Abstract
Background:
Epstein-Barr virus (EBV) seronegative solid organ transplant recipients (SOTRs) are at increased risk for post-transplant lymphoproliferative disorder (PTLD). Assays for EBV serostatus assess antibody to both EBV viral capsid antigen (VCA) and Epstein-Barr nuclear antigen-1 (EBNA-1), but PTLD risk among SOT recipients with discordant VCA and EBNA-1 results is unknown.
Methods:
We performed a retrospective, single center cohort study to determine the risk of PTLD among adult (≥ 18 years) SOTRs with discordant pre-transplant VCA and EBNA-1 IgG compared to that of SOTRs with concordantly negative or concordantly positive serology using univariable and multivariable Cox-proportional hazards models.
Results:
Of 4106 SOTRs, the number (%) who were concordantly positive, concordantly negative, and discordant was 3787 (92.2%), 149 (3.6%), and 170 (4.2%), respectively. The adjusted hazard of PTLD was significantly higher among discordant SOTRs compared to concordantly positive SOTRs (aHR 2.6, 95% CI 1.04–6.6, p=0.04) and lower compared to concordantly negative SOTRs (aHR 0.27, 95% CI 0.10–0.76, p<0.001). The adjusted hazard of EBV+ PTLD among those with discordant serology was also significantly higher compared to the concordantly positive cohort (aHR 3.53, 95% CI 1.04–12.0, p=0.04) and significantly lower compared to the concordantly negative cohort (aHR 0.23, 95% CI 0.06–0.82, p=0.02).
Conclusions:
Risk of PTLD among SOTRs with discordant VCA and EBNA-1 may be intermediate between those with concordantly positive and negative serology. If confirmed in future studies, revision of national EBV serology reporting to include both VCA and EBNA results may be needed to optimize PTLD risk stratification.
Keywords: EBV, PTLD, Epstein-Barr nuclear antigen, viral capsid antigen, serology
Graphical Abstract
Introduction
Post-transplant lymphoproliferative disorder (PTLD) is the most common extra-cutaneous malignancy in solid organ transplant (SOT) recipients and is associated with significant morbidity and mortality1. Over 50% of PTLDs are driven by abnormal expansion of Epstein-Barr virus (EBV)-infected B cells2. Although EBV establishes latency and immortalizes B cells in all hosts following primary infection, excess proliferation of EBV-infected B cells is normally limited by virus-specific cytotoxic T lymphocyte (CTL) responses. Loss of such T cell surveillance due to post-transplant immunosuppression contributes to PTLD development in SOT recipients infected with EBV prior to transplant (R+)3. Risk of PTLD is significantly higher among EBV-naïve SOT (R-) who acquire primary EBV after transplant in the context of immunosuppression, which impairs initial CTL responses and leads to permissive viral replication and an expanded reservoir of EBV-infected B cells with malignant potential4–9.
Because >90% of adult organ donors have been infected with EBV (D+), most R− SOT candidates will receive EBV-infected allografts (D+R-) and develop primary EBV infection in the early post- transplant period10–12. It is estimated that approximately 10% of EBV D+R− SOT recipients will develop PTLD during their lifetime, a 10-fold increased risk compared to EBV R+ SOT recipients, with the majority of risk within the first several years post-transplant5, 7, 8, 13, 14. Pre-transplant recipient EBV IgG serology is used to identify high-risk EBV R− SOT candidates, allowing transplant centers to target PTLD mitigation strategies (e.g., post-transplant EBV viral load surveillance, minimal intensity immunosuppression) to this high risk group. Investigational therapies such as novel prophylactic agents and adoptive EBV-specific T cells rely on pre-transplant recipient EBV serology to identify patients most likely to benefit from intervention. Most commercial EBV serology assays detect antibodies to both viral capsid antigen (VCA) and Epstein-Barr nuclear antigen-1 (EBNA-1). While both VCA IgG and EBNA-1 IgG are detectable for life in the majority of EBV-infected individuals, discordant VCA and EBNA-1 IgG results occur: 5–10% of people either never develop or develop but subsequently lose EBNA-1 IgG following primary infection, and VCA IgG is not detected in ~1–2% EBNA-1 IgG positive individuals15–17. Large epidemiologic studies that have examined the associations between EBV R− serostatus and PTLD risk have generally not specified the antigen target(s) of pre-transplant EBV IgG serology, and the implications of discordant recipient VCA and EBNA-1 IgG on PTLD risk are unknown4–7, 13, 18, 19. We hypothesized that PTLD risk would differ among those with discordant results for VCA/EBNA compared to those who are concordantly VCA/EBNA negative or positive. We performed a retrospective, single-center cohort study to assess the cumulative incidence rate of PTLD in SOT recipients with discordant pre-transplant EBNA-1 and VCA IgG (VCA+/EBNA-1- or VCA-/EBNA-1+) and to compare risk of PTLD among those with discordant serology to those with concordantly positive or concordantly negative serology.
Methods
2.1. Recipient EBV serology
This study was approved by the institutional review board at the University of Washington (STUDY00009960) with a waiver of informed consent. We identified all patients who underwent a first solid organ transplant at a single United States academic medical center between January 1, 2000 and April 30, 2018 and who were ≥ 18 years old at the time of transplant. Electronic medical records were used to extract results of EBV serology (VCA IgM, VCA IgG and EBNA-1 IgG) obtained prior to transplant for each patient. EBV serology was performed using an FDA-approved enzyme-linked-immunoassay (ELISA) system (ZEUS Scientific, Inc., Somerville, NJ from 2000–2004 and Sigma-Aldrich, St. Louis, MO from 2005–2018). SOT recipients were excluded if pre-transplant EBV serology was unavailable, incomplete (e.g., EBNA-1 IgG was not done), equivocal, or if results of two or more pre-transplant serology profiles were discrepant. SOT recipients were classified into one of three categories based on pre-transplant EBV IgG results: concordantly negative (VCA-/EBNA-1 −), concordantly positive (VCA+/EBNA-1+) and discordant (VCA+/EBNA-1+ or VCA-/EBNA-1-). Serial plasma EBV PCR monitoring (e.g., weekly for the first three months post-transplant, then monthly until month six, then again at months nine and twelve) with associated reduction in immunosuppression upon detection of EBV DNAemia was performed in VCA-/EBNA-1− recipients of organs from EBV seropositive donors at the discretion of transplant clinicians.
2.2. Donor EBV serology
Donor VCA and EBNA-1 IgG serology were not available for all recipients. To estimate the EBV serology profiles of donors relevant to the present study, we assessed VCA and EBNA-1 IgG among deceased donors evaluated in the donor testing laboratory for the local organ procurement organization (LifeCenter Northwest). EBV serology, measured using the Bio-rad enzyme immunoassay (Bio-rad, Redmond, WA), was available from donors evaluated between February 16, 2011 through April 30, 2018
2.3. Data collection and PTLD detection
Electronic medical records were used to extract demographic and clinical information and capture all cases of biopsy-proven PTLD that occurred between transplant and the study end date of April 30, 2020. April 30, 2020 was chosen as the end of study to ensure that all patients were followed through the first 2 years post-transplant, during which the incidence of PTLD is greatest3, 13, 20. We used three separate systems to comprehensively identify cases of PTLD: 1) Leaf, a self-service tool for capturing data from the electronic medical records at the University of Washington21, 2) our center’s reports of PTLD cases to the Scientific Registry of Transplant Recipients (SRTR) in accordance with Organ Procurement and Transplantation Network (OPTN) reporting requirements, and 3) PowerPath (Sunquest, Tucson, AZ), an electronic database for pathology records. Once subjects with PTLD were identified, medical records were reviewed to confirm the clinical diagnosis and extract details regarding the PTLD diagnosis date (defined as the date that diagnostic specimen was obtained), World Health Organization (WHO) classification according to 2016 definitions22, and results of tumor Epstein-Barr-virus-encoded small RNA (EBER) testing using in-situ hybridization. PTLD was considered EBV positive if EBER was detected.
2.4. Statistical Analyses
Descriptive analyses assessed clinical and demographic characteristics of SOT recipients in each EBV serostatus group using counts and percentages for categorical variables and medians (range) for continuous variables. Proportions were compared across EBV serostatus groups using Chi-squared or Fisher’s exact tests where appropriate. The cumulative incidence rates of PTLD and EBV+ PTLD were calculated for each EBV serostatus group and 95% confidence intervals were constructed using a Poisson distribution. Cumulative incidence curves were plotted using the Kaplan-Meier method. Patients were censored at death or April 30, 2020 (end of study), whichever came first.
We constructed univariable Cox proportional-hazards models to assess associations between pre-transplant EBV recipient serology and a priori selected covariates (exposures) and PTLD (outcome). Covariates including age at first transplant, year or first transplant dichotomized as 2000–2009 and 2010–2018, organ(s) transplanted and induction with anti-thymocyte globulin) were selected based on hypotheses and associations with PTLD in prior studies6, 13, 23. Age at first transplant was assessed as a continuous variable.
Multivariable Cox proportional-hazards models were constructed to include recipient pre-transplant EBV serostatus and covariates with a p-value ≤0.05 in univariable analyses, consistent with methods used in prior studies8, 24. For all analyses, p-values ≤ 0.05 were considered statistically significant. R version 4.0.2 (R Foundation for Statistical Computing) was used to perform statistical analyses.
Results
3.1. Characteristics of SOT recipients
There were 4889 adults who underwent a first SOT during the study period. Complete, unequivocal, and non-discrepant pre-transplant EBV serology results were available for 4106 (84%, Figure S1). Characteristics of SOT recipients excluded from analysis are shown in Table S1 and Table S2. One hundred forty-nine of 4016 (3.6%) had concordant negative serology, 3787 (92.2%) had concordant positive serology, and 170 (4.1%) had discordant serology (148 VCA+/EBNA-1- and 22 VCA-/EBNA-1+). Just under half (1802, 44%) of patients underwent transplant in the earlier part of the study (2000–2009). Few (221, 5.4%) SOT recipients had detectable VCA IgM pre-transplant and VCA IgM positivity did not vary significantly by IgG serostatus group (Fisher exact p=0.33, Table 1).
Table 1:
Characteristics of adult solid organ transplant recipients stratified by pre-transplant Epstein-Barr virus IgG serostatus
| VCA-/EBNA-1− (N=149) | VCA+/EBNA-1+ (N=3787) | Discordant (N=170) | |
|---|---|---|---|
| Median age at transplant, years (range) | 46.0 (18.5, 80.7) | 54.8 (18.1, 82.9) | 51.3 (18.6, 73.8) |
| Transplant year | |||
| 2000–2009 | 63 (42.3%) | 1668 (44.0%) | 71 (41.8%) |
| 2009–2018 | 85 (57.3%) | 2448 (56.0%) | 99 (58.2%) |
| Male, n (%) | 106 (71.1%) | 2448 (64.6%) | 87 (51.2%) |
| VCA IgM Positive, n (%) | 4 (2.7%) | 207 (5.5%) | 10 (5.9%) |
| ATG induction, n (%) | 103 (69.1%) | 2406 (63.5%) | 98 (57.6%) |
| Organ, n (%)a | |||
| Lung | 26 (17.4%) | 614 (16.2%) | 33 (19.4%) |
| Heart | 21 (14.1%) | 482 (12.7%) | 14 (8.2%) |
| Liver | 29 (19.5%) | 1385 (36.6%) | 43 (25.3%) |
| Kidney | 64 (43.0%) | 1191 (31.4%) | 77 (45.3%) |
| Any pancreas | 9 (6.0%) | 115 (3.0%) | 3 (1.8%) |
Abbreviations: ATG (anti-thymocyte globulin), EBNA-1 (Epstein-Barr virus nuclear antigen-1), VCA (viral capsid antigen)
Lung includes 1 lung-heart and 1 lung-liver recipient; Heart includes 14 heart-kidney recipients, Liver includes 55 liver-kidney recipients; Any pancreas includes 114 kidney-pancreas and 13 isolated pancreas recipients.
3.2. EBV serology profile of organ donors
Donor VCA and EBNA-1 IgG were not available for all SOT recipients. Of 1747 donors evaluated by a local organ procurement organization during the latter half of the study period, the majority (1618, 93%) were positive for IgG to at least one EBV antigen, and 1391 (80%) were positive for both VCA and EBNA-1 IgG (Table S3).
3.3. PTLD in SOT recipients
Fourteen (9.4%) SOT recipients with concordant negative and 49 (1.3%) with concordant positive serology developed PTLD during follow up (cumulative incidence rate 13.6/1000 person-years [95% CI 6.0–20.2–22.9] and 1.7/1000 person-years [95% CI 1.4–2.5], respectively, Table 2). Five (2.9%) SOT recipients with discordant EBV serology (four VCA+/EBNA-1- and one VCA-/EBNA-1+) developed PTLD during follow-up (cumulative incidence rate 4.1/1000 person-years [95% CI 1.3–9.5]). Sixty percent of all PTLDs among SOT recipients with discordant serology were EBV+ by histopathology. This proportion was intermediate between, but not significantly different from those with concordant negative serology (100%, p=0.07) and those with concordant positive serology (47%, p=0.66). The cumulative incidence rate of EBV+ PTLD among VCA/EBNA-1 IgG discordant SOTRs was 2.4/1000 person-years (95% CI 0.50–7.08).
Table 2:
Characteristics of post-transplant lymphoproliferative disorders in adult solid organ transplant recipients by recipient Epstein-Barr virus IgG serostatus
| VCA-/EBNA-1− (n=149) | VCA+/EBNA-1+ (n=3787) | Discordant (n=170)a | Overall (n=4106) | |
|---|---|---|---|---|
| Total cases of PTLD, n (%) | 14 (9.4%) | 49 (1.3%) | 5 (2.9%) | 68 (1.6%) |
| PTLD characteristics, n (% PTLD cases) | ||||
| Tumor EBER statusb | ||||
| Positive | 12 (100.0%) | 20 (46.5%) | 3 (60.0%) | 35 (58.3%) |
| Negative | 0 (0%) | 23 (53.5%) | 2 (40.0%) | 25 (36.8%) |
| WHO classification | ||||
| Plasmacytic hyperplasia | 1 (7.1%) | 0 (0.0%) | 0 (0.0%) | 0 (0.0%) |
| Polymorphic | 3 (21.4%) | 2 (4.1%) | 2 (40.0%) | 7 (10.3%) |
| Monomorphic | 9 (64.3%) | 41 (83.7%) | 3 (60.0%) | 53 (77.9%) |
| Hodgkin’s lymphoma | 0 (0%) | 1 (2.0%) | 0 (0%) | 1 (1.5%) |
| Unable to Classify | 1 (7.1%) | 5 (10.2%) | 0 (0%) | 7 (10.3%) |
| Cumulative incidence rate per 1000 person-years (95% confidence interval) | ||||
| All PTLD | 13.6 (6.0–20.2) | 1.71 (1.26–2.26) | 4.05 (1.32–9.47) | 2.20 (1.70–2.79) |
| EBV positive PTLDc | 11.7 (0.75–22.9) | 0.70 (0.42–1.07) | 2.42 (0.50–7.08) | 1.27 (0.79–1.57) |
Abbreviations: EBER (Epstein-Barr encoded RNA), EBNA-1 (Epstein-Barr nuclear antigen-1), EBV (Epstein-Barr virus), PTLD (post-transplant lymphoproliferative disorder), WHO (World Health Organization), VCA (viral capsid antigen)
148 (87%) of discordant SOT recipients were VCA+/EBNA-1- and 22 (13%) were VCA-/EBNA-1+
PTLD EBER status was unavailable on tumors from 2 VCA-/EBNA-1- and 6 VCA+/EBNA-1+ solid organ transplant recipients with PTLD.
Cumulative incidence of EBER positive PTLD. PTLDs in which EBER status was indeterminate or unavailable were not considered EBV positive.
Two SOT recipients with detectable pre-transplant VCA IgM developed PTLD. One was a kidney/pancreas recipient with discordant pre-transplant VCA/EBNA-1 IgG (VCA+/EBNA-1-) who developed EBV+ PTLD two years after transplant. The other was a concordantly positive liver recipient who developed non-EBV+ PTLD seven years post-transplant.
3.4. PTLD risk by Cox proportional hazards
Male sex, age at transplant and receipt of peri-transplant anti-thymocyte globulin (ATG) were not associated with PTLD in univariable analyses (Table 3). Transplant between 2010–2018 was associated with a lower hazard of PTLD compared to transplant between 2000–2009 (HR 0.54, 95% CI 0.32–0.90, p=0.02). Compared to kidney transplant recipients, PTLD occurred more frequently among recipients of non-kidney organs and was significantly higher among lung (HR 3.1, 95% CI 1.4–6.9, p=0.004) and liver (HR 2.9, 95% CI 1.5–5.8, p=0.002) recipients. Compared to concordantly negative serology, discordant EBV serology was associated with a significantly lower hazard of PTLD in univariable analysis (HR 0.29, 95% CI 0.11–0.82, p=0.02) and in multivariable analysis adjusting for organ type and year of transplant (aHR 0.27, 95% CI 0.10–0.76, p=0.01). When SOT recipients with discordant serology were compared to those with concordantly positive serology, there was a trend toward a higher hazard of PTLD in univariable analysis (HR=2.3, 95% CI 0.9–5.7, p=0.08) and a significantly higher hazard of PTLD in multivariable analysis adjusted for organ and year of transplant (aHR 2.6, 95% CI 1.04–6.6, p=0.04). Thus, the risk of PTLD among SOT recipients with discordant EBV serology was statistically different from both the concordantly negative and the concordantly positive groups.
Table 3:
Cox proportional hazards for all post-transplant lymphoproliferative disorder
| Univariable | Multivariable | |||
|---|---|---|---|---|
| HR (95% CI) | p-value | aHR (95% CI) | p-value | |
| Male | 1.33 (0.77–2.17) | 0.34 | - | - |
| Age at transplant, years | 0.99 (0.97–1.01) | 0.17 | - | - |
| Transplant in 2010–20181 | 0.54 (0.32–0.90) | 0.02* | 0.60 (0.36–1.01) | 0.06 |
| Anti-thymocyte globulin | 0.64 (0.37–1.10) | 0.10 | - | - |
| Organ (vs. Kidney) | ||||
| Lung | 3.14 (1.43–6.93) | 0.004* | 3.41 (1.54–7.53) | 0.002* |
| Heart | 1.28 (0.45–3.69) | 0.65 | 1.35 (0.47–3.90) | 0.58 |
| Liver | 2.93 (1.49–5.77) | 0.002* | 3.32 (1.66–6.64) | 0.001* |
| Any pancreas | 2.51 (0.70–9.01) | 0.16 | 2.33 (0.65–8.41) | 0.20 |
| Recipient VCA/EBNA-1 IgG serostatus | ||||
| Discordant vs. −/− | 0.29 (0.11–0.82) | 0.02* | 0.27 (0.10–0.76) | 0.01* |
| Discordant vs. +/+ | 2.28 (0.91–5.73) | 0.08 | 2.63 (1.04–6.61) | 0.04* |
Abbreviations: Adjusted hazard ratio (aHR), Confidence interval (CI), Epstein-Barr nuclear antigen (EBNA-1), hazard ratio (HR), viral capsid antigen (VCA)
Reference is transplant between 2000–2009.
3.5. Risk for the subset of EBV+ PTLD
In Cox proportional hazard analysis examining the subset of EBV+ PTLDs as the outcome, each one year increase in age at the time of transplant was associated with a decreased risk of EBV+ PTLD (HR 0.97, 95% CI 0.95–0.99, p=0.01). Sex, year of transplant and peri-transplant ATG were not associated with EBV+ PTLD, while recipients of non-kidney organs were more likely to develop EBV+ PTLD (Table 4). Compared to concordantly negative serology, discordant serology was associated with a significantly lower hazard of EBV+ PTLD in univariable analysis (HR 0.21, 95% CI 0.06–0.74, p=0.02) and in multivariable analysis adjusting for age and organ type (aHR 0.23, 95% CI 0.06–0.82, p=0.02). When compared to SOT recipients with concordantly positive serology, there was a trend toward a higher hazard of EBV+ PTLD among those with discordant serology in univariable analysis (HR 3.3, 95% CI 0.98–11.1, p=0.053) and a significantly higher hazard of PTLD in multivariable analysis adjusted for age and organ type (aHR 3.53, 95% CI 1.04–12.0, p=0.04). Unadjusted Kaplan-Meier curves showing the cumulative probability of any PTLD and EBV+ PTLD, stratified by recipient pre-transplant EBV serostatus, are shown in Figure 1.
Table 4:
Cox proportional hazards for subset of EBV+ PTLD
| Univariable | Multivariable | |||
|---|---|---|---|---|
| HR (95% CI) | p-value | aHR (95% CI) | p-value | |
| Male | 1.4 (0.7–2.8) | 0.41 | - | - |
| Age continuous | 0.97 (0.95–0.99) | 0.01* | 0.98 (0.96–1.0) | 0.09 |
| Transplant in 2010–20181 | 0.63 (0.34–1.33) | 0.23 | ||
| Anti-thymocyte globulin | 0.68 (0.33–1.4) | 0.31 | - | - |
| Organ | ||||
| Kidney | Reference | - | Reference | - |
| Lung | 3.5 (1.3–9.9) | 0.02* | 4.1 (1.5–11.6) | 008* |
| Heart | 1.9 (0.5–6.6) | 0.34 | 2.0 (0.7–8.3) | 0.27 |
| Liver | 2.0 (0.78–5.4) | 0.15 | 3.2 (1.2–8.7) | 0.04* |
| Any pancreas | 4.9 (1.2–19.7) | 0.02 | 4.5 (1.2–18.2) | 0.02* |
| Recipient VCA/EBNA-1 IgG serostatus | ||||
| Discordant vs. −/− | 0.21 (0.06–0.74) | 0.02* | 0.23 (0.06–0.82) | 0.02* |
| Discordant vs. +/+ | 3.30 (0.98–11.1) | 0.053 | 3.53 (1.04–12.0) | 0.04* |
Abbreviations: Adjusted hazard ratio (aHR), Confidence interval (CI), Epstein-Barr nuclear antigen (EBNA-1), hazard ratio (HR), viral capsid antigen (VCA
Reference is transplant between 2000–2009.
Figure 1:
Unadjusted Kaplan-Meier curves depicting cumulative probability of any PTLD and EBV+ PTLD by recipient viral capsid antigen and Epstein-Barr virus nuclear antigen IgG serostatus1
1 Reported hazard ratios (HRs) are unadjusted
Abbreviations: Epstein-Barr virus (EBV), post-transplant lymphoproliferative disorder (PTLD), hazard ratio (HR)
Three SOT recipients with discordant pre-transplant serology developed EBV+ PTLD, all of whom were VCA IgG+/EBNA-1 IgG− pre-transplant. One woman who underwent a kidney transplant at age 21 developed EBV+ diffuse large B-cell lymphoma (DLCBL) of the parotid gland ten years post-transplant. EBV DNA was not detected in the plasma at the time of diagnosis nor on any post-transplant surveillance PCRs, the most recent of which was nine years prior to PTLD diagnosis. Another 57-year-old woman developed EBV+ DLBCL arising from a paratracheal lymph node nine months after bilateral lung transplantation, with associated plasma EBV DNAemia at 330 copies/mL at the time of diagnosis. There were no prior plasma EBV PCRs on record for this patient. The third patient was a man who developed polymorphic EBV+ PTLD of the central nervous system two years after undergoing kidney/pancreas transplant at age 31. He had plasma EBV DNAemia at the time of PTLD diagnosis (65 copies/mL); no EBV DNA was detected in plasma on his most recent PCR testing one year prior to PTLD diagnosis. The third patient, but not the first two patients, had detectable VCA IgM on pre-transplant serology. There was no evidence of acute EBV infection at the time of pre-transplant serology testing for any of these three VCA/EBNA-1 IgG discordant patients who developed EBV+ PTLD.
Discussion
In this single center cohort study of adult SOT recipients, the risk of PTLD with discordant pre-transplant VCA and EBNA-1 IgG was intermediate between, and significantly different from, those with concordantly positive serology (lowest risk) or concordantly negative serology (highest risk), underscoring the potential for enhanced PTLD risk stratification provided by combined assessment of both VCA and EBNA-1 serology.
Concordantly positive VCA IgG and EBNA-1 IgG definitively indicate prior EBV infection, and concordantly negative serology reliably identify EBV-naïve individuals. The interpretation of discordant VCA IgG and EBNA-1 IgG includes several possibilities. The VCA IgG+/EBNA-1 IgG− pattern is present in a sizeable proportion of the population-- 5–10% of healthy adults and 4% of SOT candidates in this study16, 17. This pattern most likely indicates past EBV infection with failure of EBNA-1 seroconversion or loss of EBNA-1 over time, as immunoblotting can detect antibodies to non-EBNA-1 late antigens in VCA IgG+, EBNA-1 IgG− individuals15. Less commonly, recent EBV infection in the “window period” prior to EBNA-1 seroconversion may result in the VCA IgG+/EBNA-1 IgG− pattern, and a positive VCA IgM in a VCA IgG+/EBNA-1 IgG− individual would support such scenario. However, the poor specificity of IgM limits its positive predictive value in adult SOT candidates, most of whom are undergoing routine pre-transplant assessment and are therefore unlikely to have acute EBV infection16. The prevalence of IgM positivity was low in this study and did not differ between discordant and concordant individuals, suggesting that few, if any, patients with discordant serologies were recently infected but had not yet developed an EBNA-1 antibody response. A third explanation for a VCA IgG+/EBNA-1 IgG− result could be a false positive VCA IgG in an EBV naïve individual, but this has not been well characterized15. The VCA IgG-/EBNA-1 IgG+ profile is much rarer (1–2% of the general population and 0.5% of SOT recipients in this study), and it is not known whether this pattern is a marker of failure of VCA IgG seroconversion in an EBV-infected person or a non-specific EBNA-1 antibody in the absence of EBV infection16. In this study, the majority of SOT recipients with discordant serology were VCA IgG+/EBNA-1- and likely had acquired EBV infection prior to transplant but either lost or never developed antibodies to EBNA-1.
As expected, the risk of PTLD in patients with a discordant pre-transplant serology profile was significantly lower than for those with concordantly negative pre-transplant EBV serology. However, compared to SOT recipients with concordantly positive pre-transplant serology, recipients with a discordant EBV serology profile were significantly more likely to develop PTLD, including EBV+ PTLD. These findings raise the possibility that lack of EBNA-1 seroconversion or EBNA-1 IgG loss over time (i.e., absence of pre-transplant EBNA-1 IgG) represents impaired EBV-specific immunity and an associated higher risk of PTLD. EBNA-1 is the only viral protein expressed in all phases of EBV latency and in all forms of EBV-associated malignancies, and is the antigen target for novel, EBV-specific T cell therapies that are under investigation of treatment and prophylaxis of PTLD16, 25, 26 27. Functional T cell responses toward EBV-infected B cells in latency, which have a well-recognized role in PTLD surveillance, have also been shown to have potential importance in EBNA-1 antigen processing and stimulation of humoral responses28, 29. Among VCA IgG seropositive adults, EBNA-1 IgG is more likely to be absent after transplant (while receiving immunosuppression) and among those with advanced human immunodeficiency virus (HIV), supporting a relationship between EBNA-1 seronegative status and impaired EBV-specific T cell immunity16, 28. Thus, discordant VCA and EBNA-1 IgG (and specifically, VCA+/EBNA-1− serology status) may identify a subgroup of “EBV seropositive” individuals with a distinct PTLD risk profile. Future studies to confirm these findings in separate adult SOT cohorts should be a priority. If corroborated, these findings could be used to better stratify PTLD risk and to target monitoring or preventive strategies and to identify highest risk subgroups for future preventive interventional studies. Additionally, current reporting of pre-transplant EBV serology to OPTN/SRTR would need to be modified to include separate data fields for both VCA and EBNA-1 serology results.
The relatively large cohort size and number of PTLD cases, long duration of follow-up, and classification of PTLD histopathology using WHO classification are strengths of the study. Study limitations include the retrospective single center design and the small number of PTLD events among SOT recipients with discordant serology. The particularly low number of EBV+ events is an additional challenge, as EBV and associated virus-specific immune responses are likely far less relevant in the pathogenesis of EBV− compared to EBV+ PTLD3, 23. However, large epidemiological studies that described the increased risk of PTLD in EBV seronegative patients considered all PTLDs as the outcome, regardless of tumor EBER status5, 7, 8, 30. While analysis of a French kidney transplant registry found no relationship between R− status and EBV− PTLD, a large single center study did find EBV seronegative status to be associated with an increased risk of EBV− PTLD.13 EBER status was unavailable or inconclusive in a subset of patients in the present study and analysis of all PTLD, rather than EBV+ PTLD alone, captures potential cases of EBV+ PTLD that could not be definitively classified. Thus, the incidence all PTLDs, not just EBV+ PTLD, remains an important endpoint in this and other confirmatory investigations examining the implications of pre-transplant recipient EBV serology.
A small proportion of SOT recipients did not have available VCA and EBNA-1 serology and were therefore excluded from the analyses. Lung and liver transplantation, which were independent risk factors for PTLD, were more common among excluded SOT recipients than those included in analyses. Thus, the present findings may slightly underestimate the cumulative incidence of PTLD among all SOT recipients. However, adjustment for organ type in multivariable analysis mitigates the potential impact of selection bias13.
Donor EBV IgG seropositivity, indicative of latent EBV and potential transmission via the allograft, is a strong risk factor for EBV in seronegative adult SOT recipients5, 8, 13. Since donor EBV serostatus was not available for all donors, we estimated the prevalence of donor EBV IgG seropositivity in donors using contemporaneous data from our local organ procurement organization. Greater than 90% of donors had evidence of prior EBV infection (as indicated by VCA IgG positivity) consistent with population-wide seroprevalence studies12, 16. While more nuanced assessment of PTLD risk based on donor serostatus in addition to recipient VCA IgG and EBNA-1 IgG could be informative, inclusion of rare EBV-naïve donors in a study of this magnitude was unlikely to significantly impact the measured PTLD incidence.
In summary, combined assessment of VCA and EBNA-1 antibodies may improve PTLD risk stratification among adult SOT recipients. Those with a discordant pre-transplant VCA and EBNA-1 IgG serology profile (i.e., VCA+/EBNA-1-) appear to have a lower risk of PTLD than those with concordantly negative results, and a higher PTLD risk than those with concordantly positive results. Larger studies in adult SOT recipients with a greater number of EBV+ PTLD events are needed to confirm these findings. Refined risk estimates have the potential to improve PTLD risk stratification and identify individuals most likely to benefit from targeted post-transplant PTLD risk mitigation strategies.
Supplementary Material
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Acknowledgements
We gratefully acknowledge Jennifer Molta and Mark Destree, MT, MS, MBA and their colleagues at Bloodworks Northwest for providing results of EBV serology results from donors in our local organ procurement organization.
Funding:
This work was supported by the National Institute of Allergy and Infectious Diseases (T32AI118690 to M.R.H.) and the National Heart, Lung, and Blood Institute (HL143050 to C.E.F.) at the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Disclosures:
M.R.H reports receiving speaking honoraria from Cigna LifeSource. A.P.L received consulting fees from Merck, GSK, Allovir and Novartis and served on a data safety and monitoring board for Novartis.
Abbreviations:
- CTL
cytotoxic T lymphocyte
- DLCBL
diffuse large B-cell lymphoma
- EBER
Epstein-Barr-virus-encoded small RNA
- EBV
Epstein-Barr virus
- EBNA-1
Epstein-Barr virus nuclear antigen-1
- OPTN
Organ Procurement and Transplantation Network
- PTLD
post-transplant lymphoproliferative disorder
- SRTR
Scientific Registry of Transplant Recipients
- SOT
solid organ transplant
- VCA
viral capsid antigen
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