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. 2024 Sep 24:7:0475.
doi: 10.34133/research.0475. eCollection 2024.

Blind Brush Biopsy: Quantification of Epstein-Barr Virus and Its Host DNA Methylation in the Detection of Nasopharyngeal Carcinoma

Caoli Tang  1   2 , Xizhao Li  1 , Yumeng Zhang  1   2 , Ting Zhou  1 , Xiaojing Yang  1 , Ying Liao  1 , Tongmin Wang  1 , Yongqiao He  1 , Wenqiong Xue  1 , Weihua Jia  1   2 , Xiaohui Zheng  1
Affiliations

Blind Brush Biopsy: Quantification of Epstein-Barr Virus and Its Host DNA Methylation in the Detection of Nasopharyngeal Carcinoma

Caoli Tang et al. Research (Wash D C). .

Abstract

Background: The nasopharyngeal brush sampling can effectively collect samples from the nasopharynx. The blind brush sampling does not require the guidance of endoscopy, which is favorable for implementation and dissemination in the community. This study explored methylation markers for nasopharyngeal carcinoma (NPC) at both Epstein-Barr virus (EBV) and its host genome levels, aiming to construct a blind brushing diagnostic method. Methods: EBV DNA capture and methylation sequencing and GEO Illumina 450K methylation array data were used respectively for the discovery of EBV and host methylation markers. The diagnostic method was built in training cohort (n = 347) and validated in an independent validation cohort (n = 155). Results: A total of 1 EBV methylation marker (BILF2) and 6 host methylation markers (ITGA4, IMPA2, ITPKB, PI9, AMIGO2, and VAV3) were identified. Both EBV and host methylation markers were almost exclusively detected in NPC samples, with negligible detection in control samples. In validation cohort, the diagnostic method that included only the EBV BILF2 marker showed a sensitivity and specificity of 80.22% and 98.44%, respectively. When combining the EBV-derived marker BILF2 with the host-derived marker IMPA2, the diagnostic method's sensitivity increased to 84.62%, while the specificity remained unchanged (IDI = 4.4%, P = 0.0419). Conclusion: Overall, the blind nasopharyngeal brushing diagnostic method, combining EBV and host methylation markers, showed great potential in NPC detection and could promote its application in nonclinical screening of NPC.

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Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1.
Fig. 1.
The relative methylation levels and modeling ROC of intergenic region, BILF2, and Cp DMRs.
Fig. 2.
Fig. 2.
Intergroup comparison of methylation levels of CpG sites within DMRs on ITGA4 (A), WIF1 (B), and SHISA3 (C) genes in GSE52068 and GSE62336.
Fig. 3.
Fig. 3.
Intergroup comparison of methylation levels of CpG sites within DMRs on AMIGO2 (A), PI9 (B), IMPA2 (C), ITPKB (D), and VAV3 (E) genes in GSE52068 and GSE62336.
Fig. 4.
Fig. 4.
Comparison of relative methylation levels of candidate methylation markers [ITGA4 (A), IMPA2 (B), ITPKB (C), PI9 (D), AMIGO2 (E), VAV3 (F), and BILF2 (G)] between case-controls in the training set and comparison of CT values of internal reference gene ACTB (H).
Fig. 5.
Fig. 5.
Comparison of relative methylation levels of methylation markers [ITGA4 (A), IMPA2 (B), ITPKB (C), PI9 (D), AMIGO2 (E), VAV3 (F), and BILF2 (G)] between case-controls in the validation set and comparison of CT values of internal reference gene ACTB (H).
Fig. 6.
Fig. 6.
Relative methylation levels of methylation markers [ITGA4 (A), IMPA2 (B), ITPKB (C), PI9 (D), AMIGO2 (E), VAV3 (F), and BILF2 (G)] in early and advanced NPC samples.
Fig. 7.
Fig. 7.
Workflow indicating study design.
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