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Identify and develop a set of standard conditions and reagents that will help produce comparable data on EBV DNA abundance independent of time and place, including
sample type and preparation method (plasma, serum, or whole blood); standardized EBV DNA extraction and isolation method and materials (eg, sample spiking with control DNA molecule for DNA recovery efficiency and quality control, use of automated platforms); and aliquoting and short-term storage conditions.
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Focus on developing a PCR assay that
uses a standardized PCR protocol and reagents (kits); is suitable for analysis of plasma, nasopharyngeal cells, or tumor cells; uses primers and probes that target conserved sequence regions; targets two EBV sequences (amplicon size < 100 bp) in parallel, including a single-copy sequence and a multiple-copy sequence; reports in international units (IU) per mL (or for tissue testing, normalized to a human gene, such as beta globin); and is calibrated using secondary or tertiary reference materials that are traceable to the WHO reference standard for EBV DNA.
Examine whether and how much the EBV DNA quantitation can be improved by adapting next-generation sequencing-based analytical procedures or digital PCR. -
Conduct a multicenter study to evaluate the performance of the EBV DNA assay protocol,
using a panel of well-characterized clinical samples that includes positive and negative controls (eg, non-NPC H&N cancers, healthy EBV carriers), samples that cover a large range of concentrations, and several sample types; to define the analytical performance, including the limit of detection, linear range, precision, reproducibility (inter- and intra-assay variability), sensitivity, and specificity; and to establish cutoff values tailored to sample types and to the intended use (eg, prognostication of progression or recurrence, early detection, etc.)
Initiate clinical validation studies through multi-site clinical trials. |
