Using Epstein-Barr viral load assays to diagnose, monitor, and prevent posttransplant lymphoproliferative disorder

Abstract

Epstein-Barr virus (EBV) DNA measurement is being incorporated into routine medical practice to help diagnose, monitor, and predict posttransplant lymphoproliferative disorder (PTLD) in immunocompromised graft recipients. PTLD is an aggressive neoplasm that almost always harbors EBV DNA within the neoplastic lymphocytes, and it is often fatal if not recognized and treated promptly. Validated protocols, commercial reagents, and automated instruments facilitate implementation of EBV load assays by real-time PCR. When applied to either whole blood or plasma, EBV DNA levels reflect clinical status with respect to EBV-related neoplasia. While many healthy transplant recipients have low viral loads, high EBV loads are strongly associated with current or impending PTLD. Complementary laboratory assays as well as histopathologic examination of lesional tissue help in interpreting modest elevations in viral load. Circulating EBV levels in serial samples reflect changes in tumor burden and represent an effective, noninvasive tool for monitoring the efficacy of therapy. In high-risk patients, serial testing permits early clinical intervention to prevent progression toward frank PTLD. Restoring T cell immunity against EBV is a major strategy for overcoming PTLD, and novel EBV-directed therapies are being explored to thwart virus-driven neoplasia.

FIG. 1.

Histopathologic features reflect clinical subtypes of PTLD. (A) An early lesion (plasmacytic hyperplasia) contains mature polyclonal plasma cells that expand but do not replace tissue architecture. (B) In polymorphic PTLD there is a mixture of small and large lymphocytes. (C) In monomorphic PTLD there are sheets of large lymphoid cells reminiscent of diffuse large B cell lymphoma. (D) EBER in situ hybridization reveals a purple EBER signal localized to the nuclei of tumor cells. (Photomicrographs courtesy of Yuri Fedoriw, University of North Carolina at Chapel Hill; reproduced with permission.)

FIG. 2.

EBV load, as measured by quantitative PCR in whole blood or plasma, mirrors clinical status in transplant recipients. Nearly every human becomes infected during childhood or adolescence, at which time the viral load climbs until the infection is brought under control by the immune system. Humoral and cell-mediated immunity established during primary infection helps maintain viral quiescence for the remainder of the person's life, with latent EBV DNA retained for life in a small subset of B lymphocytes. Healthy carriers have measurable EBV DNA in whole blood, whereas plasma rarely contains EBV DNA at levels exceeding the lower limit of detection. When a transplant patient is iatrogenically immunosuppressed to prevent graft rejection, active viral infection results in higher baseline viral loads in both whole blood and plasma. Levels often rise before clinical diagnosis of posttransplant lymphoproliferation (PTLD), allowing preemptive intervention in high-risk patients who are routinely monitored for EBV levels. Successful intervention is marked by a return to baseline. A child or a rare adult who was never infected before onset of iatrogenic immunosuppression lacks prior immunity, placing the patient at high risk for active viral infection and progression to neoplasia.

FIG. 3.

EBV load is measured by real-time PCR. Extracted DNA is placed in a thermocycler with the reagents for real-time PCR to replicate a segment of the EBV genome in a cyclical fashion using DNA polymerase (DNA pol). To detect accumulating PCR products, the assay depicted here uses a TaqMan probe labeled with reporter (R) and quencher (Q) dyes. As DNA pol replicates the template strand, hydrolysis of the probe releases the reporter dye, which, when separated from the quencher, results in a measurable fluorochrome signal. As each PCR cycle ensues, product accumulation is measured over time in the patient sample and in each of a series of standards, allowing extrapolation of the EBV load in the patient sample. Spiking a nonhuman, nonpathogen DNA into the specimen before extraction and then amplifying the spiked DNA ensures adequacy of extraction and lack of PCR inhibition in each patient sample.

FIG. 4.

Generic laboratory report content that might accompany an EBV load result following testing and analytic interpretation by a laboratory scientist.