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 into earlier diagnoses and higher cure rates for eBL. Improved
specimen handling also has the potential to improve eBL treatment monitoring, and could extrapolate to the
diagnosis/monitoring of other EBV-associated malignancies.