Epstein-Barr Virus DNAemia and post-transplant lymphoproliferative disorder in pediatric solid organ transplant recipients.

BACKGROUND: Pediatric solid organ transplant (SOT) recipients commonly have Epstein-Barr virus (EBV) DNAemia and are at risk of developing post-transplant lymphoproliferative disorder (PTLD). EBV DNAemia has not been analyzed on a continuous scale in this population.
METHODS: All children ≤ 18 years of age who underwent SOT at a single center between January 1, 2007 and July 31, 2018 were included in this retrospective study. Transplant episodes in which PTLD occurred were compared to transplant episodes without PTLD. Multivariable logistic regression was used to identify factors associated with the development of EBV DNAemia and maximum height of EBV DNAemia. A Cox proportional hazards model was used to calculate hazard ratios for time to PTLD.
RESULTS: Of 275 total transplant recipients and 294 transplant episodes, there were 14 episodes of PTLD. Intestinal and multivisceral transplant were strongly associated with PTLD (p = 0.002). Risk factors for the development of EBV DNAemia include donor and recipient positive EBV serologies (p = 0.001) and older age (p = 0.001). Maximum level of EBV DNAemia was significantly associated with development of PTLD (p<0.0001). Every one log (log10) increase in the maximum level of EBV DNAemia was associated with a more than doubling of the hazard on developing PTLD (HR: 2.18, 95% CI 1.19-3.99).
CONCLUSIONS: Transplant type was strongly associated with development of PTLD in pediatric SOT recipients. EBV serologies and age were associated with the development of EBV DNAemia and height of DNAemia. High levels of EBV DNAemia were strongly associated with an increased hazard for PTLD.

Introduction

Post-transplant lymphoproliferative disorder (PTLD) is a significant cause of morbidity and mortality in pediatric solid organ transplantation (SOT) recipients. Epstein-Barr Virus (EBV) DNAemia plays a key role in the pathophysiology of EBV-related PTLD but characteristics of pediatric SOT recipients who develop EBV DNAemia and patterns of EBV DNAemia have not been well defined [1]. Many centers monitor EBV DNAemia with established protocols for interventional measures such as decreasing immunosuppression [2]. These protocols may differ between centers and between different types of organ transplant recipients [3]. More data is needed to guide clinical decisions in specific scenarios.

There have been many studies on risk factors for PTLD in SOT recipients [1-4]. Overall, risk factors for development of PTLD within the first 12 months of transplant (early PTLD) include: type of organ transplant, T-cell depleting therapies such as anti-thymocyte globulin (ATG), and young age. Early PTLD is also strongly influenced by donor and recipient serologies as donor-derived EBV infection is a strong risk factor for PTLD. Risk factors for development of PTLD after 12 months post-transplant (late PTLD) include length and duration of immunosuppression, and type of organ transplant. Intestinal transplant recipients have been found to have the highest risk for PTLD, followed by lung, then heart, then liver, and lastly, kidney transplant recipients.

There is a higher incidence of PTLD in pediatric SOT recipients when compared to adult SOT recipients [1]. One reason is that children are at higher risk for primary infection, and primary EBV infection has a higher risk of causing PTLD [4]. One pediatric SOT study found that EBV viral load was significantly higher in cases of PTLD, when matched to controls [5]. Another study reviewing only pediatric patients found the following risk factors to be significant in a univariate model: age at transplant, use of basiliximab, steroid intensification, ATG, median peak EBV value level, and chronically elevated EBV DNAemia [6]. However, in the multivariable model, only age, use of steroids, and peak EBV value were significant.

Many previous studies in pediatric solid organ transplants have categorized EBV DNAemia into three categories: 1) chronic high load (CHL) carriers, 2) chronic low viral load carriers, and 3) those with sustained undetectable EBV levels, with chronic defined as a time period > 6 months [7-10]. In pediatric heart transplant recipients, CHL carriers are predisposed to PTLD, and pediatric intestinal transplant recipients are at intermediate risk [7, 8]. In the pediatric liver and kidney populations, CHL carriers are not at higher risk of PTLD [9, 10]. Despite low sample sizes within studies, this difference suggests that transplant type and immunosuppression play a large role in the development of PTLD.

There have been many studies on trying to categorize cutoffs for EBV DNAemia and risk for PTLD, as well as establishing a predictive model for PTLD [6, 11, 12]. While many of these studies have tried to categorize a certain EBV quantitative cutoff level for when patients are at risk for PTLD, there are confounding risk factors such as patient age, type of transplant and type of immunosuppression and not all models have been well validated. In addition, there is controversy between the use of whole blood vs plasma samples, and there is interlaboratory variability for quantification of EBV levels from the same samples [13].

The epidemiology of PTLD has been changing in recent years [14, 15]. In addition, EBV monitoring has provided a wealth of data that has not been studied in a detailed manner. While EBV DNAemia has been categorized in previous studies, it has not yet been studied on a continuous scale and available pediatric literature has not linked maximum height of EBV DNAemia to PTLD on a granular level. Therefore, we undertook a comprehensive retrospective descriptive study at a large pediatric SOT center to specifically evaluate risk factors associated with EBV DNAemia. We also wanted to investigate the predictive value of continuous EBV monitoring for PTLD in order to demonstrate its ongoing utility.

Material and methods

We performed a retrospective study of patients ≤ 18 years of age who received a solid organ transplant at Duke University Medical Center between January 1, 2007 and July 31, 2018. Events were recorded by transplant episode. The population included heart, kidney, liver, lung, intestinal, and multivisceral transplant recipients. Characterization of the pediatric SOT population was performed using the United Network for Organ Sharing (UNOS) database, and data was also extracted from the center’s electronic health records using the program Duke Enterprise Data Unified Content Explorer (DEDUCE) [16]. The diagnosis of PTLD and lymphoma were identified by diagnosis code. The diagnoses were next verified through chart review of pathology results and progress notes in the electronic health record. The study was approved and granted a waiver of informed consent by the Duke University institutional review board (IRB). Patient health information and identifiers were available within a secure workspace, but only de-identified, anonymized data can be exported out of this workspace.

At our center, each pediatric solid organ transplant program has developed their own methods of screening for EBV DNAemia. However, in general, quantitative whole blood EBV polymerase chain reaction (PCR) studies are done monthly for the first year post-transplant, then spaced out to every 3 months for the second and third year post-transplant, then either every 6 months or yearly afterwards if there are no issues. If EBV DNAemia was detected, quantitative PCRs were obtained more frequently. All EBV PCRs included in this study were done at the central laboratory of our center, which used the same machine over the study period, the ABI PRISM 7500/7500DX Sequence Detection System (Applied Biosystems) and Qiagen EBV PCR (ASR) reagents.

EBV DNAemia values were extracted from the electronic health record, including accompanying chart review for missing values. For most of the study period, the quantitative unit of EBV DNAemia was reported as copies/μL. Starting in mid-2017, this was changed to international units/mL (IU/mL), and a conversion factor of 1 copy/μL = 113.6 IU/mL was used to normalize these values in concordance with the previous quantification. The laboratory checks this conversion every 6 months and the conversion factor has not changed over the duration of the study.

Patients who developed EBV DNAemia after transplant were compared to patients who did not develop DNAemia. EBV DNAemia was defined as any detection of EBV virus by quantitative polymerase reaction (PCR). EBV donor and recipient serologies were based on what was entered into the patient’s chart at time of transplant. Demographic and clinical characteristics were compared between the two groups, using Chi square and Fisher’s exact tests for categorical factors and Mann-Whitney U tests for continuous factors. A logistic regression was done to calculate odds of EBV DNAemia using the variables of age, race, type of transplant, type of induction immunosuppression, and donor and recipient EBV serologies. These variables were either significant in the unadjusted model or risk factors that were denoted to be clinically significant a priori based on current knowledge.

The diagnosis of PTLD was confirmed by chart review. All cases of PTLD had accompanying histologic diagnoses which included EBER staining and description of atypical cells seen on pathology. Sources of biopsy tissue included lymph nodes, tonsils and adenoids, and the transplanted organ.

For the analysis of the height of EBV DNAemia, the maximum single EBV value was identified. That value was then log10 transformed and an analysis of covariance (ANCOVA) was used to evaluate associations between the maximum EBV value and demographic and clinical factors. After categorization and taking the average of the log10 transformed values, the data was back transformed, establishing the geometric mean. Logarithmic comparison of quantitative EBV values has been well described in the literature [17, 18].

A Cox proportional hazards model was used to link clinical risk factors to time to PTLD. The event of PTLD, death, and the event of a subsequent transplant were used as censoring criteria. The last observed value was carried forward. A robust variance (sandwich) estimator was used to control for the fact that some children were included more than once, due to multiple transplants. Adjusted models incorporated variables that were significant in the unadjusted model as well as risk factors that were denoted to be clinically significant a priori.

Results

We reviewed 275 pediatric patients who underwent 294 transplant episodes (Table 1). The median (interquartile range) age of patients was 4 (IQR 0, 14) years. The most common transplant type was liver (46%), followed by heart (25%), kidney (13%), multivisceral (7%), lung (7%), and intestinal (3%). For EBV serologies, donor positive/recipient negative (D+/R-) and donor positive/recipient positive (D+/R+) were the most common (33% and 31% respectively), followed by donor negative/recipient negative (D-/R-) (14%), and then donor negative/recipient positive (D-/R+) (11%), with the remaining unknown (11%). A total of 55 children (18.7%) received T-cell depleting therapy including anti-thymocyte globulin (ATG) or alemtuzumab (Campath) at induction.

Table 1

Characteristics of all children in the study, regardless of PTLD or EBV status.

		n	%
Total number of transplants		294
Total number of children		275
Race/Ethnicity
	American Indian or Alaska Native	3	1.1%
	Asian	6	2.2%
	Black	84	30.6%
	Hispanic	35	12.7%
	Multiracial	6	2.2%
	White	141	51.3%
Gender
	Male	148	53.8%
	Female	127	46.2%
Type of Organ Transplant
	Heart	73	24.8%
	Intestine	10	3.4%
	Kidney	38	12.9%
	Liver	134	45.6%
	Lung	19	6.5%
	Multivisceral	20	6.8%
EBV Serology at Transplant
	D+/R+	98	33.3%
	D+/R-	92	31.3%
	D-/R+	32	10.9%
	D-/R-	40	13.6%
	Unknown	32	10.9%
Transplant Number
	First	274	93.2%
	Second	16	5.4%
	Third	4	1.4%
EBV Status Post Transplant (Positive EBV PCR)
	EBV Positive	160	54.4%
	EBV Negative	111	37.8%
	No testing	23	7.8%
Induction Immunosuppression
	ATG or T-cell depleting therapy	55	18.7%
	Not ATG or T-cell depleting therapy	231	78.6%
	Missing	8	2.7%
		median	(p25, p75)
Year of Transplant		2013	(2011, 2016)
Age at Transplant (years)		4	(0, 14)

All children who developed PTLD were EBV positive by quantitative PCR, and thus, only EBV positive children were included in following analyses (Table 2). Fourteen children developed PTLD after transplant, for an overall incidence rate of 4.8%. Transplant type was significantly associated with PTLD (p = 0.001). Intestinal and multivisceral transplant recipients accounted for 21% and 14% of PTLD cases respectively, while only accounting for 2% and 3% of the total population that was EBV positive. There was also an association between PTLD and race (p = 0.04) as there were a total of six Asian children in the study who were EBV positive, and two developed PTLD. Asian children comprised 14% of the PTLD cases, but only 2.2% of the study population. Induction immunosuppression and age were not associated with PTLD in our study (p = 0.18 and p = 0.17, respectively). For the nine patients who developed PTLD within the first year, EBV values were plotted over time (Fig 1). For most patients, the general pattern was a steady increase in EBV values, followed by diagnosis of PTLD, and then a rapid decline.

Table 2

The characteristics of children who had PTLD at any time post-transplant are compared to children who had no PTLD.

This is limited to the children who had any EBV DNAemia after transplant. The incidence of PTLD is shown for each demographic group.

		PTLD		No PTLD
		n	%	n	%	p-value
Total		14		146
Type of Organ Transplant
	Heart	1	7.1%	36	24.7%	0.001*+
	Intestine	3	21.4%	3	2.1%
	Kidney	3	21.4%	22	15.1%
	Liver	4	28.6%	77	52.7%
	Lung	1	7.1%	3	2.1%
	Multivisceral	2	14.3%	5	3.4%
EBV Serology at Transplant
	D+/R+	4	28.6%	53	36.3%	0.47
	D+/R-	4	28.6%	56	38.4%
	D-/R+	1	7.1%	14	9.6%
	D-/R-	2	14.3%	10	6.8%
	Unknown	3	21.4%	13	8.9%
Race/Ethnicity
	American Indian/Alaskan	0	0%	5	3.4%	0.04*+
	Asian	2	14.3%	2	1.4%
	Black	2	14.3%	50	34.2%
	Hispanic	3	21.4%	19	13.0%
	Multiracial	0	0%	4	2.7%
	White	7	50.0%	66	45.2%
Gender
	Female	3	21.4%	67	45.9%	0.078
	Male	11	78.6%	79	54.1%
Transplant Number
	1	14	100%	133	91.1%	0.51+
	2	0	0%	10	6.9%
	3	0	0%	3	2.1%
Induction Immunosuppression
	ATG or T-cell depleting therapies	4	28.6%	26	15.4%	0.18
	Not ATG or T-cell depleting therapies	9	64.3%	136	80.5%
	Unknown/Missing	1	7.1%	7	4.1%
Age (years) at transplant	Median (IQR)	2 (0, 9)		4 (0, 14)		0.17

*p<0.05

+ By Fisher’s-Exact Test

Fig 1

Risk factors for EBV DNAemia were examined (Table 3). Significant risk factors for EBV DNAemia included: EBV serology at the time of transplant, age, and type of transplant in the unadjusted model, and only EBV serology and age in the adjusted model (Table 4). Heart transplant recipients were at lower odds for EBV DNAemia, with OR 0.44 (95% CI 0.22–0.89). Lung transplant recipients were similarly at lower odds, but sample size was limited. Donor positive/recipient negative (D+/R-) and donor positive/recipient positive (D+/R+) transplant recipients were at increased odds of developing EBV DNAemia compared to donor negative/recipient negative (D-/R-) transplant recipients, with OR 3.90 (95% CI 1.55–9.80), and OR 4.83 (95% CI 2.02–11.55), respectively. Older children were also associated with an increased risk, with every one year increase in age associated with an OR of 1.10 (1.04–1.16).

Table 3

The characteristics of children who had any EBV DNAemia after transplant are compared to those who did not have any EBV DNAemia.

		EBV		No EBV		p-value
		N	%	n	%
Total		160	59%	111	41%
Type of Organ Transplant
	Heart	37	23.1%	31	27.9%	0.14+
	Intestine	6	3.8%	3	2.7%
	Kidney	25	15.6%	12	10.8%
	Liver	81	50.6%	47	42.3%
	Lung	4	2.5%	7	6.3%
	Multivisceral	7	4.4%	11	9.9%
EBV Serology at Transplant
	D+/R+	57	35.6%	29	26.1%	0.001*
	D+/R-	60	37.5%	28	25.2%
	D-/R+	15	9.4%	15	13.5%
	D-/R-	12	7.5%	26	23.4%
	Unknown	16	10.0%	13	11.7%
Race
	American Indian/Alaskan	5	3.1%	0	0.0%	0.11
	Asian	4	2.5%	0	0.0%
	Black	52	32.5%	33	29.7%
	Hispanic	22	13.8%	14	12.6%
	Multiracial	4	2.5%	1	0.9%
	White	73	45.6%	63	56.8%
Sex
	Female	70	43.8%	50	45.0%	0.83
	Male	90	56.3%	61	55.0%
Transplant Number
	1	147	91.9%	106	95.5%	0.28
	2	10	6.3%	5	4.5%
	3	3	1.9%	0	0.0%
Induction Immunosuppression
	ATG or High Dose Steroids	27	17.0%	25	22.5%	0.26
	Not ATG or High Dose Steroids	131	83.0%	86	77.5%
		Median	(p25, p75)	median	(p25, p75)
Year of Transplant		2014	(2011, 2016)	2012	(2010, 2015)	0.58
Age at Transplant (years)		7	(1, 14)	1	(0, 10)	0.001*

*p<0.05

+By Fisher’s-Exact Test

Table 4

The association between demographic characteristics and EBV DNAemia.

Only significant risk factors are included in this table.

		EBV (n)	No EBV (n)	p-value	OR of EBV DNAemia (95% CI)	p-value
Total		160	111
Type of Organ Transplant
	Heart	37	31	0.14	0.44 (0.22, 0.89)	0.02
	Intestine	6	3		2.07 (0.44, 9.76)	0.36
	Kidney	25	12		0.57 (0.22, 1.43)	0.23
	Liver	81	47		Reference
	Lung	4	7		0.08 (0.02, 0.36)	0.001
	Multivisceral	7	11		0.47 (0.16, 1.36)	0.16
EBV Serology at Transplant
	D+/R+	57	29	0.001	3.90 (1.55, 9.80)	0.004
	D+/R-	60	28		4.83 (2.02, 11.55)	0.0005
	D-/R+	15	15		2.48 (0.85, 7.19)	0.096
	D-/R-	12	26		Reference
	Unknown	16	13		2.50 (0.87, 7.17)	0.089
		Median (IQR)	Median (IQR)
Age at Transplant		5.6 (0.8, 15.1)	4.1 (0.8, 13.8)		1.10 (1.04, 1.16) (Per Every One Year Increase)	0.0005

An analysis of the geometric mean of the highest value of EBV DNAemia (Table 5) showed that in the unadjusted model, liver transplant recipients had a higher geometric mean of the maximum EBV value 79.8 units/uL (95% CI 47–135.5) compared to heart transplant recipients at 14.6 units/uL (95% CI 6.6–32.1), and kidney transplant recipients 8.6 units/uL (95% CI 3.3–22.6), p = 0.007 and p<0.001 respectively. In the adjusted model, liver transplant recipients had a higher geometric mean of the maximum EBV value at 53 units/μL (95% CI 26.3, 106.9) compared to intestinal transplant recipients at 6.4 units/μL (95% CI 1.2, 31.0), p = 0.005. Lastly, children who developed PTLD had higher geometric mean values of EBV, 211.6 (56.7, 790.1) copies/μL, compared to children without PTLD, who had a geometric mean EBV value of 27.3 (18.0, 41.2) copies/μL (p<0.0001). Type of transplant was not significant in the adjusted model (p = 0.14).

Table 5

The geometric mean maximum EBV value (Mean of Max EBV) in copies/uL is shown for each demographic group in an unadjusted model, and an adjusted model.

		Unadjusted Model			Adjusted Model
		Mean of Max EBV	95% CI	p-value	Mean of Max EBV	95% CI	p-value
Type of Organ Transplant
	Heart	14.6	(6.6, 32.1)	0.007	39.8	(17.7, 89.5)	0.99
	Intestine	8.2	(1.2, 56.7)	0.224	6.4	(1.2, 31.0)	0.005
	Kidney	8.6	(3.3, 22.6)	<0.001	34.5	(13.3, 89.3)	0.17
	Liver	79.8	(47, 135.5)	Ref.	53	(26.3, 106.9)	Ref.
	Lung	7.4	(0.5, 114.7)	0.095	60	(5.0, 722.3)	0.5
	Multivisceral	50	(8.3, 300)	0.622	20.6	(4.4, 97.0)	0.58
EBV Serology at Transplant
	D+/R+	11.2	(5.9, 21.2)	0.88	14.9	(6.5, 34.5)	0.605
	D+/R-	100.3	(54.1, 185.9)	0.056	62.8	(29.0, 136.0)	0.07
	D-/R+	42.9	(12.1, 152.5)	0.52	45.6	(13.4, 155.0)	0.26
	D-/R-	23.3	(5.9, 91.4)	Ref.	23.2	(6.3, 84.9)	Ref.
	Unknown	21.7	(6.6, 71)	0.94	20	(6.6, 60.9)	0.82
Age at Transplant (For every 1 year increase)		0.8	(0.76, 0.84)	<0.001	0.81	(0.76, 0.86)	<0.001
Induction Immunosuppression
	ATG or High Dose Steroids	22.4	(8.5, 59.3)	Ref.	47	(17.5, 126.1)	Ref
	Not ATG or High Dose Steroids	34.1	(21.8, 53.3)	0.44	17.7	(9.32, 33.5)	0.1
PTLD
	PTLD +	211.6	(56.7, 790.1)	Ref.	177.6	(58.6, 538.0)	Ref.
	PTLD -	27.3	(18.0, 41.2)	<0.001	15.2	(7.4, 31.2)	<0.001

A final analysis was performed evaluating the association between risk factors and time to PTLD, and the adjusted analysis is included (Table 6). Every log10 increase in maximum EBV value was associated with a hazards ratio (HR) of 2.18 for development of PTLD (95% CI 1.19–3.99, p = 0.02). Transplant year was also significant in this analysis. For every one year increase in transplant year, there was a decrease in the hazard ratio, denoted at 0.71 (95% CI 0.53–0.95, p = 0.02). Kidney transplant recipients also had higher hazards ratios for PTLD compared to liver transplant recipients, HR 6.15 (95% CI 1.36–27.73, p = 0.02). There was also a trend toward association between type of induction immunosuppression, with ATG and high dose steroids, with a hazards ratio of 10.1 (95% CI 0.994–103.8, p = 0.0506), which was just out of the range of significance.

Table 6

Adjusted hazards ratios (HR) for significant covariates for the outcome time to PTLD.

Covariate		Reference	HR	95% CI	p-value
EBV DNAemia level		For every log10 increase in max level of EBV DNAemia	2.18	(1.19, 3.99)	0.02
EBV Serology
	D+/R+	D-/R-	0.37	(0.05, 2.92)	0.34
	D+/R-	D-/R-	0.21	(0.02, 3.00)	0.21
	D-/R+	D-/R-	N/A
Type of Organ Transplant
	Heart Transplant	Liver Transplant	N/A
	Intestine Transplant	Liver Transplant	1.34	(0.29, 6.25)	0.72
	Kidney Transplant	Liver Transplant	6.15	(1.36, 27.73)	0.02
	Lung Transplant	Liver Transplant	43.58	(0.97, 1957)	0.051
	Multivisceral Transplant	Liver Transplant	0.2	(0.01, 2.93)	0.24
Transplant Year		For every year increase	0.71	(0.53, 0.95)	0.02
Age at transplant (years)			0.98	(0.85, 1.13)	0.78
Induction Immunosuppresion	ATG or high dose steroids	No T-cell depleting agents	10.1	(0.994, 103.8)	0.0506

Additional data on testing broken down by type of organ transplant and by year is available (Tables 7 and 8). The trends in these tables show that multivisceral and intestinal transplant recipients were tested the most frequently. The number of transplant recipients who were EBV positive by PCR post-transplant did not seem to change over time. In addition, while the number of tests for liver transplant recipients during the first year decreased over the study time period, the number of tests done during the first year for non-liver transplant recipients (heart, kidney, lung, intestinal, and multivisceral transplants) combined did increase overall which suggests better adherence to EBV PCR monitoring over time.

Table 7

Total number of EBV tests 4614				Median number of tests per patient 12
Type of organ transplant		Total number of subjects	Missing	Deceased < 1 yr post-transplant	Median number of tests for all patients	Median number of tests for subjects EBV+ by PCR	Median number of tests for subjects EBV- by PCR
	Heart	73	5	5	11	12.5	7
	Intestine	10	1	0	43	50	27
	Kidney	38	1	0	10	10.5	10
	Liver	134	5	1	14	19	8
	Lung	19	8	1	1.5	3	1
	Multivisceral	20	0	0	18.5	34	15

Table 8

Median number of tests done per subject, stratified by year and only including tests done within first year post-transplant.

Year	Total including deceased	Total excluding deceased	EBV- by PCR	EBV+ by PCR	Liver transplant only	Non-liver transplants
2007	6	7.5	2	9	16	2
2008	6	6	0	6	10.5	4
2009	5	5	4	5	9	2
2010	7	8	6	8	10.5	2.5
2011	8	8	9	8	12.5	4.5
2012	7	7	6	7.5	11	4
2013	11	11	10	13	13	10
2014	8.5	9	7	11	11	9
2015	8	8	7.5	8.5	9.5	7
2016	7	7	6.5	8	6.5	10
2017	6	6.5	5	9	6	11
2018	8	8	7	9.5	6	12

Discussion

This is the first study to fully describe characteristics of post-transplant EBV DNAemia in the pediatric solid organ transplant population. In a raw analysis of 275 SOT pediatric recipients, type of transplant and race were significantly associated with higher risk for PTLD. Intestinal and multivisceral transplant recipients had the highest risk of developing PTLD, which is supported by the literature. We interpret the findings of race with caution, as the overall sample size is small, and only two out of four Asians with EBV DNAemia developed PTLD. We were unable to find any other risk factors that were significantly associated with PTLD in our population, including type of induction immunosuppression, EBV donor and recipient serologies, or age. This may have been due to distinctions within our population or to limited sample size.

When we examine risk factors for EBV DNAemia, and for magnitude of EBV DNAemia, some common themes emerge. For EBV DNAemia, EBV serologies of the donor and the recipient are an obvious risk factor. Age is also a practical risk factor as the risk of being exposed to EBV increases with age. Type of transplant is not associated with risk of EBV DNAemia in the adjusted model, but within the category, we see that EBV DNAemia tended to be more common in liver transplant recipients.

Age was the most resilient predictor for the maximum height of EBV DNAemia, as it changed the least from the unadjusted to the adjusted model. Intestinal transplants did not have a significantly different height of EBV DNAemia, yet they still had a higher risk association with PTLD, which suggests that other factors such as length and duration of immunosuppression should be considered. Ultimately, those who developed PTLD had significantly higher maximum levels of EBV DNAemia, which has been seen in previous studies.5 However, stratifying by type of organ transplant does matter; liver transplant recipients were more likely to have a higher maximum level of DNAemia compared to heart and intestinal transplant recipients but were not more likely to develop PTLD. This may be due to immunosuppression or organ-specific risk factors for PTLD. Interestingly, the association of EBV serostatus D+/R- is often associated with the highest risk for PTLD, and our findings in Tables 2 and 6 do not reinforce this. This may have been limited by sample size, or other unmeasurable factors in our population.

Our final analysis is significant in that higher levels of EBV DNAemia are associated with decreased time to PTLD, even when adjusted for other risk factors. It is also important to note that an earlier date of transplant was associated with a higher hazards ratio for PTLD. This may have been due to better EBV monitoring over time and the development of protocols which have standardized practice at our center. The significance of a higher hazards ratio for kidney transplants is not clear at this time. We did have much more liver transplant recipients and less kidney transplant recipients which is a limitation of this study and this may have affected the hazards ratio. Lung transplants did not have a high incidence of DNAemia as indicated by Table 3, but did have high hazard ratios for PTLD. This again may be due to a small sample size of lung transplant recipients within our population. In addition, many lung transplant recipients from our center are referred back to more local home institutions and EBV PCRs may be obtained externally. Those who stay at our center often have many complications such as rejection, and of course, the development of PTLD. Lastly, type of induction immunosuppression may be important given the trend towards decreased time to PTLD with the use of alemtuzumab, ATG, or high dose steroids. These findings may help with stratifying patients who may be at higher risk for PTLD.

It is not immediately quite clear why risk factors associated with PTLD (Table 2) were different than risk factors associated with increased hazards of PTLD (Table 6). For the analysis of hazard ratios, we did add an extra variable of the maximum level of EBV DNAemia and the Cox regression analysis was adjusted for different variables, and this may have changed the analysis. There may also be statistical differences when examining development of PTLD vs time to PTLD.

Risk factors associated with increased odds of EBV DNAemia were slightly different than risk factors associated with increased hazards of PTLD. This may reflect the fact that while EBV DNAemia is a risk factor for PTLD, how clinical providers manage the EBV DNAemia may influence the development of PTLD. Also, for example, liver transplant recipients were more likely to have EBV DNAemia, but they are less likely to develop PTLD so other factors such as type of organ transplant and immunosuppression may be more important in the development of PTLD.

All pediatric SOT studies for PTLD and EBV are limited by small sample sizes and this single center study is no exception. The overall incidence of PTLD was relatively low. This may have be due to the quick responsiveness of clinicians to high EBV levels. One major limitation is that we were unable to adjust for time-varying exposures to immunosuppressive medications. Higher periods of immunosuppression could have led to increased incidence of PTLD, EBV DNAemia, and higher maximum EBV levels. On the other hand, increased EBV levels may have led clinical providers to decrease immunosuppression, which could have prevented PTLD. However, there is no clear way to delineate all the changes to maintenance immunosuppression as changes occur often and it is very difficult to quantitate such a time-varying risk factor in our analysis.

We did have access to a wealth of data due to frequent EBV monitoring and were able to examine multiple risk factors for EBV DNAemia and the maximum height of EBV DNAemia. However, this study was not adequately powered to examine the numerous interactions between risk factors that may occur in predicting PTLD. Another issue may be the introduction of testing by indication bias, where transplant recipients who had positive and higher levels of EBV were tested more frequently than others. We did note that for the most part, while those who did test positive for EBV by PCR were more likely to receive a higher number of total tests, those who tested negative for EBV by PCR also received a fair number of tests, which denotes that teams were adhering to their protocols (S1 Data).

Other limitations include identifying PTLD and lymphoma cases by diagnosis code and while we did verify each diagnosis, we may have undercounted the number of cases as we may have missed some cases if they were not coded a certain way. Our center uses whole blood sampling for EBV which is much more sensitive but not as specific. While other centers may have moved towards plasma sampling, the more sensitive measure may have led to less cases of PTLD. We did not establish specific cutoffs for the risk of PTLD, but our findings have been shown in a way that is much more useful to individual centers as our final analysis looks at the risk of PTLD associated with each change in log of the quantitative level. We only included EBV quantitative PCRs done at the central laboratory at our center, and we may have missed values that were obtained externally. The decision was made to not include external values in this study as that may have introduced interlaboratory variability to this study. In addition, EBV results from these external laboratories would have been very hard to find in our electronic records, as our center utilized paper charting prior to 2014 and they may not have made their way into each scanned chart from that era.

Despite the study limitations, we did gain some valuable insights into the nature of EBV DNAemia and PTLD in our study population. Further prospective or multicenter studies are needed to link significant clinical factors and EBV DNAemia with PTLD and other clinical outcomes.

Conclusion

Type of transplant and age were associated with PTLD in pediatric SOT recipients. Age and EBV donor and recipient serology remain key risk factors in the development of EBV DNAemia and the maximum height of EBV DNAemia. Maximum level of EBV DNAemia, and year of transplant were associated with time to PTLD in our population, and there was a trend towards type of induction and time to PTLD. While this study has shed some light on risk factors for EBV DNAemia in pediatric SOT recipients, further studies are needed in order to fully characterize the relationship between EBV levels and PTLD.

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11 Jul 2022

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Reviewer #1: Overall, I appreciate the investigators’ efforts to quantify the relative risk for PTLD with increasing EBV DNA loads. I have several comments to improve the manuscript.

1. Introduction, line 52 of pdf: does “type of transplant” mean “type of organ transplant”, as stated in line 56? If yes, please make both sections consistent.

2. Methods, lines 93-94: Billing diagnosis codes are notorious for under-capture of PTLD cases. In the older ICD-9 coding that was in use for a large part of this study’s time period, a diagnosis code for PTLD not even initially present. Did the authors make an additional initial search of their center’s pathology or cancer databases to capture more PTLD cases, using the search term PTLD or its full form, or post-transplant lymphoma? What about from the Problem list of your electronic health record?

3. Methods, line 96: your EBV DNA assay is from whole blood, while most centers have moved to plasma assays. The results from the two sources do not correlate well with each other, with whole blood running higher levels, higher sensitivity but much lower specificity than plasma (see multiple reviews by Preiksaitis J et al). I suggest to add in your Discussion limitations paragraph about the lower generalizability of your results since they are based on whole blood assays only.

4. Methods, lines 100-102: was your EBV PCR DNA assay performed locally or at an outside lab through the 11 year study period? Were there no changes in the assay or the lab used over the 11 years? State in the manuscript what was the conversion factor used from copies/mL to IU/mL.

5. Methods, lines 105-106: why were these specific covariates only used in the logistic regression? Compare with a later paragraph, where you do mention how covariates were selected, but not here. Also, since EBV DNA replication can occur at varying points in time and your patients would have varying periods of follow up, how could you use logistic regression here, versus a Cox proportional hazards model?

6. Results, lines 128-129: your population is skewed heavily towards liver transplants, with very few kidney transplants, unlike the proportions at most pediatric transplant centers. This should also be added in your Discussion limitations paragraph as a factor that lowers generalizability of your results.

7. Table 1, row heading EBV status post-transplant: does this refer to whether recipients turned PCR DNA positive or not? If yes, please make the heading more specific. Similarly, in the text on line 136 “All children who turned PTLD were EBV positive” – is this referring to recipient EBV seropositivity at time of transplant, or recipients who turned EBV PCR DNA positive post-transplant?

8. Table 2: all prior studies have shown that donor recipient EBV seromismatch (D+/R-) associates to an elevated risk for PTLD, and often this risk factor has the highest magnitude of all risk factors (see PMID 29178667). The incongruence of this result is highlighted further in Tables 3 and 4, where D+/R- is the biggest risk factor for EBV DNAemia. You need to state in your Discussion that your result is in discord with prior literature.

9. Results line 155: I think you inverted in the text sentence the ORs, since D+/R- is mentioned first and Odds Ratio 3.90 is mentioned first. (Should be D+/R- with OR 4.80 and D+/R+ with 3.90).

10. Why are the results for risk factors for PTLD (Table 2) different for risk factors for TIME to PTLD (Table 6)?

11. The authors have available to them granular single center data – you should also therefore be able to look at maintenance immunosuppression changes in response to EBV DNA positivity or acute rejection episodes – these would affect your results.

Reviewer #2: Major comments:

1. The authors have described the general algorithm for EBV screening in the cohort, but a more detailed description of EBV PCR tests actually performed during follow up would be helpful, i.e how many tests were performed in total and stratified by transplant type, what was the median number of samples performed during the first vs later years, and were there children who did not have any screening performed during follow up? This is due to the risk of introducing a testing by indication bias. Thus, the difference in the association between EBV DNAemia and risk of PTLD in the different transplant types could be due to different sampling regimens. Recipients with EBV DNAemia may be tested more intensively compared to patients who do not have EBV DNAemia and thus a potential positive test may be missed in those patient and you may then also miss a potential increase in viral load in these patients. Further, EBV serology may be an indicator for the screening plan and thus again risk underestimating positive and high EBV levels.

2. The potential difference in immunosuppression treatment regimens during follow-up and between departments could have an impact on PTLD incidence and thus introduce a bias. Thus, the potential reduction of IS dosage whenever a positive EBV is detected could prevent PTLD occurrence and thus underestimate this diagnosis in some groups. This could be discussed in the limitation section.

3. it is interesting that it seems that you find a lower risk of EBV DNAemia in lung and heart tx sompared to fx kidney. This is in contrast to must studies where lung and heart are more heavily immunosuppressed and thus have a higher risk of developing EBV DNAemia. This could be discussed in the discussion section.

Furter, it is interesting that although Lung tx have very low risk of EBV DNAemia, they have the highest risk of PTLD. How do you explain this?

4. your write that “It is also important to note that an earlier date of 212 transplant was associated with a higher hazards ratio for PTLD. This may have been due to better EBV 213 monitoring over time and the development of protocols which have standardized practice at our center.”

In this case you would also expect an increase in the patients who were tested positive for EBV DNA. Did you see this?

Minor comments:

Table 1: EBV status post transplant: Revise to EBV DNAemia detected posttransplant.

Line 136: all PTLD were EBV positive. Was EBV detected prior to or at time of diagnosis? Some information about the time relation between the detected EBV and PTLD diagnosis would be helpful.

Line 138: Confidence intervals to incidence of PTLD would be helpful. Median time to PTLD?

Table 6: there seems to be a typo in the confidence interval for lung tx where the upper limit is 1957.

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9 Aug 2022

We have also responded to the following points made by the reviewers.

Reviewer #1:

1. In line 52, the term “organ transplant” has been changed to “type of organ transplant” to correspond to similar language in line 56. We have also changed the headings in all of our tables from “Type of Transplant” to “Type of Organ Transplant” in order to stay consistent.

2. Methods, line 93-94: We did identify diagnosis codes for lymphoma in addition to PTLD and thus, this has been added to the manuscript. As for the comment that there was not an ICD diagnosis code for PTLD, there was one from 2008 forward, and thus, we went 1 year previous to this and started our study in 2007. We did not look at a cancer registry as we did not have access to one and we did not look at problem lists as they are notoriously hard to pull from the charts and also come with their shortcomings. We do realize that there are limitations of identifying PTLD and lymphoma by diagnosis code, and have added this to the discussion section. In the end, we wanted to make sure that the cases of PTLD were indeed true cases of PTLD, proven by biopsy.

3. Methods, line 96: We did use whole blood quantitative EBV PCRs, which as one reviewer mentions is more sensitive but less specific. Indeed, there is much intervariability of quantitative PCRs, and we have included the reference mentioned. In the end, our data is presented not as a specific cutoff but as an association between serial levels using the same laboratory and this may be more universally applicable. This has all been added to the Discussion section as a limitation of this study.

4. Methods, line 100-102: We used only quantitative EBV PCRs from our electronic health system which only pulled in values done at our central laboratory. In checking with the laboratory, they used the same machine over the time period of the study. We also confirmed with them the exact conversion factor, 1 copy/µL = 113.6 IU/mL which has not changed over the study period. All this was added into the manuscript under Methods.

5. We have taken out language regarding logistic regression for PTLD in order to clarify the methods, especially as we do not show these results. Logistic regression was used for the odds of EBV DNAemia, and this is detailed further. In response to how a logistic regression was OK for EBV DNAemia despite the fact that patients develop EBV DNAemia over time, we did notice that a pattern that those transplants who were EBV donor positive/recipient positive (D+/R+) and donor positive/recipient negative (D+/R-) were more likely to develop EBV DNAemia. There were also increased odds of EBV DNAemia for donor negative/recipient positive (D-/R+) subjects although this was not significant. In all of these patients, we noticed that the EBV DNAemia occurred pretty quickly after transplant, and thus, the time to EBV DNAemia was not as variable. As shown in Table 4, not many variables were significantly associated with odds of EBV DNAemia, and the ones that were significant were not time dependent.

6. Results, line 128-129: We take into account the excellent point made on our patient population. We have added the observation that our sample population is heavy in liver transplant recipients, and has less kidney transplant recipients than the typical pediatric center in the Discussion section.

7. EBV positive clarification: We added PCR positive to clarify our definition of EBV positive in Table 1. As further clarification, we also added EBV positive by quantitative PCR to the description “All patients who developed PTLD were EBV positive” on line 136.

8. The reviewer states that in the literature, often D+/R- is one of the highest risk factors for PTLD, and this was not the case in our study. We have added it to the limitations section of this study. We are not sure why our data does not match this, it could be a sample size issue, something intrinsic to our population, or the fact that providers much more carefully followed patients if they were D+/R-.

9. Results line 155: The odds ratios for EBV D+/R- and D+/R+ were indeed switched and we have edited this to correctly correspond with the proper EBV serostatus category.

10. We have added more in the Discussion section as to why there are differences between risk factors for PTLD (Table 2), and risk factors for time to PTLD (Table 6). There is an additional variable in the analysis in Table 6, the log¬10 transformed maximum level of EBV. Also, in the analysis in Table 6, each variable is adjusted against the others. Lastly, with the analysis of time to PTLD, we do realize that we cannot study all the variables that vary over time such as immunosuppression and this may confound the results as well. I think it is interesting to see that Table 2 presents the systematic overview of associations and is a good frame of reference, and Table 6 presents variables that may be more important for individualistic decision-making.

11. While we do have granular EBV quantitative levels, there are too many changes in the patients’ immunosuppressive maintenance regimens over time and we could not study changes in all the patients. While we can pull out T cell depleting agents such as anti-thymocyte globulin (ATG) and alemtuzumab (Campath), it is very hard to categorize all the small changes to maintenance immunosuppression. In our center, changes to medications could reflect a change in levels or an actual adjustment in immunosuppression level. This would require a massive chart review as clinical notes would be the gold standard here, in order to generate a detailed summary of immunosuppression changes, which would be extremely time intensive. Patients do obtain troughs but these values are also variable and do not always reflect true goals. We would have to do a chart review looking at clinical notes and as patient immunosuppressive levels change over time, it would be hard to categorize changes. We do state this as a major limitation of this study.

Reviewer #2:

1. We understand the issue of testing by indication bias, and have added this to the limitations. This is a very important point. We have additional tables in the Supplementary Data section that does confirm this. Whether stratified by year or type of organ transplant, for the most part, those patients who are EBV positive by PCR have a higher median number of tests when compared to those who are EBV negative PCR. Per our protocol and guidelines, those who have higher levels of EBV DNAemia are monitored more closely and yes, there is a lower threshold to decrease immunosuppression in these patients. However, to the point of sudden EBV DNAemia (primary infection) resulting in PTLD, as you can see from Figure 1, the vast majority of patients who developed PTLD had high EBV levels for about 2 months or so before developing PTLD. There weren’t a lot of patients where the development of PTLD was caught by surprise. We do realize that a better summary of how EBV testing was done can help frame our reference, and we have thus included this extra data in the Supplemental Data section.

2. We have added the specific comment that teams could decrease immunosuppression as a response to high EBV levels and thus reduce risk for PTLD to our Discussion section. There is no easy way to delineate all the changes in maintenance immunosuppression at our center and as previously mentioned, we have stated this as a major limitation.

3. Lung transplants did not have much EBV DNAemia but had high hazards ratio for PTLD. This is likely due to testing nuances at our center. Lung transplants are followed for only a short time period at our center, and most of them receive EBV levels as external labs which we cannot capture easily. We do have a biased sample of this population because those who stay at our center often are those who are sicker, gone through rejection, and of course, they stay or become referred back into our care if they are diagnosed with PTLD.

4. We did not see more EBV over time. We do have some raw data on this. What we did see was more frequent monitoring at our center in the heart and kidney transplant recipients, which likely led to immunosuppression adjustments over time. Multivisceral/intestinal transplant recipient EBV monitoring was kept constant, and monitoring in liver transplant recipients actually decreased over time, but we did not see as much PTLD in liver transplant recipients. Please see the Supplemental Data section for this.

Minor Comments:

Table 1: In order to clarify the phrase “EBV positive post-transplant”, we added the words “PCR positive” was added

Line 136: To clarify the phrase “all cases of PTLD were EBV positive”, this was edited to refer to PCR positivity prior to PTLD diagnosis.

Line 138: For the comment on confidence intervals, it is very hard to put a confidence interval on time to PTLD, the curve is non-parametric. Also, with the hazard ratios that had smaller sample size, the confidence interval may be extremely wide and harder to interpret.

Table 6: The 1957 on the CI of the hazards ratio for lung transplants is not an error. Due to the very limited sample size of lung transplants (we only had one positive PTLD case amongst 19 cases, and if you looked at the EBV data for lung transplants, it was very sparse as most are referred back to a closer home institution after transplant for EBV monitoring there.

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22 Sep 2022

Epstein-Barr Virus DNAemia and Post-Transplant Lymphoproliferative Disorder in Pediatric Solid Organ Transplant Recipients

PONE-D-22-15480R1

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7 Oct 2022

PONE-D-22-15480R1

Epstein-Barr Virus DNAemia and Post-Transplant Lymphoproliferative Disorder in Pediatric Solid Organ Transplant Recipients

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