Role of plasma EBV-DNA load and EBER status on newly diagnosed peripheral T-cell lymphoma

Peripheral T-cell lymphomas (PTCLs), a group of heterogeneous T-cell neoplasms, originate from post-thymic lymphocytes and constitute approximately 15% of non-Hodgkin lymphomas, including peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), nodal T-follicular helper (TFH) cell lymphoma (PTCL-TFH), systemic anaplastic large cell lymphoma (sALCL), among others (Alaggio et al. 2022). ALCLs, classified based on the anaplastic lymphoma kinase (ALK) status, comprise two groups. Apart from ALK + ALCLs, most PTCLs exhibit a grim prognosis, with a 5-year overall survival (OS) ranging from 10 to 51% (Zhang et al. 2016; Rodríguez et al. 2021; Mina and Pro 2022).

Recognizing patients at high risk and selecting appropriate therapeutic regimens are essential. Epstein–Barr virus (EBV) is a ubiquitous DNA virus belonging to the gamma subfamily of herpesviruses, associated with several lymphoma subtypes, such as Burkitt's lymphoma (BL) and extranodal NK/T-cell lymphoma (ENKTL) (Bednarska et al. 2024; Damania et al. 2022; Lopez et al. 2022; Xiong et al. 2020). EBV-associated lymphomas are characterized by the presence of EBV-encoded RNA (EBER) in tumor tissue. However, the effect of EBV on PTCL remains controversial. Some studies (Dupuis et al. 2006; Weisenburger et al. 2011; Kim et al. 2021) have demonstrated poor survival outcomes in patients with EBV infection, while others have presented contrary views (Haverkos et al. 2017; Shen et al. 2022). Additionally, only a few studies have compared different EBV detection methods, such as EBER using in situ hybridization (ISH) in neoplasms and EBV DNA polymerase gene in peripheral blood using polymerase chain reaction (PCR) (Kim et al. 2021; Shen et al. 2022; Zeng et al. 2022).

The optimal first-line chemotherapy regimen for PTCL remains undefined. The addition of etoposide to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP/E) has been proposed to enhance clinical benefit, but its therapeutic efficacy is disputed (Deng et al. 2019; Liu et al. 2018; Jia et al. 2016). Some studies have indicated that EBV infection typically leads to extensive methylation, contributing to immune escape and disease progression in conditions such as gastric cancers, nasopharyngeal carcinoma, and breast cancer (Matsusaka 2014; Usui et al. 2021; Peng et al. 2016; Abdallah et al. 2018). Hypomethylating agents, such as azacytidine (AZA), have the potential to reduce genomic methylation levels and reactivate hypermethylated tumor suppressor genes. Therefore, targeting DNA methylation may represent a novel therapeutic strategy for EBV-associated diseases (Zhang et al. 2022).

This study aimed to investigate the frequency, clinical features, and survival outcomes of PTCL with EBV, compare the use of two methods for detecting EBV (EBER vs. EBV DNA), and monitor dynamic changes in EBV DNA. This study also found evidence suggesting that CHOP plus AZA may offer promising treatment efficacy for EBV-associated patients, along with relative genetic analysis.

To the best of our knowledge, this is the largest study to examine the value of EBV in PTCL patients using ISH plus PCR and the first to describe the molecular characteristics of EBV-associated PTCL. We retrospectively enrolled 262 PTCL patients and compared survival outcomes based on the pretreatment EBER status, pretreatment, and post-treatment EBV DNA levels, supporting the association of EBER, DNA1, and DNA4 with survival. Our findings show that CHOP plus AZA may improve survival outcomes for EBV-associated patients. The genetic basis of PTCL revealed that EBER-positive and partially positive patients were more likely to harbor DNMT3A (P = 0.002), RHOA^G17V (P = 0.023), and TET2 mutations (P = 0.032). It is plausible that AZA reverses methylation caused by EBV infection via TET2 and/or DNMT3A mutations.

The relationship between EBER and EBV DNA has been explored extensively. Haverkos et al. 2017 found that EBER-positive patients had elevated DNA1 levels, but DNA1 failed to identify all EBER-positive patients (100% specificity; 53% sensitivity). Zhao et al. observed a high level of DNA1 associated with EBER positivity (Zhao et al. 2022). In this study, we observed poor consistency (kappa = 0.254) between EBER and DNA1, with a significant difference between EBER-positive and -negative patients in DNA1 (P = 0.000) but only a slight difference in DNA4 (P = 0.075). This indicates that EBER-positive patients tend to have higher DNA levels at diagnosis, but this tendency can change after treatment.

Our observations showed that patients with EBER negativity and lower levels of DNA1 achieved overall response rates easily (P = 0.015 and P = 0.078, respectively). However, EBER failed to predict relapse in competing risk models (P = 0.496), which is inconsistent with another report (Zhao et al. 2022). Overall, EBV infection impacted the disease response, but further research is still needed.

The impact of EBV on the prognosis of PTCLs has remained controversial. Previous studies have found EBV status to be either associated or not associated with a poorer prognosis (Dupuis et al. 2006; Weisenburger et al. 2011; Kim et al. 2021; Haverkos et al. 2017; Shen et al. 2022). In our study, EBER-positive patients exhibited dismal OS (P = 0.047) and PFS (P = 0.009). Univariate analysis revealed that DNA1 and DNA4 were significantly associated with inferior OS (P = 0.001; P = 0.005). Multivariate analysis further demonstrated that DNA4 had an independent effect on the prognosis of PTCL for OS. Consistent with previous studies by Song et al. (2022), Shen et al. (2022), and Qiu et al. (2022), EBV DNA emerged as an effective prognostic marker. It appears that EBV DNA may be superior to EBER, possibly because EBER is a qualitative diagnostic index derived from tissue samples, whereas EBV DNA is a quantitative measurement analyzed from plasma samples. This superiority of EBV DNA over EBER is reflected in the diagnostic test results: an inconsistent relationship (kappa = 0.254). Thus, monitoring EBV DNA levels may be recommended for predicting survival.

The optimal treatment regimens for PTCLs remain undefined, particularly for those with EBV infection who typically have a poor prognosis. This may be attributed to EBV infection leading to extensive methylation, resulting in immune escape and disease progression (Zhang et al. 2022; Ambinder et al. 1999). Our study found that the CHOP plus AZA regimen achieved promising OS for EBER-positive patients (Fig. S2; P = 0.046) and EBER-positive plus partially positive patients (Fig. 5; P = 0.192). This trend was also observed in the AITL cohort, which constituted the largest population (Fig. S3, P = 0.052; Fig. S4, P = 0.286). Despite the limited sample size, these cases provide insights into treatment, suggesting that EBV-infected lymphomas may respond well to hypomethylating agents. Falchi et al. conducted a phase II clinical trial combining AZA and romidepsin, where 48% of patients achieved complete remission (Falchi et al. 2021). Additionally, a patient with AITL and chronic myelomonocytic leukemia preceded by an EBER-positive large B-cell lymphoma showed a favorable response to AZA (Saillard et al. 2017). These findings resonate with our results, supporting the notion that targeting DNA methylation abnormalities could be an effective strategy for patients with EBV-associated lymphomas.

The molecular paradigm of EBV was also investigated. We observed that EBER-positive and partially positive patients were more likely to harbor DNMT3A (P = 0.002), RHOA^G17V (P = 0.023), and TET2 mutations (P = 0.032) (Fig. 6). Previous reports have indicated a correlation between TET2 and/or DNMT3A mutations and DNA methylation in hematological tumors (Xie et al. 2020; Lemonnier et al. 2018, Woods and Levine 2015). However, the association of EBV with methylation in PTCL has not been extensively studied. Considering the observed clinical benefit of AZA in EBV-related patients, it is conceivable that hypomethylating agents like AZA could target methylation caused by EBV through DNMT3A or TET2 to improve prognosis. Nonetheless, the detailed genetic features of EBV in PTCL remain unclear. Further studies with larger cohorts are warranted to explore this aspect, especially considering the partial overlap between the subjects of survival analysis and genetic analysis in our study.

Several limitations should be acknowledged in our study. Firstly, its retrospective nature and single-center design with varied therapeutic regimens may introduce bias, despite efforts to mitigate this through comparable clinical characteristics and statistical adjustments. Secondly, the limited sample size of EBV-infected or AZA-treated patients warrants validation in larger prospective cohorts with homogenous treatment approaches. Thirdly, while molecular analysis related to DNA methylation was conducted, there was not complete consistency between the subjects of survival analysis and genetic analysis.

In conclusion, EBER positivity, elevated DNA1, and elevated DNA4 were significantly associated with inferior survival, highlighting the importance of monitoring EBV DNA in newly diagnosed PTCL. Front-line treatment with CHOP plus AZA is possibly good for EBER partially positive and positive patients, who may exhibit high levels of methylation due to DNMT3A or TET2 mutations. However, these speculations require further exploration in larger cohort studies.