Can a blood test monitor lymphoma?

Disease monitoring biomarkers are parameters that reflect the progress of a disease. Ideally, a successful lymphoma biomarker would be inexpensive, easy to measure, non-invasive, highly specific and sensitive, as well as accurate and reproducible. Unsurprisingly, establishing a lymphoma biomarker is not easy. Indeed, very few successful disease monitoring biomarkers have been incorporated into wide clinical practice for any disease in the last decade.

For lymphoma, clinicians currently assess treatment response via computed tomography (CT) or positron emission tomography (PET) scan. Although often effective, these methods have their limitations. CT is able to identify anatomic abnormalities but cannot differentiate between tumor tissue and residual fibrosis. Although PET scans can make this differentiation, it can also be difficult to interpret, as it is positive in patients with inflammation, infection, and other co-existing malignancies [1]. Furthermore, both methods are expensive and not always readily available when required, especially in rural or third-world settings. In Australia, government funding is restricted to pre-therapy and post-therapy assessment except in the context of a clinical trial. Thus, an accurate and specific blood disease response biomarker would be of great assistance in therapeutic decision-making.

Epstein–Barr virus (EBV) is associated with a number of malignancies including a variety of B, T, and NK (natural killer) cell lymphomas [2]. The presence of the virus within the malignant cell means that EBV has the potential to be a highly specific biomarker. Several groups have clearly demonstrated that there is elevated cell-free, tumor-derived DNA in the plasma and serum of cancer patients [3–5]. The release of cell-free tumor-specific DNA into the peripheral blood exposes the possibility of a blood biomarker that could reflect tumor load. In accordance with this theory, several groups have shown that serum/plasma EBV DNA is increased in the peripheral blood of patients with a variety of EBV-associated diseases, including Hodgkin lymphoma (HL), post-transplant lymphoproliferative disorder (PTLD), extra-nodal NK–T cell lymphoma (ENKTL), and nasopharyngeal carcinoma (NPC) [6–9]. In contrast, results from cell-associated EBV can be misleading, particularly in the context of the B-cell depleting-agent rituximab [10]. It is yet to be established how the EBV DNA load changes during the course of treatment. Serial monitoring of EBV DNA load throughout treatment of different EBV-associated diseases is required to determine how the viral load evolves during treatment and in response to treatment, and to define a relationship between disease response and viral load.

The biology of EBV DNA in the peripheral blood is still a matter of debate; however, work from our group and others has shown that the presence of the virus is not from viral reactivation [6,7,11,12]. Figure 1 illustrates the potential sources of cell-free and cell-associated EBV DNA in the peripheral blood of patients with lymphoma. Sources of cell-associated EBV DNA include benign latently infected B-cells or neoplastic cells that have migrated into the peripheral blood. Cell-free EBV DNA presumably arises from necrotic or apoptotic infected benign and neoplastic cells in the peripheral blood or at the tumor site. As previously mentioned, there is no evidence that lytic viral replication is occurring; thus, EBV DNA from benign B-cells undergoing viral reactivation is not a likely source. Furthermore, there is little if any cell-free EBV DNA in the peripheral blood of seropositive healthy individuals and seropositive lymphoma patients with EBV-negative tumors, suggesting that the main source is the necrotic neoplastic cell [7]. Interestingly, there is recent evidence that the release of viral nucleic acids might serve a biological purpose and influence tumor infiltrating cells [13].

Figure 1.  Sources of EBV DNA in the peripheral blood of patients with lymphoma.

In this issue, Machado et al. [14] present a prospective study of 30 cases of pediatric B-non-Hodgkin lymphoma (B-NHL; 28 with Burkitt lymphoma), all uniformly treated. Samples were taken at diagnosis, during treatment (on the day of the next cycle), and post-treatment. Total cell-free DNA and cell-free EBV DNA were determined using real-time polymerase chain reaction (PCR) to quantify β-globin and the EBV nuclear antigen-1, respectively. In accordance with previous studies, Machado et al. found that the total cell-free DNA was elevated in the patient cohort compared to healthy controls. However, although the cell-free DNA decreased to healthy control levels in 7/10 of the patients in complete remission, the cell-free DNA did not decrease in all patients at remission, and, thus, Machado et al. concluded it was not an appropriate biomarker to measure clinical response. This supports the use of a tumor-specific target. Also in accordance with previous studies, Machado et al. found that cell-free EBV DNA was elevated in all of the EBV-positive patients (7/30) prior to treatment and became undetectable at complete remission. Cell-free EBV DNA was absent from the EBV-negative patients and healthy controls. Interestingly, in all patients who responded to therapy, the cell-free EBV DNA became undetectable during therapy (defined as 2–4 months post-induction of chemotherapy). This provides novel evidence that cell-free EBV DNA might be a useful biomarker to monitor disease response during the course of treatment. However, as it appears that complete remission was established in all seven patients, no data were provided on viral load levels during therapy in patients who were non-responsive to treatment. Notably, one patient did relapse after establishing complete remission and a viral load increase was seen.

Although this study by Machado et al. is an important contribution to the field, there are still areas to be addressed. It is likely that the timing of samples will be critical. Theoretically, a decrease in viral load suggests a decrease in tumor burden; however, if samples are taken immediately after treatment, it is plausible that the EBV DNA load will spike as tumor cells apoptose. For instance, we observed that EBV DNA viral load significantly increased in patients with PTLD immediately post-adoptive T-cell therapy and then returned to undetectable levels prior to subsequent infusions [15]. The kinetics of EBV DNA viral load is unknown, and a more thorough understanding is required. It is quite plausible that a rapid increase of viral load within a short time frame post-treatment will indicate treatment efficacy, while a decrease in viral load at a later time point post-treatment also demonstrates treatment efficacy. Further logistical issues need to be addressed such as defining which blood specimen is optimal to assay (cell-free, cell-associated, or whole blood), establishing a common quantification method including standards and cut-offs, and determining if a common method is applicable to all EBV-associated lymphomas. Well designed, large, prospective studies including serial samples at defined time points are still required to address these issues and definitely show the value of EBV DNA monitoring.

It has been well established that positive EBV tissue status is a poor prognostic indicator for patients with lymphoma; however, EBV-encoded RNA in situ hybridization (EBER-ISH) is still not routinely done by many hospitals. This does not offer much encouragement for successfully implementing EBV DNA monitoring in clinical practice. Although there is a real need for additional therapeutic monitoring biomarkers, until a large, comprehensive, and definitive study is conducted (possibly including a cost–benefit analysis), it is unlikely that hospitals will implement EBV DNA load as therapeutic monitoring of patients with EBV-positive lymphoma.

Declaration of interest: The Clinical Immunohaematology Laboratory is funded by the NHMRC (Australia), Cancer Council of Queensland, and the Queensland Smart State. K.J. is supported by the Leukaemia Foundation of Queensland.