Elevated Epstein-Barr virus (EBV) antibody levels are associated with risk of nasopharyngeal carcinoma (NPC) and risk scores based on these antibodies can predict NPC risk with high accuracy. The immune mechanisms underlying this association remain unclear. We evaluated whether individuals with elevated EBV-NPC antibody risk scores had a reduced T-cell response to EBV proteins within a NPC multiplex family study in Taiwan. In total, 79 cancer-free individuals from families with a history of NPC had blood tested for (i) EBV antibodies using a multiplex serology assay and (ii) T-cell responses to 7 EBV proteins (EBNA1, LMP1, LMP2, BZLF1, BRLF1, BARF1, gp350) using interferon-γ ELISPOT. Excluding individuals with low phytohemagglutinin proliferative responses, no statistically significant association was observed between lower EBV T-cell responses and higher EBV antibody-based NPC risk scores (P = 0.21). However, our findings did trend in the a-priori hypothesized direction, with lower EBV T-cell responses noted among those with higher EBV antibody-based NPC risk scores. Compared to individuals who responded to at least 3 EBV-specific T-cell peptide pools by ELISPOT, those who responded to 1–2 peptide pools had a 10

Epstein-Barr virus (EBV) causes most nasopharyngeal carcinoma (NPC), a malignancy endemic in certain regions, presenting with nonspecific symptoms and lacking a robust minimally invasive biomarker. To assess previously developed multiplex Luminex assay EBV antibody signatures in a nonendemic population, we analyzed and compared 98 NPC sera and 142 matched controls from the nonendemic Head and Neck 5000 clinical cohort against an endemic Taiwan case-control cohort. All models, except the 2-antibody IgA panel, performed similarly, achieving 90%-99% accuracy in identifying EBV-positive NPC. Our results showed that EBV antibody panels can accurately define EBV-positive NPC regardless of endemicity.

Background Epstein-Barr virus (EBV) is a major cause of nasopharyngeal carcinoma (NPC) and measurement of different EBV antibodies in blood may improve early detection of NPC. Prospective studies can help assess the roles of different EBV antibodies in predicting NPC risk over time.Methods A case-cohort study within the prospective China Kadoorie Biobank of 512 715 adults from 10 (including two NPC endemic) areas included 295 incident NPC cases and 745 subcohort participants. A multiplex serology assay was used to quantify IgA and IgG antibodies against 16 EBV antigens in stored baseline plasma samples. Cox regression was used to estimate adjusted hazard ratios (HRs) for NPC and C-statistics to assess the discriminatory ability of EBV-markers, including two previously identified EBV-marker combinations, for predicting NPC.Results Sero-positivity for 15 out of 16 EBV-markers was significantly associated with higher NPC risk. Both IgA and IgG antibodies against the same three EBV-markers showed the most extreme HRs, i.e. BGLF2 (IgA: 124.2 (95% CI: 63.3-243.9); IgG: 8.6 (5.5-13.5); LF2: [67.8 (30.0-153.1), 10.9 (7.2-16.4)]); and BFRF1: 26.1 (10.1-67.5), 6.1 (2.7-13.6). Use of a two-marker (i.e. LF2/BGLF2 IgG) and a four-marker (i.e. LF2/BGLF2 IgG and LF2/EA-D IgA) combinations yielded C-statistics of 0.85 and 0.84, respectively, which persisted for at least 5 years after sample collection in both endemic and non-endemic areas.Conclusions In Chinese adults, plasma EBV markers strongly predict NPC occurrence many years before clinical diagnosis. LF2 and BGLF2 IgG could identify NPC high-risk individuals to improve NPC early detection in community and clinical settings.

Knowledge about the underlying causes of the individual occurrence of symptoms during acute COVID-19 disease and during the post-acute sequelae of COVID-19 is limited. In a German COVID-19 follow-up study, we assessed whether elevated antibody responses to herpesviruses were associated with symptom occurrence in acute COVID-19 disease (n = 96 participants) and during 20 months of follow-up (n = 62 participants). Serum samples were analyzed for their antibodies to herpes simplex virus (HSV)-1 and -2, Epstein–Barr virus (EBV), and Cytomegalovirus (CMV) using fluorescent bead-based multiplex serology. The association of herpesvirus antibodies with symptom occurrence (fatigue, fever, dyspnea, decrease in taste, concentration problems) was assessed using multivariate logistic regression models. High EBV antibody levels were significantly associated with a more than fourfold increased odds of experiencing fatigue during acute COVID-19 disease and during follow-up. High CMV antibody levels were significantly associated with a more than threefold increased odds of experiencing concentration problems and a decrease in taste during the follow-up. The HSV-1 and -2 antibody levels were not elevated in the individuals that experienced symptoms. In conclusion, our findings indicate that herpesvirus infections, specifically EBV and CMV infections, might play a role in symptom development during acute and post-acute COVID-19 disease. It remains to be elucidated whether the elevated EBV and CMV antibodies determined in our study are indicators of herpesvirus reactivation.

Nasopharyngeal carcinoma (NPC) risk prediction models based on Epstein-Barr virus (EBV)-antibody testing have shown potential for screening of NPC; however, the long-term stability is unclear. Here, we investigated the kinetics of two EBV-antibody NPC risk scores within the Taiwan NPC Multiplex Family Study. Among 545 participants with multiple blood samples, we evaluated the stability of a 2-marker enzyme-linked immunosorbent assay score and 13-marker multiplex serology score using the intra-class correlation coefficient (ICC) by fitting a linear mixed model that accounted for the clustering effect of multiple measurements per subject and age. We also estimated the clustering of positive tests using Fleiss's kappa statistic. Over an average 20-year follow-up, the 2-marker score showed high stability over time, whereas the 13-marker score was more variable (p < .05). Case-control status is associated with the kinetics of the antibody response, with higher ICCs among cases. Positive tests were more likely to cluster within the same individual for the 2-marker score than the 13-marker score (p < .05). The 2-marker score had an increase in specificity from ~90% for single measurement to ~96% with repeat testing. The 13-marker score had a specificity of ~73% for a single measurement that increased to ~92% with repeat testing. Among individuals who developed NPC, none experienced score reversion. Our findings suggest that repeated testing could improve the specificity of NPC screening in high-risk NPC multiplex families. Further studies are required to determine the impact on sensitivity, establish optimal screening intervals, and generalize these findings to general population settings in high-risk regions.

A meeting of experts was held in November 2021 to review and discuss available data on performance of Epstein-Barr virus (EBV)-based approaches to screen for early stage nasopharyngeal carcinoma (NPC) and methods for the investigation and management of screen-positive individuals. Serum EBV antibody and plasma EBV DNA testing methods were considered. Both approaches were found to have favorable performance characteristics and to be cost-effective in high-risk populations. In addition to endoscopy, use of magnetic resonance imaging (MRI) to investigate screen-positive individuals was found to increase the sensitivity of NPC detection with minimal impact on cost-effectiveness of the screening program.

Epstein-Barr virus (EBV) IgA and IgG antibodies in serum from nasopharyngeal carcinoma (NPC) patients are well-established markers for EBV-positive NPC. Luminex-based multiplex serology can analyze antibodies to multiple antigens simultaneously; however, the detection of both IgA and IgG antibodies requires separate measurements. Here we describe the development and validation of a novel duplex multiplex serology assay, which can analyze IgA and IgG antibodies against several antigens simultaneously. Secondary antibody/dye combinations, as well as serum dilution factors, were optimized, and 98 NPC cases matched to 142 controls from the Head and Neck 5000 study (HN5000) were assessed and compared to data previously generated in separate IgA and IgG multiplex assays. EBER in situ hybridization (EBER-ISH) data available for 41 tumors was used to calibrate antigen-specific cut-offs using receiver operating characteristic (ROC) analysis with a prespecified specificity of ≥90%. A directly R-Phycoerythrin-labeled IgG antibody in combination with a biotinylated IgA antibody and streptavidin-BV421 reporter conjugate was able to quantify both IgA and IgG antibodies in a duplex reaction in a 1:1000 serum dilution. The combined assessment of IgA and IgG antibodies in NPC cases and controls from the HN5000 study yielded similar sensitivities as the separate IgA and IgG multiplex assays (all > 90%), and the duplex serological multiplex assay was able to unequivocally define the EBV-positive NPC cases (AUC = 1). In conclusion, the simultaneous detection of IgA and IgG antibodies provides an alternative for the separate IgA/IgG antibody quantification and may present a promising approach for larger NPC screening studies in NPC endemic areas.

Background: IgA antibodies against few Epstein-Barr virus (EBV) proteins are established serological markers for nasopharyngeal carcinoma (NPC).We recently validated a novel, comprehensive EBV marker panel and showed that IgA, but also IgG antibodies against multiple EBV proteins are highly sensitive and specific for EBV-positive NPC at diagnosis.However, data about these novel biomarkers as prospective markers for NPC are sparse.Methods: This study included 30 incident NPC cases and 60 matched controls from the Norwegian Janus Serum Bank.For 21 NPCs, molecular EBV and human papillomavirus (HPV) status were assessed by EBER-ISH and HPV DNA/RNA testing by PCR, respectively.IgA and IgG serum antibodies against 17 EBV antigens were analyzed in prediagnostic sera of cases (median lead time 14 years) and controls using multiplex serology.Sensitivities were calculated using receiver operating characteristic analysis pre-specified to yield 90% specificity in the control group.From 10 cases, serial samples were available.Results: Quantitative EBV antibody levels were significantly elevated among all cases (p<0.05) for three IgA and six IgG antibodies.The highest sensitivities for defining 12 EBER-ISH-positive NPCs were observed for BGLF2 IgA (67%) and BGLF2 IgG (83%).Increased IgA and IgG antibody levels between the first and last draw before diagnosis were observed for EBER-ISH positive, but not for EBER-ISH negative NPCs.Among 21 molecularly analyzed NPCs, 4 EBER-ISH negative NPCs showed concomitant positivity to HPV typespecific DNA and RNA; 3 NPCs were HPV16 and 1 NPC was HPV18 positive.Conclusion: Both, EBV IgA and IgG antibody levels are significantly elevated many years before diagnosis of EBV-positive NPCs in Norway, an NPC low-incidence region.This study provides insights into one of the largest available prospective sample collections of NPCs in a non-endemic country.

Abstract Background Long COVID is defined as the persistence of symptoms beyond 3 months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To better understand the long-term course and etiology of symptoms we analyzed a cohort of patients with COVID-19 prospectively. Methods Patients were included at 5 months after acute COVID-19 in this prospective, noninterventional, follow-up study. Patients followed until 12 months after COVID-19 symptom onset (n = 96; 32.3% hospitalized, 55.2% females) were included in this analysis of symptoms, quality of life (based on an SF-12 survey), laboratory parameters including antinuclear antibodies (ANAs), and SARS-CoV-2 antibody levels. Results At month 12, only 22.9% of patients were completely free of symptoms and the most frequent symptoms were reduced exercise capacity (56.3%), fatigue (53.1%), dyspnea (37.5%), and problems with concentration (39.6%), finding words (32.3%), and sleeping (26.0%). Females showed significantly more neurocognitive symptoms than males. ANA titers were ≥1:160 in 43.6% of patients at 12 months post–COVID-19 symptom onset, and neurocognitive symptom frequency was significantly higher in the group with an ANA titer ≥1:160 versus <1:160. Compared with patients without symptoms, patients with ≥1 long-COVID symptom at 12 months did not differ significantly with respect to their SARS-CoV-2 antibody levels but had a significantly reduced physical and mental life quality compared with patients without symptoms. Conclusions Neurocognitive long-COVID symptoms can persist ≥1 year after COVID-19 symptom onset and reduce life quality significantly. Several neurocognitive symptoms were associated with ANA titer elevations. This may indicate autoimmunity as a cofactor in etiology of long COVID.

Serological testing for nasopharyngeal carcinoma (NPC) has recently been reinvigorated by the implementation of novel Epstein-Barr virus (EBV)-specific IgA and IgG antibodies from a proteome array. Although proteome arrays are well suited for comprehensive antigen selection, they are not applicable for large-scale studies. We adapted a 13-marker EBV antigen signature for NPC risk identified by proteome arrays to multiplex serology to establish an assay for large-scale studies. Taiwanese NPC cases ( n = 175) and matched controls ( n = 175) were used for assay validation.

Epstein-Barr virus (EBV) causes nasopharyngeal carcinoma (NPC) in endemic regions, where almost every tumor is EBV-positive. In Western populations, NPC is rare, and human papillomavirus infection (HPV) has been suggested as another viral cause. We validated multiplex serology with molecular tumor markers, to define EBV-positive, HPV-positive and EBV-/HPV-negative NPCs in the United Kingdom, and analyzed survival differences between those groups. Sera from NPC cases (n = 98) and age- and sex-matched controls (n = 142) from the Head and Neck 5000 clinical cohort study were analyzed. IgA and IgG serum antibodies against 13 EBV antigens were measured and compared with EBER in situ hybridization (EBER-ISH) data of 41 NPC tumors (29 EBER-ISH positive, 12 negative). IgG antibodies to EBV LF2 correctly diagnosed EBV-positive NPCs in 28 of 29 cases, while all EBER-ISH negative NPCs were seronegative to LF2 IgG (specificity = 100%, sensitivity = 97%). HPV early antigen serology was compared to HPV molecular markers (p16 expression, HPV DNA and RNA) available for 41 NPCs (13 positive, 28 negative). Serology matched molecular HPV markers in all but one case (specificity = 100%, sensitivity = 92%). EBV and HPV infections were mutually exclusive. Overall, 67% of the analyzed NPCs were defined as EBV-positive, 18% as HPV-positive and 14% as EBV/HPV-negative. There was no statistical evidence of a difference in survival between the three groups. These data provide evidence that both, EBV-positive and HPV-positive NPCs are present in a low incidence country, and that EBV and HPV serum antibodies correlate with the viral status of the tumor.

Background: Adolescent/young adult (AYA) Hodgkin lymphoma (HL) is one of the most common cancers in this age group in developed countries. High socioeconomic status, small sibship size and lack of crowding in childhood are risk factors. We also found that among twins, the twin who had more oral exposures in early life was 80% less likely to develop AYA Hodgkin lymphoma. These risk factors are compatible with the hygiene hypothesis. We wanted to further explore additional risk factors consistent with this etiologic model. Methods: In a case-family control study, we examined past exposure to 12 infections in serum samples from 291 AYA HL cases and 194 of their unaffected family members. Cases were ascertained from the USC International Twin Registry (n=36) and the USC Cancer Surveillance Program (n=255). We measured antibodies to HSV1, HSV2, VZV, EBV, HHV7, HCMV, HHV6, KSHV, T. gondii, H. pylori, Rubella virus and Parvovirus B19 using a multiplex serology bead array (Luminex) (Waterboer Laboratory, German Cancer Research Center). A positive history of exposure to each infection was determined by a titer threshold specific for each infection. We did not examine the association between antibody levels and risk because of the possible impact of AYA HL diagnosis or treatment. We made the assumption that disease or treatment would not affect detection of a low level of antibody indicating previous exposure. A logistic regression model was used to calculate the odds ratio (OR) and 95% Confidence Interval (95% CI) adjusting for age at blood draw, sex and family. The sample was mostly non-Hispanic white, thus we did not adjust for race/ethnicity. We performed stratified analysis for histologic subtype where possible. Linear regression was used to calculate the p-for trend for 0, 1 or 2 fecal-oral or respiratory infections, with 0 infections as the reference. Results: The mean age at diagnosis in AYA HL cases was 30 years and the mean age at blood draw was 36 years. 57% of the cases were female. Out of the 186 cases with EBV tumor status, 78% were EBV-. Out of the 242 cases with histologic subtype, 72.5% were nodular sclerosis. The mean age of the 194 family controls at blood draw was 53 years and 63.4% were female. Evidence of past HHV6 infection was associated with an increased risk of AYA HL (OR= 1.80; 95% CI= 1.06-3.07; for nodular sclerosis only: OR= 1.92, 95% CI=1.05-3.42). Having antibodies to H. pylori was associated with a decreased risk (OR= 0.49; 95% CI= 0.25-0.98; for nodular sclerosis only: OR= 0.33, 95% CI= 0.14-0.77). An increasing number of fecal-oral infections (H. pylori, T. gondii) was associated with a decreasing risk of AYA HL (ptrend=0.015), while an increasing number of respiratory transmitted infections (Rubella virus, Parvovirus) was not significantly associated (ptrend=0.43). After adjusting for family, infection with Parvovirus was strongly and significantly associated with AYA HL (OR=3.8,1.06-13.6; for nodular sclerosis only: OR=10.5, 95% CI=1.71-65.25). Past infection with EBV was not associated with risk of overall HL, or with EBV- or EBV+ AYA HL separately, probably because of the high frequency of this infection in the overall population. Conclusions: We found continuing evidence for an inverse association between fecal-oral transmitted agents and risk of AYA HL, supporting the hypothesis that a deficit of early life fecal-oral transmitted microbes may be associated with susceptibility. HHV6 and Parvovirus were associated with an increased risk, possibly due to a subclinical immune deficiency or another unknown mechanism Disclosures No relevant conflicts of interest to declare.

Abstract Background: Adolescent/young adult (AYA) Hodgkin lymphoma (HL) risk is increasingly linked to a deficit of microbial exposure in early life, especially from fecal-oral transmission. Methods: In this cross-sectional study, we examined past exposure to 15 infections in serum samples from 291 AYA HL cases and 194 of their unaffected family members. We measured antibodies to HSV1, HSV2, VZV, EBV, HHV7, HCMV, HHV6, KSHV, S. gallolyticus, F. nucleatum, T. gondii, H. pylori, Rubella virus, and Parvovirus B19 using a multiplex serology bead array (Luminex). A positive history was determined by a titer threshold specific for each infection. A logistic regression model was used to calculate the odds ratio (OR) of AYA HL risk for each infection separately adjusted for sex and age at sampling. We then applied the same logistic regression model, adding an adjustment for family. Results: Evidence of past HHV6 infection was associated with an increased risk of AYA HL (OR= 1.80; 95% CI= 1.06-3.07). Having antibodies to H. pylori was associated with a decreased risk (OR= 0.49; 95% CI= 0.25-0.98). An increasing number of fecal-oral infections (H. pylori, T. gondii) was associated with a decreasing risk of AYA HL (ptrend<0.001), while an increasing number of respiratory-transmitted infections (Rubella virus, Parvovirus) was not significantly associated (ptrend=0.062). After adjusting for family, infection with Parvovirus was the only significant risk factor for AYA HL (OR=3.8,1.06-13.6). Conclusions: We show preliminary evidence for an inverse association between fecal-oral transmitted agents and risk of AYA HL, supporting the hypothesis that a deficit of early life fecal-oral transmitted microbes may be associated with increased risk. HHV6 and Parvovirus were associated with an increased risk, possibly due to a subclinical immune deficiency or an unknown mechanism. In the final presentation, we will add data from 40 additional unaffected family members currently missing age, and results by histology and EBV status. Citation Format: Maryam Saleh, Amie Hwang, Julia Simon, Thomas M. Mack, Tim Waterboer, Wendy Cozen. Past infection and risk of adolescent/young adult HL [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-44.

IgA antibodies targeting Epstein-Barr virus (EBV) have been proposed for screening for nasopharyngeal carcinoma (NPC). However, methods differ, and the antigens used in these assays differ considerably between laboratories. To enable formal comparisons across a range of established EBV serology assays, we created a panel of 66 pooled serum samples and 66 pooled plasma samples generated from individuals with a broad range of IgA antibody levels. Aliquots from these panels were distributed to six laboratories and were tested by 26 assays measuring antibodies against VCA, EBNA1, EA-EBNA1, Zta, or EAd antigens. We estimated the correlation between assay pairs using Spearman coefficients (continuous measures) and percentages of agreement (positive versus negative, using predefined positivity cutoffs by each assay developer/manufacturer). While strong correlations were observed between some assays, considerable differences were also noted, even for assays that targeted the same protein. For VCA-IgA assays in serum, two distinct clusters were identified, with a median Spearman coefficient of 0.41 (range, 0.20 to 0.66) across these two clusters. EBNA1-IgA assays in serum grouped into a single cluster with a median Spearman coefficient of 0.79 (range, 0.71 to 0.89). Percentages of agreement differed broadly for both VCA-IgA (12% to 98%) and EBNA1-IgA (29% to 95%) assays in serum. Moderate-to-strong correlations were observed across assays in serum that targeted other proteins (correlations ranged from 0.44 to 0.76). Similar results were noted for plasma. We conclude that standardization of EBV serology assays is needed to allow for comparability of results obtained in different translational research studies across laboratories and populations.