The development of lymphoid proliferations after transplantation has been recognized for more than a quarter of century as an important morbidity factor (1). The post-transplant lymphoproliferative disorder (PTLD) refers to a heterogeneous group of lymphoproliferative diseases, which vary from uncomplicated, self-limiting infectious mononucleosis, to malignant lymphoma. The histological characterization varies from reactive-appearing, polyclonal lymphoid infiltrates or undifferentiated cells that are morphologically indistinguishable from malignant lymphoma or plasma cell myeloma (2–4).

PTLD is relatively rare; nevertheless, it is the most frequent malignant disease early after transplantation, with the majority of cases being reported in the first year after transplantation (3,5,6). Risk factors for PTLD development include young age and age over 50 years at transplantation, white race, unrelated or HLA-mismatched graft, Epstein-Barr virus (EBV)-seronegative status prior to transplant, primary EBV infection, type of organ transplant, intensity of immunosuppression and the occurrence of concomitant cytomegalovirus disease (3,7).

Not all PTLD cases are EBV-related, but consistent data recognize primary EBV infection as the most important risk factor for PTLD development (8,9). Indeed, the immunosuppression after transplantation in an EBV-seropositive patient reduces the activity of the patients' EBV-specific cytotoxic T-cell surveillance, which increases the probability of uncontrolled proliferation of EBV-infected B-cells and subsequent progression to PTLD (10). Moreover, transplant recipients experiencing primary EBV infection during the early post-transplant period seem to be particularly susceptible to develop PTLD of B-cell origin, reflecting their lack of any preexisting EBV-specific T-cell immunity (3,10).

The overall incidence of PTLD varies from 1 to 22% depending on the presence of risk factors, namely the transplanted organ, patient age, EBV serostatus from recipient and donor, aggressiveness of immunosuppression (11). The cumulative incidence of PTLD in allogeneic hematopoietic stem cell transplantation (aHSCT) recipients is approximately 1.0% (range 0.5–1.8%), with slightly higher rates in the pediatric population (1,12). Survival rates depend mainly on the type of PTLD, extent of disease and patient age: While pediatric patients and those with localized disease seem to have a better prognosis, monomorphic PTLDs are the most aggressive forms (5,7,13).

The purpose of this study was to examine the clinical and pathologic characteristics, as well as the outcome of PTLD after aHSCT, in patients diagnosed at the Portuguese Oncology Institute of Porto (Porto, Portugal) between 2005 and 2012.

The study included a total of 15 patients, 8 females (53.3%) and 7 males (46.7%), with median age of 10 years-old (range 3–38)- Table I. Patients had a median follow-up time of 14 months (range: 2–72). Primary diagnoses of patients included in this study included paroxysmal nocturnal hemoglobinuria (n=1), primary immunodeficiency (n=1), acute lymphocytic leukemia (n=6), acute myelogenous leukemia (n=4), chronic myelogenous leukemia (n=1), myelodysplastic/myeloproliferative syndrome (n=1) and congenital amegakaryocytic thrombocytopenia (n=1). Most of patients had mismatched/unrelated donors (73.3%) and the collection of cells was mainly performed by peripheral blood stem cells (80.0%). Myeloablative conditioning was used in 14 patients and ATG in 12 patients. Transplant-related information for each patient is described in Table II.

Regarding the clinical presentation of patients, 2 presented with fever, 12 had increased liver enzymes, adenomegalies were observed in 2 patients and 12 patients had also increased lactate dehydrogenase. EBV serological status prior to transplantation were evaluated according to presence of IgM and IgG titers in plasma samples. Serological status was divided in three groups: susceptible (absence of IgM and IgG), active infection (presence of IgM and/or IgG) and finally, past infection (absence of IgM and presence of IgG).

The development of EBV infection was present in all of 15 patients, with a median time of diagnosis after transplant of 68 days (range 29–464 days), with 80% (n=12) of them detected <180 days after transplant, and with a median viral load of 4.75 log₁₀ copies/ml (range 3.30–6.26 log₁₀ copies/ml; Fig. 1). PTLD diagnosis occurred approximately in the same period where EBV infection occurred (mean 135, median 75 days and range 25–485 days vs. mean 130 days, median 68 days and range 29–464 days, respectively). PTLD classification was available for only 5 patients and included monomorphic-type PTLD (n=3), polymorphic PTLD (n=1) and reactive plasmocytic hyperplasia (early lesions) (n=1) (Table III; Fig. 2). The remaining cases were not histological confirmed, and diagnosis was established by considering all clinical findings.

We observed graft-vs.-host disease (GVHD) in 13 patients (93.3%): 3 with acute GVHD (20.0%), 2 with chronic GVHD (13.3%) and 8 with both (53.3%). Considering the grade of acute GVHD, all patients with clinical information had a grade of II or higher. Regarding chronic GVHD, 3 patients had an evolution of acute-to-chronic, while 7 had a de novo chronic GVHD; two patients experienced extensive disease and 5 had only limited disease (Table II).

As for the overall outcome, 8 patients are still alive (53.3%), 5 with no signs of disease (33.3%) and 3 with evidence of disease (20.0%); and 7 patients have died (46.7%), 4 from complications associated with the transplant (26.7%) and 3 from progression of disease (20.0%) (Table II). A Kaplan-Meier plot was obtained by evaluating the cumulative survival of these patients, in months (Fig. 3).

PTLD is one of the most serious complications of immunosuppression in patients who undergo hematopoietic stem cell transplantation, with high impact on morbidity and mortality (14). EBV infection has been strongly associated with the development of PTLD, and this association is widely described in the literature (15,16).

In this retrospective analysis, we verified that PTLD affects individuals of all age groups and with several types of hematological malignancies, the majority having had unrelated donors. Our patients had different types of pre-conditioning regimens (myeloablative in 14 patients), with predominance of busulfan and cyclophosphamide. Since types of regimens are varied, they appear not to have a direct correlation with the development of PTLD. ATG was used in almost all patients except for two, and without absolute prevalence date it is difficult to confirm if its use is directly correlated with PTLD development. GVHD prophylaxis was performed mainly with tacrolimus, and concomitant with MTX, and still patients have developed some type of GVHD which indicates that altering prophylaxis regimen should be taken into consideration.

In our case series, EBV infection was diagnosed at a median of 68 days after transplant. EBV infection is frequently associated with the intermediate period after aHSCT, mainly between 3 weeks to 3 months after transplant (17). Viral infection during this period is correlated with delayed or incomplete reconstitution of specific immunity, or patients experiencing GVHD (18). Regarding PTLD, frequently, the median onset of development is 3 months, with a range of 2–5 months after transplantation (13), which is consistent with our data. Symptoms are quite nonspecific, with patients presenting with fever, malaise, enlarged lymph nodes and high levels of LDH, which were the factors for clinical PTLD suspicion in our patients (2). All patients that developed PTLD had an EBV infection at some point after transplantation. EBV positivity is directly related to PTLD development since its infection, or increase in viral load up to 2,000 copies/ml, occurs mainly at the same time PTLD is diagnosed. PTLD is more frequent in EBV-seronegative patients receiving allografts from EBV-seropositive donors and in patients with delayed immune reconstitution due to T-cell-depletion or HLA-mismatched donor. In a study conducted by Brunstein et al (19), 15 of 335 patients developed a EBV-related complication, at a median of 133 days (range 52–407 days), which is consistent with our results.

As previously described by Bhatia et al (20), PTLD has mortality rates reaching up to 70–90%, which is higher than our results (46.7%). Survival rates depend on age and stage of disease at the time of diagnosis, with pediatric and patients with localized disease showing the best prognosis (5). In our study, overall patient survival was not affected by the development of PTLD.

This is the first study to describe PTLD cases in HSCT patients in Portugal, combining data from several years at a reference transplantation center. This study demonstrates that EBV infection occurs mainly between 2 and 4 months after transplant and precedes the development of PTLD, and especially the viral load may be important for the monitorization and early diagnosis of PTLD. Thus, the study shows the importance of identify high-risk patients for PTLD development and to provide them a frequent monitorization of EBV viral load as suggested by recent guidelines (21,22).