Incidence and risk factors of Epstein–Barr virus reactivation and post-transplant lymphoproliferative disorder after allogeneic hematopoietic stem cell transplantation: A 985-patient retrospective cohort analysis

Abstract Background: Epstein-Barr Virus (EBV) reactivation frequently occurs following allogenic stem cell transplantation (allo-HSCT). In a subset of patients, uncontrolled EBV reactivation can progress to post-transplant lymphoproliferative disease (PTLD), a potentially life-threatening condition that driven by impaired T cell activity and unchecked EBV-infected B-cell expansion. Established clinical risk factors include T cell depletion, pharmacologic immunosuppression, EBV serology mismatch, cytomegalovirus (CMV) reactivation, and the presence of graft versus host disease (GVHD). Despite these known risks, critical limitations persist in current risk stratification models, particularly the oversimplification of immune reconstitution dynamics and inconsistent incorporation of quantitative EBV virologic thresholds for PTLD risk prediction. Methods: To identify critical factors that governing EBV reactivation and EBV-to-PTLD progression, we retrospective analyzed patients who underwent allo-HSCT at our center between 2020 and 2022. The viral load for EBV was monitored weekly for the first 3 months after transplantation, biweekly from the fourth to the sixth month post-transplant, and monthly from the seventh to the twelfth month post-transplant. EBV reactivation was identified when the viral load surpassed 1×103 copies/ml from whole blood samples. The diagnosis of PTLD was made in accordance with the guidelines established by the ECIL-6. Results: Out of 985 patients, 447 (45.4%) experienced EBV reactivation. The median time to EBV reactivation from transplantation was 55 days, with a range of 6 to 231 days. We observed that patients who received grafts from matched sibling donors (MSD) had a significantly lower incidence of EBV reactivation compared to those with other donor types (MSD 13.5% vs HRD 50.9% vs MURD 35.2% vs MMURD 42.6%, p<0.001). This difference coincided with substantial variation in anti-thymocyte globulin (ATG) dosing: MSD recipients received no ATG (0 mg/kg), whereas HRD, MURD, and MMURD groups received 6, 4.5, and 6 mg/kg, respectively. CMV viremia (52.4% vs 33.8%, p<0.001) and older donors (≥40 years old) (50.6% vs 42.4%, p=0.012) were also associated with a higher risk of EBV reactivation. Multivariate analysis confirmed MSD as a protective factor against EBV reactivation (HR=0.704, 95%CI: 0.595-0.832, p<0.001), while CMV viremia (HR=2.234, 95%CI: 1.695-2.946, p<0.001) and older donors (HR=1.510, 95%CI: 1.158-1.970, p=0.002) were identified as independent risk factors. In this cohort of 447 EBV-infected transplant recipients, viral loads stratified as follows: 334 (74.7%) at 103–10⁵ copies/mL, 87 (19.5%) at >10⁵ copies/mL, and 26 (5.8%) at >10⁶ copies/mL. PTLD incidence demonstrated a nonlinear escalation with increasing viral burden: only 0.9% (3/334) of patients in the 10³–10⁵ copies/mL group developed PTLD, compared to 9.2% (8/87) in the >10⁵ copies/mL group (10-fold increase, p<0.001) and 26.9% (7/26) in the >10⁶ copies/mL group (3-fold increase vs. >10⁵ group, p=0.003). Regarding donor type, patients with MSD and MURD grafts exhibited lower PTLD incidence compared to those with HRD and MMURD grafts (3.4% vs 4.0% vs 7.1% vs 12.5%, p=0.094). Notably, patients receiving thiotepa experienced a significantly higher PTLD rate than those who did not (3.4% vs 27.3%, p=0.007). In the multivariate analysis, HRD (p=0.008, HR=54.6), high viral loads of EBV (＞105copies/mL: p=0.002, HR=29.7; ＞106copies/mL: p=0.002, HR=220.1) were corelated with higher rates of PTLD. We also analyzed the relationship between immune reconstitution and PTLD. At day 30 post-transplantation, patients who later developed PTLD exhibited lower median levels of CD3⁺ T cells (19 vs 118 cells/µL, p=0.009) and CD3⁺CD8⁺ T cells (11 vs 81 cells/µL, p=0.011), alongside higher median levels of C3 (130 vs 110 mg/dL, p=0.006) and NK cells (286 vs 109 cells/µL, p=0.046) compared to those without PTLD.Conclusion: Our study identified three independent factors significantly associated with increased risk of EBV reactivation: receipt of matched sibling donor (MSD) grafts, CMV viremia, and advanced donor age. Furthermore, haploidentical related donor (HRD) transplantation and high EBV viral load (>10⁵ copies/mL) were directly associated with elevated risk of post-transplant lymphoproliferative disorder (PTLD). Notably, delayed immune reconstitution, particularly low CD3⁺CD8⁺ T cell counts, demonstrated a compelling association with PTLD development.