A New Diagnostic Marker for Secreted Epstein-Barr Virus–Encoded LMP1 and BARF1 Oncoproteins in the Serum and Saliva of Patients with Nasopharyngeal Carcinoma

Abstract

Purpose: EBV has been associated with nasopharyngeal carcinomas (NPC). In North Africa, the incidence is bimodalöthe first peak occurring at f20 years of age and the second peak occurring at f50 years. Standard diagnostic tests based on immunofluorescence using anti-IgA EBV have shown that young North African patients have a negative serology compared with older patients.We are interested in two EBV-encoded oncoproteins, LMP1and BARF1, which have thus far not been studied in terms of their potential as diagnostic markers for NPC. These two viral oncoproteins have been detected in cell culture media, so we tested whether they could be detected in the serum and saliva of patients with NPC. Experimental Design: LMP1 and BARF1 proteins were analyzed in the sera and saliva of young patients and adult patients with NPC from North Africa and China. We then examined whether the secreted proteins had biological activity by analyzing their mitogenic activity. Results: Both LMP1 and BARF1 were present in the serum and saliva from North African and Chinese patients with NPC. All young North African patients secreted both proteins, whereas 62% and 100% of adult patients secreted LMP1 and BARF1, respectively. From animal studies, the secreted LMP1was associated with exosome-like vesicles. These secreted EBV oncoproteins showed a powerful mitogenic activity in B cells. Conclusion: Both proteins will be a good diagnostic marker for NPC whereas BARF1is a particularly promising marker for all ages of patients with NPC. Their mitogenic activity suggests their implication in the oncogenic development of NPC. Nasopharyngeal carcinoma (NPC) is a human malignancyderived from the epithelium of the nasopharyngeal cavity. It is one of the most striking examples of a human malignancy that is consistently associated with a virus (1 -3). The EBV genome is contained in all malignant NPC cells and it encodes viral proteins that contribute to the malignant phenotype (4 -6). Even though infection with EBV is ubiquitous in humans, the incidence of NPC is extremely variable, depending on the geographic area. Whereas the incidence of NPCs in the Chinese population peaks at f50 years of age, there are two peaks of incidence in North Africa-one at f20 years of age and the second at f50 years of age (6). Because of the close association of EBV with NPC, detection of EBV anti-IgA, anti-EA, or anti-VCA by immunofluorescence tests in serum from patients with NPC is used in most Asian countries. However, this test is almost always negative for young North African patients (6). Recent data showed a successful diagnosis of NPC by molecular serology based on EBV-encoded proteins, DNase, thymidine kinase, and p16 VCA used as viral antigens (7 -10). Virus load in patient blood has been used as a diagnostic marker for NPC (11,12), but high levels have been reported in nonneoplastic disorders, gastrointestinal malignancies, and for lymphoproliferative disease (13,14). We therefore need a more reliable, simpler, and specific diagnostic test for NPC.Seve...

Human tumor virus utilizes exosomes for intercellular communication

“…Importantly, LMP1 was also detected in exosomes purified from the serum of mice carrying the C15 tumor (Fig. 1C, serum), consistent with previous work on NPC patient serum (16).…”

“…Exosomes containing LMP1 have been detected in the serum of NPC patients, and NPC serum has been shown to have mitogenic activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (16). To evaluate the properties of exosomes produced by NPC cells, exosomes were harvested from the media of the NPC C666 cell line, an NPC cell line that has retained EBV but expresses low to undetectable levels of LMP1 (17).…”

Endoplasmic Reticulum Stress Triggers XBP-1–Mediated Up-regulation of an EBV Oncoprotein in Nasopharyngeal Carcinoma

“…LMP1 protein is weakly expressed or undetectable in most of EBV-infected NPC cell lines in normal culture condition in vitro (21,22,34). Interestingly, considerable increase of LMP1 expression has been observed when an EBVpositive NPC cell line is injected into nude mice to develop solid tumors (35). Because ER stress readily occurs under hypoxia or nutrition insufficiency in solid tumors (4), we propose that ER stress/UPR can be a key factor that up-regulates LMP1 in the in vivo condition of NPC tumors.…”

Exosomes as the Promising Biomarker for Epstein-Barr Virus (EBV)-Associated Cancers

“…There have been several lines of evidences suggesting that EBV oncoproteins can be targeted for cancer diagnostics. For instances, Houali and colleagues showed that both EBV oncoproteins, LMP1 and BARF1, could be detected in serum and saliva of NPC patients, and the secreted LMP1 was highly associated with exosome-like vesicles [54]. Both EBV oncoproteins were presented with high mitogenic activity that supported their implication in oncogenic development of NPC.…”