Nasopharyngeal carcinoma patients from Norway show elevated Epstein-Barr virus IgA and IgG antibodies prior to diagnosis

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Introduction

Nasopharyngeal carcinoma (NPC) is a malignant tumor, which is distributed heterogeneously throughout the world. NPC is endemic in Southeast Asia, where incidence rates are as high as 20–30 per 100.000 person-years (PY) in men in highly affected regions [1] . Epstein-Barr virus (EBV) is a major risk factor for NPC and is uniformly associated with NPC development in endemic areas, although EBV infection alone is not sufficient to cause NPC [2] .

In regions where NPC is not endemic, incidence rates are below 1 per 100,000 PY [3] and NPCs are not uniformly associated with EBV infection. Approximately two out of three NPCs are positive for EBV 45678 . Whereas NPCs within NPC endemic areas show a characteristic incidence peak at the age of 50–59 years and decline afterwards, NPC incidence in NPC low-incidence areas is virtually absent below an age of 30, starts increasing from 40 years of age onwards and continuously rises with increasing age without any characteristic incidence peak [9] .

EBV serum antibodies in NPC patients have been extensively studied since 1966 [10] . Immunofluorescence was used in early case control studies to examine IgA antibodies against the viral capsid antigen (VCA), early antigen (EA) and Epstein–Barr nuclear antigen 1 (EBNA1) 111213 . Evidence for the presence of EBV IgA antibodies more than 5 years before NPC diagnosis was provided in a prospective study with around 10,000 Taiwanese men [14] . IgA antibodies against EBNA1 and VCAp18 have been shown to be the markers best differentiating between NPC cases and controls [15] and are currently investigated for screening purposes in a cluster randomized controlled trial in Southern China [16] .

Serological markers other than IgA antibodies against these few antigens have never been examined until 2018, when an EBV proteome array was utilized for novel antigen discovery [17] . The newly discovered EBV antibody risk stratification signature consisting of 13 IgA and IgG markers against 11 antigens showed a significant improvement for predicting NPCs in comparison to EBNA1/VCAp18 IgA antibodies alone. We successfully validated the expanded EBV antigen panel for the use in multiplex serology, a bead-based high-throughput serological assay [18] . Not only IgA, but also IgG antibodies performed well in defining EBV-positive NPCs. Two IgG antibodies alone, LF2 and BGLF2, have been shown to be highly specific markers for EBV-positive NPCs, both in high and low incidence regions [818] . However, there is little knowledge about EBV serology as a predictive biomarker in low incidence regions since NPCs are very rare and therefore, NPC screening is not feasible.

As a unique characteristic of NPCs in low incidence regions, specific human papillomavirus (HPV) types have been shown to be present in those NPCs that are negative for EBV. Five studies from the UK, the US and Finland reported HPV attributable fractions in NPCs of 9–18% 45678 . The characterization of HPV status, especially in EBV negative NPCs, helps to understand NPC etiology in non-endemic regions, where not all NPCs are positive to EBV infection.

The aim of our study was to characterize a broad panel of EBV antibodies in prediagnostic sera from NPC patients in a non-endemic region, and compare those to the molecular EBV and HPV tumor status. This is challenging, due to the rarity of NPC, and requires a large population-based biobanking infrastructure maintained over several decades. In the present study, we utilized serum of incident NPC cases up to 41 years prior to diagnosis matched to controls from a large prospective Norwegian study, the Janus Serum Bank [19] . EBV and HPV status were determined from tumor tissue and prediagnostic serum samples were examined for IgA and IgG antibodies against a comprehensive EBV antigen panel. The availability of serial samples allowed us to describe the kinetics of EBV antibody levels years prior to NPC diagnosis.