# Enrichment for Tumor-derived Cell-free EBV DNA: Towards a Diagnostic Assay for Endemic Burkitt Lymphoma > **NIH NIH U01** · JOHNS HOPKINS UNIVERSITY · 2024 · $357,595 ## Abstract PROJECT SUMMARY Endemic Burkitt lymphoma (eBL) outcomes in children with advanced stage disease are markedly inferior to those in children with early-stage disease. eBL is one of the most rapidly growing tumors. As a result, diagnostic delays of days-to-weeks contribute to late presentations, and directly affect stage and curability of disease. BL is the most common childhood cancer in Uganda. A child with a large jaw mass will be readily recognized as having eBL, and will typically be treated before histologic confirmation. However, a child with a lesser jaw swelling, or an abdominal mass, may be treated with empiric antimicrobials or observed for weeks. Even after procedures are performed for suspected BL, final histopathology is typically not available for another two weeks. EBV is consistently associated with eBL, and is detectable in blood and saliva of eBL patients. Although EBV DNA holds promise as an early detection marker for eBL, diagnostic utility is limited by poor specificity. EBV DNA not related to tumor is present in blood in a small fraction of latently infected normal lymphocytes and EBV virions may be present in plasma. EBV virions are also frequently detectable in saliva. We hypothesize that the specificity for malignancy would be markedly enhanced by enriching specimens for circulating tumor-derived EBV DNA. This may be accomplished by modifying preanalytical variables in specimen preparation, including upfront cell stabilization to limit viral DNA leakage from latently infected cells, elimination of normal lymphocytes (and epithelial cells in saliva) that may harbor latent EBV DNA, and enrichment for CpG methylated DNA, which effectively reduces virion DNA since that is never CpG methylated. Enrichment for methylated EBV DNA is a particularly innovative approach to improve assay specificity. We will test these hypotheses in two aims. In Aim 1 (blood), PBMC-derived viral DNA will be reduced by analyzing plasma, as opposed to whole blood or buffy coat cells, collected in cell-stabilizing tubes to minimize ex vivo cell lysis. Virion EBV DNA will be excluded via CpG methylated DNA separation. The effect of specimen transport temperature, time, and other preanalytical variables on cell-free DNA (cfDNA) recovery and EBV DNA measurements will be evaluated. In Aim 2 (saliva), collection techniques that minimize oral epithelial cells and lymphocytes and minimize ex vivo cell lysis along with exclusion of virion EBV DNA will be assessed. The effect of specimen transport temperature, time, and additive on cell-free DNA (cfDNA) recovery and EBV DNA measurements will be also be evaluated. These studies aim to define the optimal specimen preparation methods that will be feasible in African settings, and elsewhere, and can serve as the basis for future standards for the biospecimen science community at large. Improved diagnostic specificity of EBV DNA measurements achieved by optimizing preanalytical procedures should translate directly i... ## Key facts - **NIH application ID:** 10803713 - **Project number:** 1U01CA271252-01A1 - **Recipient organization:** JOHNS HOPKINS UNIVERSITY - **Principal Investigator:** RICHARD Frederick AMBINDER - **Activity code:** U01 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $357,595 - **Award type:** 1 - **Project period:** 2024-04-01 → 2029-03-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10803713 ## Citation > US National Institutes of Health, RePORTER application 10803713, Enrichment for Tumor-derived Cell-free EBV DNA: Towards a Diagnostic Assay for Endemic Burkitt Lymphoma (1U01CA271252-01A1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10803713. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*