Shiraz Organ Transplant Center (Shiraz, Iran) is a leading transplant center in Iran, with considerable annual cases of liver transplantation for both adult and pediatric patients. A cross-sectional survey was conducted among the adult and pediatric patients (< 18 years) who underwent liver transplantation at Shiraz Transplant Center between August 2004 and March 2015. Clinical and laboratory data of patients were collected using a data gathering form containing information regarding age, sex, underlying liver disease, type of allograft (deceased donor, living related donor, split liver transplantation), time of liver transplantation and time of PTLD development, survival of patients from date of liver transplantation, survival after PTLD diagnosis, immunosuppressive regimen and dosage, rejection episodes, EBV status before and after transplantation, presenting sign and symptoms, PTLD histology, multi-organ involvement, modality of treatment, response to therapy, and serum level of calcineurin inhibitors (including tacrolimus and cyclosporine). All patients received intravenous methylprednisolone as induction of immunosuppression. Patients received tacrolimus, cyclosporine, mycophenolate mofetil and prednisolone as immunosuppressive therapy during their follow-up. Serum tacrolimus level was measured periodically during follow-up for each patient and the last measured serum tacrolimus levels before diagnosis of PTLD were recorded and analyzed for each patient. Patients were treated with rituximab or chemotherapy based on grade, type and invasiveness of PTLD. Change from tacrolimus to sirolimus was applied for all of the patients diagnosed with PTLD. Thirteen patients with positivity for cytomegalovirus (CMV)-DNA were treated with ganciclovir or valganciclovir.

Diagnosis of PTLD was confirmed by tissue biopsies reviewed by expert pathologists. World Health Organization (WHO) classification for tumors of lymphoid tissue was used for PTLD classification[16]. While diagnosis of PTLD was confirmed, patients underwent staging work-up, including CT scans (abdomen, chest and pelvis) and bone marrow aspiration and biopsy to detect possibility of multiple organ involvement. Frozen section or paraffin immunoperoxidase staining or flow cytometry were applied for immunophenotyping of B-cell- and T-cell-associated antigens, as previously described.

Whether PTLD is monoclonal or polyclonal was determined by flow cytometry on fresh cell suspensions checking immunoglobulin light chain restriction, by immunoperoxidase staining on frozen or paraffin-embedded tissues or by southern blot on frozen tissues checking immunoglobulin or T-cell receptor gene rearrangements[17].

Based on these classification, one patient had nodular sclerosis Hodgkin disease, one had plasmacytoma, and others had polymorphic and monomorphic B cell lymphoma.

The study protocol was approved by the institutional review board of Shiraz University of Medical Sciences. The study protocol was carried out in accordance with the Helsinki Declaration as revised in Seoul 2008. Written informed consent was obtained from patients.

Comparisons of continuous variables were performed with the Student’s t-test, and categorical variables were compared using the chi-square test. Non-parametric Mann-Whitney test was used when appropriate. Data were presented using mean ± standard deviation for numeric variables, and percent and counts for categorical variables. Kaplan-Meier estimates were used for analysis of time to PTLD development and survival after PTLD diagnosis. Kaplan-Meier and Cox regression analyses were used to calculate the influence of probable risk factors on PTLD development and survival. Rejection episodes were considered a time varying statistical variable, and rejections that occurred after PTLD development were excluded. Statistical analysis was performed with SPSS 16.0 (SPSS Inc., Chicago, IL, United States). A P value of < 0.05 were considered statistically significant.

Overall, 53 patients were diagnosed with PTLD. There were 40 cases of PTLD in the pediatric age group and 13 cases in the adult group. The incidence of PTLD was 6.25% in the pediatric patients and 1.18% in the adult liver transplant recipients. The baseline characteristics of PTLD patients are outlined in Table 1.

The mean overall (adult and pediatric) post-PTLD survival was 66.29 ± 11.86 mo. The post-PTLD survival of patients at 6 mo was 75.1% ± 6%, at 1 year was 68.9% ± 6.5% and at 5 years was 39.2% ± 14.2% (Figure 1A).

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Figure 1 Post-transplant lymphoproliferative disorder survival. A: Pediatric and adult patients; B: Adult patients; C: Pediatric patients. PTLD: Post-transplant lymphoproliferative disorder.

The mean post-PTLD survival in adult patients was 82.94 ± 18.58 mo. The post-PTLD survival of adult patients at 6 mo was 83.9% ± 10.4%, at 1 year was 74.6% ± 12.8% and at 5 years was 59.7% ± 16.8% (Figure 1B).

The mean post-PTLD survival in pediatric patients was 42.61 ± 6.1 mo. The post-PTLD survival of pediatric patients at 6 mo was 72.4% ± 7.1%, at 1 year was 67.1% ± 7.5% and at 5 years was 24.1% ± 18.6% (Figure 1C).

When both pediatric and adult patients were analyzed altogether, multi-organ involvement was significantly associated with lower post-PTLD survival (104.25 ± 9.08 mo vs 27.13 ± 6.30 mo, P = 0.002) (Figure 2A). EBV-positive patients with PTLD had significantly higher mean survival compared to EBV-negative PTLD patients (60.58 ± 7.62 mo vs 16.72 ± 5.66 mo, P = 0.018) (Figure 2B). Other variables including sex, CMV status, rejection episodes, time to PTLD before or after 1 year, and type of allograft had no significant effect on post-PTLD survival (Table 2).

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Figure 2 Post-transplant lymphoproliferative disorder survival of pediatric and adult patients. A: Impact of multi-organ involvement; B: Impact of EBV status. EBV: Epstein-Barr virus; PTLD: Post-transplant lymphoproliferative disorder.

We also analyzed the influence of different risk factors on pediatric PTLD patients separately. Multi-organ involvement and EBV negativity were significantly associated with lower mean post-PTLD survival in pediatric patients (Table 3). Higher serum tacrolimus level was associated with lower post-PTLD survival in pediatric patients (OR = 1.07, 95%CI: 1.006-1.15, P = 0.032) (Table 4).

Patients were divided into those who developed PTLD in ≤ 1 year and those who developed PTLD in ≥ 1 year. Age, post-PTLD survival, serum tacrolimus level, tacrolimus dose and prednisolone dose were not correlated with time to PTLD development in pediatric patients (P > 0.05) (Table 5). However, multi-organ involvement was more common in patients who developed PTLD within 1 year after liver transplantation (P = 0.007) (Table 6). Multi-organ involvement was also more common in pediatric patients who developed PTLD within 1 year after liver transplantation (P = 0.007) (Table 6).

Multi-organ involvement was associated with increased mortality after PTLD development (P < 0.05) (Table 7). EBV-positive patients with PTLD had lower mortality when compared to EBV-negative patients (P < 0.05) (Table 7).

Multi-organ involvement was not associated with age, serum tacrolimus level, tacrolimus dose, prednisolone dose in univariate analysis (Table 5). EBV-positive patients were less likely to have multi-organ involvement in comparison with EBV- negative patients (P = 0.008) (Table 8). Pediatric patients who received liver allograft from deceased donors were more likely to develop PTLD with multi-organ involvement when compared to those receiving liver allograft from living donors (P = 0.019) (Table 8).

To estimate a cutoff point value for tacrolimus level in relation to post-PTLD survival in pediatric patients, we used receiver operating characteristic (ROC) curve analysis. A serum tacrolimus of over 11.1 ng/mL was predictive of post-PTLD survival (sensitivity = 90%, specificity = 52%, area under the curve = 0.738, P = 0.035).

Post-transplant lymphoproliferative disorder (PTLD) is one of the complications after liver transplantation and may threaten both graft and patient survival. This study aimed to investigate incidence and survival of PTLD patients after liver transplantation.

Few studies with considerable number of patients have reported survival of PTLD patients after liver transplantation. This study aimed to investigate incidence, risk factors (including impact of immunosuppressive regimen) and survival of PTLD patients after liver transplantation in Iranian patients.

Multi-organ involvement was associated with a lower post-PTLD survival and increased mortality. Epstein-Barr virus (EBV)-positive patients had higher mean post-PTLD survival in comparison with EBV-negative subjects. EBV status was not associated with time of PTLD development in our study. This finding is in contrast with previous reports showing that EBV-negative PTLD occurs later after liver transplantation when compared to EBV-positive PTLD patients. We showed that a serum tacrolimus cutoff value of 11.1 ng/mL is associated with post-PTLD survival.

Adjustment of tacrolimus level to lower than 11.1 ng/mL may help improve post-PTLD survival of patients.

The reviewer has read with interest the manuscript entitled, “Post-transplant lymphoproliferative disorder after liver transplantation: incidence, long-term survival and impact of serum tacrolimus level”. Eshraghian and colleagues performed a retrospective single-center study with a wide recruitment period, including 53 liver transplant patients who developed PTLD (40 pediatric and 13 adult cases). The authors evaluated the risk factors affecting post-PTLD survival of patients. They found that EBV-negative recipients and multi-organ involvement are the two main risk factors of lower post-PTLD survival. They further found within a pediatric recipient cohort that higher serum tacrolimus level was associated with poor survival after PTLD development.