious and cost effective in lowering disease recurrence (284). The majority of current vaccines currently target EBV latent proteins such as EBNA1, LMP1, LMP2 (284). Several trials have also explored the potential of chimeric antigen receptor T cell (CAR-T) and T cell receptor-engineered T cell therapy (TCR-T) immunotherapy, with the latter more effective for solid tumors (284).

With EBV being a commonality behind the pathogenesis of EBV related epithelial cancers, we speculate the possibility of a universal off-the-shelf cell therapies targeting EBV-driven cancers and diseases, and this is an avenue for future research. This is especially relevant since recent studies noted clinical improvement in EBV-CTL treated EBV-driven MS, in which B-cells are proposed to be involved (285). With PPLELC and NPC sharing similar biological and molecular characteristics, it is unsurprising that treatments involving the same therapeutic targets have shown efficacy.

Of the epithelial cancers associated with EBV, NPC and PPLELC have a predominant distribution in South China and Southeast Asia compared to the western population. On the other hand, EBVaGC is more prevalent in the western countries such as Germany and the United States (15-18%) (22). The oncogenic properties of EBV in NPC are well documented, and its latent genes LMP1, LMP2A, EBNA1, and BART transcripts, as well as its lytic genes, such as BZLF1, are known to participate in multiple pathways promoting tumor growth and metastasis, as well as imparting immuno-evasive characteristics. An overview of the characteristics of NPC, EBVaGC and PPLELC is shown in Table 1 . While the connection between EBV and NPC has been well documented, its precise mechanisms remain elusive.

EBV infection is the common oncogenic pathogen underlying several seemingly anatomically unrelated cancers, in particular, NPC, EBVaGC and PPLELC. Even though EBV is spread through droplets, it is often absent in nasopharyngeal epithelium even though EBV titres are high (48). This led to hypotheses that EBV could remain latent in other anatomical sites (48). This postulation is bolstered by the suggestion that one of the variants of NPC originates from the basal cells of the respiratory epithelium despite NPC’s squamous origin (286).

Although EBV is involved in NPC, EBVaGC and PPLELC, it enters different latency programs as evidenced by their distinct molecular profiles. EBVaGCs express LMP2A, BARTs, miR-BARTs, EBNA1, and EBER (57, 58), however, they express significantly lower levels of EBNA2 and LMP1 compared to NPC. This suggests that EBVaGC exhibits a type I latency program (287), and is more similar to burkitt’s lymphoma and distinct from NPC (30, 287, 288). On the other hand, EBV latent genes expressed in NPC include EBNA1, LMP1, LMP2, EBER, BARF, BARTS and miR-BARTs, characteristic of a type II latency program. While the type of EBV latency exhibited in LELC still remains to be uncovered, expressions of LMP1 and LMP2 strongly suggest a Type II or III EBV latency program, akin to that of NPC (122). Understanding the pathogenesis of NPC may guide current understanding of PPLELC pathogenesis.

Due to the rarity of PPLELC, there is a paucity of studies examining the role of EBV in PPLELC and our understanding is still in its infancy. Histological similarities between NPC and PPLELC suggest a closely related pathogenesis, and current perspectives of the role of EBV in NPC pathogenesis can potentially guide our future understanding of EBV in PPLELC and. Despite different cell lines of origin in NPC and PPLELC, the mutational landscape in PPLELC implies similar driver mutations in NF-κB, CDKN2A, JAK/STAT pathway. They also have similar p53 and PD-L1 regulatory patterns. Its expression of latent phase gene products such as LMP1 and LMP2 also suggests that it has a type II latency program similar to NPC. The parallels in genomic and molecular profiles of EBV positive NPC and PPLELC raise the possibility for common therapeutic strategies for advanced NPC and PPLELC. With its distribution mainly in Asia as well as its good prognosis, future studies investigating the mechanisms driving the PPLELC can potentially guide therapeutic targets. Given the histopathological and molecular similarities between NPC and PPLELC, it is unsurprising that therapeutics proven effective for NPC have demonstrated efficacy in treating PPLELC as well. This convergence in treatment strategies particularly between NPC and PPLELC could potentially extend to other EBV-related epithelial cancers in future. The development of a universal EBV-directed therapeutic approach in future holds promise to effectively treat a wide range of EBV-positive epithelial cancers, including but not limited to NPC and PPLELC. However, because of the limited studies in PPLELC, studies on EBV CTL or other novel EBV related therapies on PPLELC can help guide our understanding in this area.

The complex interplay between genetic mutations with local inflammation of the nasopharyngeal epithelium is thought to establish latent EBV infection, which is the primary driver of NPC pathogenesis (289). Despite the molecular similarities between NPC and PPLELC, more studies are required to better understand the distinct in vivo microenvironments of their respective host stromal components. The role of the in vivo microenvironment in pathogenesis is reinforced by the challenges in replicating the microenvironment in vitro when creating NPC cell lines, as well as the continuous loss of EBV episomes in NPC cells in vitro ( 289). Our group is currently investigating the spatial transcriptomic differences between NPC and PPLELC. A clearer understanding of the evolutionary advantages occurring in vivo in both NPC and PPLELC pathogenesis can elucidate fresh avenues for therapeutic intervention.

Though the role of EBV in the pathogenesis of NPC is well-described in existing literature, there is still much to be understood about the role of EBV in the pathogenesis of LELC. In this review, we summarized the genetic and environmental factors that influence susceptibility to NPC, and the role of EBV in the stepwise pathogenesis of NPC. We also summarized the differences and similarities of the roles of EBV in both NPC, EBVaGC, and postulated its role in LELC pathogenesis. With increasing understanding of the role of EBV in LELC pathogenesis, better therapeutics can be developed, and novel more specific drug targets can be elucidated to improve the treatment options of EBV driven cancers.

YL and CL contributed equally to manuscript writing and share first authorship. AC contributed to manuscript writing. DP contributed to the illustration of the manuscript. SH and HT both contributed to conception, guidance, and manuscript writing. All authors contributed to the article and approved the submitted version.