Developing a multiplex serology panel to detect and quantify Epstein-Barr virus infection

Epstein-Barr virus (EBV) accounts for more than 200,000 cases of cancer each year and 1.8% of all cancer deaths are due to EBV-attributable malignancies including nasopharyngeal carcinoma (NPC) and gastric carcinoma which have higher incidence in low-middle-income-countries (LMICs), e.g. NPC in southern parts of china with >25 times higher incidence than the rest of the world. However, the mechanisms of cancer causation by EBV remain an area of active investigation and controversy. Since the clinical symptoms of NPC are nonspecific and no known pre-cursors exist, diagnosis is often delayed until the advanced stages of disease when five-year survival is less than 50%, as opposed to 90% survival when NPC is identified at an early stage. To date, there are no specific effective treatments for EBV-associated diseases, thus identification of different biomarkers present in subsets of EBV-positive individuals to aid early detection of disease is particularly important. In addition to well-established biomarkers, ten more EBV antigens that best discriminated NPC status have recently been identified through protein microarray technology. However, the output from this technology does not directly translate to an exact amount of antibody circulating in the blood. The proposed study will develop and validate a panel capable of measuring and quantifying the amounts of EBV biomarkers, using a new technology that is high-throughput with low-cost and low volume of samples required. We also propose to use this panel in a pilot case-cohort study including 200 each of NPC, gastric cancer cases and population-based subcohort individuals in the well-established China Kadoorie Biobank (CKB) - a prospective study of 0.5 million individuals recruited at age 30-79 years from 10 diverse regions in rural and urban China, with stored blood samples. The aim of this pilot case-cohort study is to evaluate whether the EBV serology measured several years prior to disease diagnosis could be used to identify high-risk individuals and also to assess the association between EBV biomarkers, their joint relationships with other lifestyle and environmental risk factors and the risk of NPC and gastric cancer. Given the high throughput and low cost features of the technology together with preliminary data identifying high-risk populations provided by this research, this EBV panel may be suitable for large epidemiology studies which can comprehensively investigate EBV-related disease mechanisms; and could potentially be used for early detection/screening among high-risk individuals who experienced other lifestyle or environmental risk factors such as smoking or with a family history of cancer. It may then help to inform EBV-related disease prevention and control policy making by treating infection or vaccinating high-risk individuals, i.e. those infected with highly-disease-associated EBV biomarkers also with other lifestyle and environmental risk factors such as family history of cancer or smoking. The improved cancer prevention/control policy will benefit not only LMICs but also global populations. We will also compare the results with our ongoing project using a panel of 46- biomarkers which was initially designed for the UK population in which only 4 EBV biomarkers are included. It will provide us with more information about the seroprevalence for those disease related EBV biomarkers in a Chinese population. It will help us to design an improved infectious pathogens panel which is suitable for Chinese populations. The improved panel will be then used in our future studies of multiple infectious pathogens conducted in the entire CKB cohort, in order to understand how multiple infectious pathogens can cause different diseases, and will have benefits for disease prevention and control worldwide.

Epstein-Barr virus (EBV) accounts for >200,000 cancer cases each year and 1.8% cancer deaths are due to EBV-attributable malignancies, some with an exceptionally high incidence in low-middle-income countries (LMICs), e.g. NPC in Southeast Asia and North Africa. The mechanisms of cancer causation by EBV remain an area of active investigation and controversy. Since EBV-related cancers are often diagnosed at advanced stages with poor survival rates, identification of biomarkers present in subsets of EBV-positive individuals to aid early detection of disease is particularly important. Twelve EBV antigens best predicting NPC risk have recently been identified by protein microarray technology, but the output from that technology cannot directly translate to an exact amount of circulating antibody. Using the identified 12 EBV biomarkers, the proposed study will develop/validate a Luminex-based EBV-Monoplex panel capable of measuring/quantifying the amounts of serum EBV biomarkers. Due to the high-throughput, low-cost and low volume of samples required of the technology, this EBV-Monoplex panel may be suitable for large epidemiology studies for investigation of disease mechanisms and as a potential early detection/screening tool among high-risk individuals. The 12-EBV-Monoplex panel will then be used in a pilot case-cohort study including 200 each of NPC, gastric cancer cases and subcohort in the China Kadoorie Biobank (CKB) prospective study. It will provide preliminary data on the aetiological role of EBV biomarkers in EBV-related diseases. Using this Luminex technology, we will develop a 46-Multiplex panel (closely followed the panel designed for the UK Biobank with only four EBV antigens included) for assessing multiple infectious pathogens in risk of gastrointestinal cancer. The proposed study will compare the EBV antibody levels measured by both panels to identify potential EBV biomarkers for an improved CKB Multiplex panel design that is more suitable for Chinese.

The potential impact of the proposed pilot study will be to develop a high-throughput and low-cost EBV-Monoplex serology panel that requires low sample volume and that is potentially suitable for large epidemiological studies of EBV-related diseases. It will provide pilot data on the quantitative level of the immune response to different EBV antigens in CKB participants, thus this EBV-Monoplex panel may have potential value for early detection/screening of EBV-related diseases among high-risk individuals. Combined with the results from the ongoing project assessing the role of H. pylori and other major infectious pathogens in risk of gastrointestinal cancers by using a 46-Multiplex panel, this project will also help to inform the design of an improved multiplex serology panel to be used in the entire CKB cohort.

Who will benefit from this research and how?

The key beneficiaries are:
- Public, with improved awareness of the health consequences of EBV infections, this may lead to behavioural changes (e.g. cease smoking, improved dietary structure); and with improvements in cancer prevention and control policy (see below), will experience reduced morbidity and mortality associated with EBV infections.
- Cancer prevention/control policy makers and clinicians, who can exploit reliable estimates of EBV biomarkers and risk of NPC, using the validated EBV-Mononplex panel which may potentially be used as an early detection/screening tool; and to guide EBV-related disease prevention and control policy actions for reducing the disease burden from EBV infection by treating infection or vaccinating high-risk individuals, i.e. who infected highly-disease-associated EBV biomarkers also with other lifestyle and environmental risk factors such as family history of cancer or smoking.
- Scientists, who will acquire a validated cost-effective EBV-specific serological panel to simultaneously quantify 12 EBV biomarkers, which may suitable for large epidemiological studies of the comprehensive investigation of the EBV-related diseases. Through assessing the levels of different EBV antibodies and their relationship with lifestyle and environmental risk factors in risk of NPC in the proposed study, scientists will be able to exploit reliable preliminary data for future large epidemiological studies that can investigate NPC and other EBV-related disease mechanisms.

All CKB data including those newly collected/generated through this proposal will be incorporated into the central database and made available to internal and external research collaborators. Study findings will be disseminated to the research community/funders and policymakers through peer reviewed publications and presentations at appropriate national/international conferences and targeted workshops. The dissemination of study findings to the wider public will be chiefly through the CKB study website and mass media.