Epstein-Barr virus infection and clinical outcome in breast cancer patients correlate with immune cell TNF-α/IFN-γ response

Abstract

Background: For nearly two decades now, various studies have reported detecting the Epstein-Barr virus (EBV) in breast cancer (BC) cases. Yet the results are unconvincing, and their interpretation has remained a matter of debate. We have now presented prospective data on the effect of EBV infection combined with survival in patients enrolled in a prospective study.

Methods: We assessed 85 BC patients over an 87-month follow-up period to determine whether EBV infection, evaluated by qPCR in both peripheral blood mononuclear cells (PBMCs) and tumor biopsies, interacted with host cell components that modulate the evolution parameters of BC. We also examined the EBV replicating form by the titration of serum anti-ZEBRA antibodies. Immunological studies were performed on a series of 35 patients randomly selected from the second half of the survey, involving IFN-γ and TNF-α intracellular immunostaining tests performed via flow cytometry analysis in peripheral NK and T cells, in parallel with EBV signature. The effect of the EBV load in the blood or tumor tissue on patient survival was analyzed using univariate and multivariate analyses, combined with an analysis of covariance.

Results: Our study represents the first ever report of the impact of EBV on the clinical outcome of BC patients, regardless of tumor histology or treatment regimen. No correlation was found between: (i) EBV detection in tumor or PBMCs and tumor characteristics; (ii) EBV and other prognostic factors. Notably, patients exhibiting anti-ZEBRA antibodies at high titers experienced poorer overall survival (p = 0.002). Those who recovered from their disease were found to have a measurable EBV DNA load, together with a high frequency of IFN-γ and TNF-α producing PBMCs (p = 0.04), which indicates the existence of a Th1-type polarized immune response in both the tumor and its surrounding tissue.

Conclusions: The replicative form of EBV, as investigated using anti-ZEBRA titers, correlated with poorer outcomes, whereas the latent form of the virus that was measured and quantified using the EBV tumor DNA conferred a survival advantage to BC patients, which could occur through the activation of non-specific anti-tumoral immune responses.

Figure 1

Quantification of EBV DNA in peripheral blood mononuclear cells (number of copies/μg) from the 85 BC patients. The detection threshold was 5 and 10 copies EBV DNA/μg for PBMCs and tumor biopsies, respectively. Comparison with EBV load in tumors (number of copies/μg) (A) and with anti-ZEBRA antibody titers (in absorbance of 450 nm) (B) (an optical density of 1 corresponds to a titer of 1000).

Figure 2

Relative effect of EBV infection in PBMCs and tumor tissue versus no EBV on survival as a function of time for different relapses. Relapse 0 means “no relapse”; relapse 1 means “relapse diagnosed”.

Figure 3

Effect of EBV-T and EBV-P on the increase in patient survival as a function of time for different relapses. At 60 months post-diagnosis, the increase in survival is 32% and 8%, respectively, without and with relapse for “EBV-T” patients, versus 12% and 6%, respectively without and with relapse for “EBV-P” patients. Relapse 0 means “no relapse”; relapse 1 means “relapse diagnosed”. EBV-T + and EBV-P + represent patients with detectable EBV DNA in tumor tissue and PBMCs, respectively.

Figure 4

Clinical outcome of 35 patients with BC (out of all 85 patients) and correlation with frequency of peripheral NK cells and cytokine production (16 healthy individuals enrolled as negative controls). Overall survival (A) in patients with TNF-α expression (MIF) by NK cells > the control group (solid green line) and < the control group (red dashed line). In this group of 35 patients, copies of EBV genomes were detected in PBMCs in 66%, and in tumor tissues in 17%. The clinical outcome of the 35 patients with BC and correlation with TNF- α expression by peripheral T cells. Overall survival (B) in patients with TNF-α expression (MIF) by T cells > the control group (solid green line) and < the control group (red dashed).

Figure 5

Synthetic diagram analyzing the impact of the IFN-γ production by peripheral blood mononuclear cells on the clinical outcome according to EBV status in blood and tumor tissue. PBMC EBV^- means <5 EBV DNA copies/μg, PBMC EBV⁺ means >5 EBV DNA copies/μg. Tumor EBV^- means <10 EBV DNA copies/μg and tumor EBV⁺ means >10 EBV DNA copies/μg. The intensity of the color is proportional to the amount of cytokine (IFN- γ or TNF-α) production (the black color indicates negative or control basal level, the red color indicates positive or high level).