Cancer Virology Program, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania.
Cancer Epidemiology and Prevention Program, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania.
Clin Cancer Res. 2024 Nov 15;30(22):5207-5217. doi: 10.1158/1078-0432.CCR-24-1142.
Antibodies to select Epstein-Barr virus proteins can diagnose early-stage nasopharyngeal carcinoma (NPC). We have previously shown that IgA against Epstein-Barr virus nuclear antigen 1 (EBNA1) can predict incident NPC in high- and intermediate-risk cohorts 4 years before diagnosis. Here, we tested EBNA1 variants, with mutants, to define the sequence requirements for an NPC risk assay.
Mammalian-expressed constructs were developed to represent EBNA1 variants 487V and 487A, which can differ by ≥15 amino acids in the N- and C-termini. Denatured lysates were evaluated by a refined IgA and IgG immunoblot assay in a case-control study using prediagnostic NPC sera from two independent cohorts in Singapore and Shanghai, the People's Republic of China.
At 95% sensitivity, 487V yielded a 94.9% specificity compared with 86.1% for 487A. EBNA1 deleted for the conserved glycine-alanine repeats (GAr) reduced false positives by 22.8%. NPC sera reacted more strongly to the C-terminus than healthy controls, but the C-terminal construct (a.a. 390-641) showed lower specificity (84.8%) than the EBNA1 GAr-deleted construct (92.4%) at 95% sensitivity.
Although EBNA1 IgA was present in healthy sera, most epitopes localized to the immunodominant GAr. We conclude that a refined EBNA1 antigen deleted for the GAr, but with residues consistently detected in Southeast Asian NPC tumors, is optimized for risk prediction with an extended sojourn time of 7.5 years. Furthermore, distinct EBNA1 serologic profiles enhanced the utility of the EBNA1 IgA assay for risk stratification. This illustrates the importance of serologically relevant EBNA1 sequences for NPC risk prediction and early detection.
针对特定 EBV 蛋白的抗体可用于诊断早期鼻咽癌（NPC）。我们之前的研究表明，针对 EBV 核抗原 1（EBNA1）的 IgA 可在诊断前 4 年预测高危和中危人群 NPC 的发生。在此，我们检测了 EBNA1 变体及其突变体，以确定 NPC 风险检测的序列要求。
构建了哺乳动物表达的构建体来代表 EBNA1 变体 487V 和 487A，它们在 N 端和 C 端可以有≥15 个氨基酸的差异。在使用来自新加坡和中国上海两个独立队列的 NPC 诊断前血清进行的病例对照研究中，通过改良的 IgA 和 IgG 免疫印迹检测评估变性裂解物。
在 95%的灵敏度下，与 487A 相比，487V 的特异性为 94.9%。缺失保守甘氨酸-丙氨酸重复序列（GAr）的 EBNA1 减少了 22.8%的假阳性。NPC 血清与健康对照相比对 C 端反应更强，但 C 末端构建体（a.a.390-641）在 95%的灵敏度下特异性（84.8%）低于 EBNA1 GAr 缺失构建体（92.4%）。
尽管 EBNA1 IgA 存在于健康血清中，但大多数表位定位于免疫显性 GAr。我们得出结论，一种经过改良的 EBNA1 抗原，缺失 GAr，但保留在东南亚 NPC 肿瘤中一致检测到的残基，最适合用于风险预测，潜伏期延长至 7.5 年。此外，独特的 EBNA1 血清学特征增强了 EBNA1 IgA 检测在风险分层中的效用。这说明了 EBV 相关 EBNA1 序列对于 NPC 风险预测和早期检测的重要性。
