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Association Between Cytomegalovirus and Epstein-Barr Virus Co-Reactivation and Hematopoietic Stem Cell Transplantation

The co-reactivation of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in patients undergoing hematopoietic stem cell transplantation (HSCT) has been found. Research has shown that the reactivation of CMV or EBV is closely related to poor HSCT outcomes. In this study, we describe the clinical characteristics of HSCT patients with co-reactivation of CMV and EBV. We retrospectively reviewed the medical records of 327 patients who underwent HSCT at the Peking University People’s Hospital Institute of Hematology. Co-reactivation of CMV and EBV was observed in a total of 75 patients (22.9%) who also had a higher incidence of hemorrhagic cystitis (P=0.000). HSCT patients with CMV and co-reactivation of CMV and EBV had a significantly lower 1-year overall survival (OS; P=0.050). Further, COX regression analysis showed that viral infection was a risk factor for 1-year OS (HR, 12.625 for co-reactivation vs. no reactivation, p=0.021, and HR 13.580 for CMV reactivation vs. no reactivation, P=0.013). In conclusion, the patients with CMV reactivation had poorer outcome after HSCT regardless of EBV reactivation.

CMV and EBV are the most closely related clinical viruses with a clear therapeutic association (Einsele et al., 2020;Yoshimori et al., 2021). There seems to be a bidirectional relationship between the two viruses, and a high incidence/low clearance rate of CMV infection and a high incidence and delayed immune reconstitution of EBV-PTLD are key to all of these findings (Green et al., 2013;Chiereghin et al., 2019). Therefore, we speculate that the co-activation of CMV and EBV after HSCT might lead to worse clinical outcomes in transplant patients compared to those with single virus reactivation. However, few studies have investigated the effects of CMV and EBV coreactivation following HSCT. Our study analyzed the clinical characteristics of HSCT patients who developed co-reactivation of CMV and EBV, investigated the influence of CMV and EBV co-reactivation on HSCT outcomes, and analyzed the underlying risk factors.

Study Population
In total, 327 patients who underwent allo-HSCT for the first time between January 2018 and January 2019 at the Peking University People's Hospital Institute of Hematology were included in our study. We performed a retrospective review of their medical records. The Ethics Committee of the Peking University People's Hospital approved our study. Moreover, informed consent was waived owing to the retrospective nature of this study.

Virus Therapy and Monitoring
CMV and EBV reactivation was tested twice per week using plasma samples with real-time quantitative polymerase chain reaction (PCR). All patients were given ganciclovir between days −9 and −2 (Huang et al., 2009). Pre-emptive therapy with either intravenous foscarnet (90 mg/kg/day) or intravenous ganciclovir (5 mg/kg, twice daily) was started when the patients had confirmed CMV viremia reactivation, and therapy was continued until the CMV DNA was not detected on two consecutive tests. Antiviral drugs, such as foscarnet, were given to patients with EBV reactivation. Moreover, rituximab was administered when EBV viremia persisted or deteriorated to EBV disease. The salvage therapy was EBV-specific CTL.

Definitions
Our study defined myeloid engraftment as the first day of three continuous days with an absolute neutrophil count ≥0.5×10 9 /L, and platelet engraftment was recognized as the first day of seven consecutive days with a platelet count ≥20×10 9 /L without blood transfusion. Viral pneumonia mainly includes lung infections caused by influenza A, influenza B, paramyxoviruses, CMV, EBV, respiratory syncytial virus, adenovirus, and rhinoviruses. CMV and EBV reactivation were defined as the first of two continuous viral DNA tests in which the viral DNA reached or surpassed 1,000 copies/mL and 500 copies/mL, respectively. Coreactivation of CMV and EBV was defined as a test of the EBV load and CMV load over 1 year after HSCT. The duration of viremia was defined as the number of days between the first day of viremia and the first day when the virus load was no longer detected Time to relapse was defined as the days between the date of HSCT and the date of disease relapse. Non-relapse mortality (NRM) was defined death as from all causes other than those directly related to the blood disease itself, occurring at any time after transplantation. Overall survival (OS) was defined as the days from HSCT to death from any cause. Leukemia-free survival (LFS) was defined as the days from HSCT to disease progression after HSCT.

Statistical Analyses
Categorical variables between the groups were compared using the c 2 test or Fisher's exact test. Continuous variables were compared using the Mann-Whitney U test. Multivariate Cox congress models were applied to test the hazards assumption and nonlinearity of the time-by-covariate interaction. The competing risk model was suggested to calculate the cumulative incidence of virus reactivation. IBM SPSS 25.0 statistical software (IBM SPSS Statistics, USA) and SAS (version 9.4; SAS institute Inc) were used in this research.

Co-Reactivation of CMV and EBV Affects Clinical Outcomes
Patients were divided into four groups according to CMV and EBV reactivation based on the following definitions: (1) coreactivation group, (2) CMV reactivation group, (3) EBV reactivation group, and (4) no reactivation group. The characteristics of the four groups are presented in Table 1. Neutrophil engraftment was comparable among the four groups (100% vs. 100% vs. 100% vs. 97.0% for the co-reactivation, CMV reactivation, EBV reactivation, and no reactivation groups, respectively, P=0.375). However, neutrophil engraftment seemed to be delayed in patients with no virus reactivation, in contrast to that in the other groups (13 vs. 13 vs. 13 vs.14 days for co-reactivation, CMV reactivation, EBV reactivation, and no reactivation, respectively, P=0.010). According to platelet engraftment, the ratio of patients (61.3% vs. 63.6% vs. 54.5% vs. 71.3% for co-reactivation, CMV reactivation, EBV reactivation, and no reactivation, respectively, P=0.460) and days of engraftment (15 vs. 15 vs. 20 vs.13 days for co-reactivation, CMV reactivation, EBV reactivation, and no reactivation, respectively, P=0.080) were comparable among the four groups. The morbidity of acute GVHD was very higher in the reactivation group compared to that in the no reactivation group (56.3% vs. 61.5% vs. 55.6% vs. 37.0% for co-reactivation, CMV reactivation, EBV reactivation, and no reactivation, respectively, P=0.005). Patients in the reactivation group were more likely than those in the other groups to progress to viral pneumonia (4.0% vs. 4.5% vs. 0% vs. 1.1% for co-reactivation, CMV reactivation, EBV reactivation, and no reactivation, respectively, P=0.484), and a similar trend was observed for viral enteritis (2.7% vs. 1.3% vs. 0% vs. 0% for co-reactivation, CMV reactivation, EBV reactivation, and no reactivation, respectively, P=0.473). Hemorrhagic cystitis was more common in the reactivation group than in the other groups (37.5% vs. 35.2% vs. 14.1% vs. 14.1% for co-reactivation, CMV reactivation, EBV reactivation, and no reactivation, respectively, P=0.000, Table 2). Co-reactivation and CMV reactivation were found in 22.9% (n=75) and 47.1% (n=154) of patients, respectively. In the coreactivation group, the time of first CMV viremia reactivation after HSCT preceded that in the CMV reactivation group (40 [31, 47] vs. 40 [33, 49] days, respectively, P=0.370). The duration of CMV viremia in the co-reactivation group was longer than that in the CMV reactivation group (21 [15,26] vs.18 [11,24], days, respectively, P=0.007). The highest CMV viral load in the CMV reactivation group was higher than that in the CMV reactivation group (1.71 [1.14,3.68] vs.1.69 [0.77,3.50] ×10 3 copies/mL, respectively, P=0.315). EBV reactivation was observed in 3.3% of the patients (n=11). The differences in the time of first EBV viremia reactivation after HSCT and the duration of EBV viremia were not statistically different between the two groups. Moreover, the highest EBV viral load was also not statistically different between the two groups.
The 1-year OS was lower in the reactivation groups than in the no reactivation group (86.7% vs. 85.1% vs. 90.0% vs. 95.4% for co-reactivation, CMV reactivation, EBV reactivation, and no reactivation, respectively, P=0.050). There were no significant differences in 1-year LFS among these groups. Viral reactivation was a risk factor for 1-year OS (Figure 1). Further Cox regression analysis showed that viral infection was a risk factor affecting the 1-year OS ( Table 3; HR, 12.625 for co-reactivation vs. no reactivation, P=0.021, and HR, 13.580 for CMV reactivation vs. no reactivation, P=0.013). However, viral reactivation was not a risk factor for LFS (Figure 2; p=0.348). The GVHD was a risk factor affecting the 1-year LFS (HR, 2.099 for GVHD vs. no GVHD, P=0.016). The 1-year OS and 1-year LFS hazards are summarized in Table 3.

Co-Reactivation of CMV and EBV Association With Risk Factors
Patients with co-reactivation of CMV and EBV were compared with all other patients to identify risk factors associated with coreactivation. The competing risk model showed that age(>=40years vs. <40years)was one risk factor for co-reactivation of CMV and EBV (

DISCUSSION
In our study, we proved that CMV and EBV co-reactivation in HSCT patients were associated with OS and LFS. Our findings are thus clinically relevant. CMV reactivation is known to be significantly associated with EBV reactivation (Fan et al., 2016), but CMV and EBV co-reactivation is relatively less widespread compared to that of the other double-stranded DNA viruses. In our study, CMV and EBV co-reactivation was found in 22.9% of patients after HSCT (n=75). This is consistent with a previous study that reported CMV-EBV coinfection, in which the rates of co-infection post-transplantation varied between 3.0% and 32.7% (Anderson-Smits et al., 2020). In the study reporting the lowest rate (3.0%,12/404), the total study population included patients who were not included in the groups at a high risk of EBV-related complications, in high-stakes patients, and in those who were not high stakes but had improved moderate-to-poor   -Cadenas et al., 2015). Although the proportion of patients with CMV-EBV coreactivation was reported with EBV-PTLD (post-transplant lymphoproliferative disorders), it is unclear whether this occurs for all cases of co-reactivation (i.e., CMV-EBV coreactivation without PTLD), which might have helped to decrease the rate of co-reactivation. In the report citing the highest rate of CMV-EBV co-reactivation (32.7%; 33/101), all cases of co-reactivation involved both EBV and CMV (defined by viral load) (Zallio et al., 2013), and a similar definition of coreactivation was used to that in our study. Another study reported CMV-EBV coinfection rates of 22.7% (10/44) (Fan et al., 2016) with a similar incidence of co-reactivation under the same definition.

steroid-refractory GVHD (Garcia
Our study proved that CMV and EBV co-reactivation were related to a decreased 1-year OS. However, the causes of death (NRM or relapse) did not significantly differ among the groups (P>0.05). This result is partly in accordance with that of a previous study. Co-reactivation of CMV and EBV was found to be related to a decreased 1-year OS, which was mainly because of an increase in NRM (Zhou et al., 2020). With co-reactivation, the 1-year NRM was higher, in contrast to that found by two other groups, the difference was not remarkably significant (P=0.053), and no deaths occurred due to relapse (Zhou et al., 2020). Another report showed that a CMV and EBV co-reactivation group had a significantly higher 6-month non-relapse mortality than the other groups with CMV or EBV reactivation alone (Song et al., 2014). According to our findings, CMV reactivation alone after HSCT was also associated with 1-year OS; however, EBV reactivation alone after HSCT was not. One meta-analysis showed that CMV reactivation is associated with an increased risk of overall mortality and NRM in allo-HSCT recipients (Gimenez et al., 2019).
According to our results, the duration of CMV viremia was found in the co-reactivation group as compared to that in the other reactivation groups, reflecting the effect of EBV reactivation on CMV reactivation. CMV induces NKG2C + CD57 + KIR+ natural killer (NK) cell expansion 3-6 months after HSCT (Zuo and Zhao, 2021). NK cells are the earliest reconstituting immune cells, achieving normal numbers within  weeks after patients undergo HSCT and helping in the graftversus-tumor function along with T cells (Farag et al., 2002). Meanwhile, the cytokine-producing and cytotoxic functions of NK cells were found to be lower until 3-6 months after HSCT (Foley et al., 2011) and reached common reactivity levels at the first year and were maintained during later times (Haas et al., 2011). Immunoreactivation of one virus mediated by another virus has been previously reported with both HSCT and solid organ transplantation. This could also demonstrate poor immune reconstitution as previously shown with poor CD3+ and CD4+25 + cell counts (on day 30) (Zhou et al., 2020). CMV and EBV coreactivation induces an increase in CD56dim/NKG2A+/CD57 +NK cell numbers, which remain elevated up to 6 months after reactivation and leads to a decrease in the absolute quantity of immature CD56bright/CD16− NK cells in the blood (Lunemann et al., 2013;Hendricks et al., 2014). One study showed that CMV is an especially active inducer of some members of the herpesvirus family and implied that the interplay between CMV and EBV occurs unidirectionally in vivo (Aalto et al., 1998). However, this study was not based on HSCT patients. Our study identified HLA match (unrelated donor and haploidentical vs. Identical) as an independent risk factor for CMV and EBV co-reactivation. Compared with HLA identical sibling transplantation, patients undergoing HLA-haploidentical stem cell transplantation(haploSCT) usually receive more intensive immunosuppressors to guarantee engraftment and later prevent graft-versus-host disease (GVHD) (Luo et al., 2021). Previous studies have shown that risk factors for CMV reactivation after HSCT include a donor or recipient seropositive for CMV, mismatched or unrelated donors, pre-allo-HSCT viremia, and use of alemtuzumab (Sousa et al., 2014). Another findings show patients undergoing hematopoietic stem cell transplantation or solid organ transplantation can experience post-transplant lymphoproliferative disorders due to dysfunction or suppression of host's immune system, or uncontrolled proliferation of EBV-infected cells (Fujimoto and Suzuki, 2020). Some study uncovered a significant correlation of recovered Vd2 with EBV reactivation following haploSCT (Liu et al., 2018). HSCT is a process comprising total immune reconstruction, and the interplay of various viruses in this process makes the condition of HSCT patients more complicated and diverse.
There are several limitations to this study. First, the retrospective nature of the study has inherent risks of bias; nevertheless, the patient characteristics and the HSCT complications did not differ significantly from those reported in prospective research. Second, we did not study other herpesviruses, which might have had a significant effect on these findings. However, we studied the two most clinically significant viruses that have defined treatment options. Despite these limitations, we believe that this study provides clinical hematologists with scientific evidence of CMV and EBV co-reactivation.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of the Peking University People's Hospital. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
SL and MS: design of the study; SL and NZ: data acquisition, analysis, and interpretation; MJ: drafted the article and critically revised the manuscript; MS: gave final approval for the version to be submitted. All authors contributed to the article and approved the submitted version.

FUNDING
This study was approved by the National Natural Science Foundation of China to MS (Grant No. 81870196).