Association Between Epstein-Barr Virus Seroconversion and Immunohistochemical Changes in Tonsils of Pediatric Solid Organ Transplant Recipients

Transcription

1 The Laryngoscope VC 2011 The American Laryngological, Rhinological and Otological Society, Inc. TRIOLOGICAL SOCIETY CANDIDATE THESIS Association Between Epstein-Barr Virus Seroconversion and Immunohistochemical Changes in Tonsils of Pediatric Solid Organ Transplant Recipients Nina L. Shapiro, MD; Christopher Guan-Zhong Tang, MD; Neil Bhattacharyya, MD Objectives/Hypothesis: Assess whether changes in Epstein-Barr virus (EBV) serology status relates to immunohistochemical evidence of active EBV infection in tonsillar tissue of pediatric organ transplant recipients. Children who seroconvert from EBV seronegative to seropositive status following organ transplantation are more likely to develop evidence of active EBV tonsillar infection than those who do not seroconvert. Active EBV infection is a potential early precursor of post-transplantation lymphoproliferative disorder (PTLD), a life- and/or organ-threatening disease. Study Design: Retrospective analysis of pathology specimens and EBV serology titers of pediatric organ transplant recipients at a tertiary care academic medical center. Methods: Immunohistochemical staining of tonsillectomy specimens and analysis of EBV serology titers were performed on 47 post-transplantation children. Eligible patients included those under age 21 years referred for tonsillectomy who had undergone solid organ transplantation. Data reviewed included gender, age at transplantation, age at tonsillectomy, interval between organ transplantation and tonsillectomy, and type of organ transplanted. EBV was detected in tonsil specimens by in situ hybridization for EBV-encoded small nuclear RNA (EBV-EBER), an indicator of active EBV infection, and EBV-encoded latent membrane protein (EBV-LMP), an indicator of latent EBV infection. Immunohistochemical staining of B-cell clonality was performed for kappa and lambda light chains. EBV serologies pre- and post-transplantation were obtained from electronic medical record data. Results: Forty-seven children having tonsillectomy following solid organ transplantation were included. Of the 47 patients, 25 (53.5%) were EBV seronegative both before transplantation and at the time of tonsillectomy, eight (17.0%) were EBV seronegative prior to transplantation but converted to EBV seropositivity in the early (6 months) post-transplantation period, eight (17.0%) were seronegative prior to transplantation but converted to EBV seropositive in the late (6 months) post-transplantation period, and six (12.8%) were EBV seropositive prior to transplantation and remained so at the time of tonsillectomy. Overall, 20 specimens (42.6%) were positive for EBV-EBER, and three specimens (6.4%) were positive for EBV- LMP. There was significant difference in the percentage of EBV-EBER positive specimens among the four groups (P ¼.020). One out of eight (12.5%) of the early seroconversion group had specimens showing EBV-EBER, wheras seven out of eight (87.5%) of the late seroconversion group had tonsil specimens showing EBV-EBER (P ¼.005). There was no significant difference in presence of EBV-LMP among the four groups (P ¼.667). Conclusions: Almost one half of post-transplantation children undergoing tonsillectomy have evidence of active EBV tonsillar infection, which was more likely in children who seroconverted from EBV-seronegative to EBV-seropositive in the late post-transplantation period than those who seroconverted in the early post-transplantation period. Based on these novel findings, continued surveillance of EBV status and adenotonsillar hypertrophy in this population should include monitoring of pretransplantation EBV status and timing of seroconversion. Key Words: Tonsil, pediatric, transplantation, Epstein-Barr virus. Level of Evidence: 2b. Laryngoscope, 121: , 2011 From the Division of Head and Neck Surgery, Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, California (N.L.S.); and Kaiser Permanente Oakland Medical Center, Oakland, California (C.G-.T.);, Department of Otology, Rhinology, and Laryngology, Harvard Medical School, Boston, Massachusetts, USA (N.B.). Editor s Note: This Manuscript was accepted for publication April 8, Nina Shapiro, MD, is a consultant for ArthroCare ENT. The authors have no other funding, financial relationships, or conflicts of interest to disclose. Send correspondence to Nina L. Shapiro, MD, Division of Head and Neck Surgery, UCLA CHS, LeConte Avenue, Los Angeles, CA nshapiro@ucla.edu DOI: /lary INTRODUCTION Organ transplantation is an increasingly viable treatment option for multiple chronic medical conditions, with improvements in long-term survival attributable to advanced surgical techniques and closely monitored immunosuppressant regimens. However, with these improvements, increased concern has arisen regarding immunosuppressant-related sequelae, most notably those related to Epstein-Barr virus (EBV) infection. Recognition of EBV infection in the pediatric organ transplant population is especially crucial, because primary EBV infection is associated with potential 1718

2 development of post-transplantation lymphoproliferative disorders (PTLD). 1 PTLD is a spectrum of EBV-related lymphoid dysplasias due to polyclonal immortalization of EBV-infected B lymphocytes, which may lead to monoclonal lymphomas. Clinical outcomes of PTLD improve with early detection and treatment. Failure to do so may lead to loss of transplanted organ and high mortality rates. 2 In immunosuppressed patients, PTLD represents a spectrum of clinical and morphologic abnormal proliferations of lymphoid cells, and is associated with EBV infection in up to 80%. 3 5 For early PTLD lesions, treatment usually involves reduction of immunosuppressant therapy, which puts the recipient at risk for organ rejection. More aggressive PTLD requires use of chemotherapeutic agents, surgical debulking of the involved tissue, as well as immunosuppressant reduction or cessation. Aggressive PTLD carries a mortality rate of 40% to 50%. 6 Risk factors for PTLD in the solid organ transplant population include type of organ transplanted (with lung and small bowel being at higher risk than renal, heart, and liver), frequency of rejection episodes, need for T-cell antibody therapy, EBV seronegativity at the time of transplant, and younger age at the time of transplant. 3,5,7 The incidence of PTLD ranges from 1% to 5% in the low-risk groups to 10% to 30% in the higher-risk groups. 5 However, regardless of type of organ transplanted, children are considered to be at relatively high risk (4% 15%) for development of PTLD compared to adults due to the higher rates of initial EBV seronegativity and young age at the time of transplantation PTLD may be seen throughout the body as lymphomatous lesions in the allograft itself, or as a fulminant disseminated disease similar to septic shock. 5 In children, Waldeyer s ring is frequently involved, as the B cells in the palatine and nasopharyngeal tonsils tend to be the main target of acquired EBV infection. This is thought to be secondary to the virus predilection of B cells, which are the predominant lymphocytic population of Waldeyer s ring. 12,13 EBV is a common B-lymphotrophic herpesvirus that can develop as a latent infection in resting memory B cells in healthy individuals. In fact, greater than 90% of adults have latent EBV infection. 14 In the immunosuppressed host, Epstein-Barr viral infection can lead to EBV-related lymphoid hyperplasia and B-cell proliferation, which is manifested as adenotonsillar enlargement. Such enlargement in transplant recipients with no pretransplantation exposure to EBV may be an early sign of active EBV infection. 7 In transplant recipients who receive immunosuppressants to decrease cytotoxic T-cell activity to prevent graft rejection, EBV-mediated B-cell proliferation can proceed unchecked, potentially leading to the neoplastic condition known as PTLD. 2 EBV seronegativity at the time of organ transplant may have an impact on an individual s future development of EBV-related illness and PTLD. 7,15 18 EBV transmission can occur from an EBV-seropositive donor organ, or from a community-acquired EBV infection, leading to host seroconversion. Such EBV exposure can infect the host, allowing for uncontrolled proliferation of TABLE 1. Classification of Postransplantaion Lymphoproliferative Disorders (PTLDs)*. Categories of PTLD Early lesions PTLD Polymorphic Monomorphic Other Subtypes Plasmacytic hyperplasia and infectious mononucleosis-like Polyclonal and monoclonal B-cell and T-lymphocyte lymphoma T-lymphocyte rich or Hodgkin disease, plasmacytoma-like, and myeloma *Based on the Society for Hematopathology Workshop 1997 recommendations (Harris). B lymphocytes in EBV-infected tissue such as tonsils. This infection can, in turn, lead to a wide spectrum of PTLD-related diseases, from polyclonal lymphoid hyperplasia to monoclonal malignant lymphoma (Table I). 4,19 Therefore, early detection of EBV infection, which can first be manifested in the tonsils, may enable earlier treatment of PTLD, thereby mitigating progression to more aggressive forms of disease. In a prior study, immunohistochemical and in situ hybridization techniques were used to analyze Epstein- Barr viral proteins and RNA in tonsil specimens of pediatric transplant recipients compared with those of healthy controls (tonsil specimens of immunocompetent children). Transplant recipients were found to have a significantly higher rate of active EBV infection (manifested as presence of EBV-encoded small nuclear RNA [EBV-EBER] in the tonsil specimens) (P <.001), and a higher incidence of EBV-related lymphoid hyperplasia, considered to be on the spectrum of PTLD. 2 However, there were several limitations to this study. EBV serology titers were not analyzed in these patients, either at the time of transplantation or at the time of tonsillectomy. Thus, the presence of EBV-EBER in these specimens could have been due to either primary acute EBV infection, or reactivation of latent disease. In addition, EBV titers were not correlated with pathologic findings, which would help to indicate the severity of infection and risk for development of PTLD. In this study, we investigated the association between pre- and post-transplantation EBV serologic status in relation to immunohistochemical detection of active EBV infection (EBV-EBER) in tonsil specimens from pediatric organ transplant recipients. No such study has been carried out previously. We hypothesized that children who became EBV seropositive in the late posttransplantation period (>6 months post-transplantation) were more likely to demonstrate immunohistochemical evidence of active tonsillar EBV infection. The implications of EBV seronegativity at the time of solid organ transplantation, timing of seroconversion, and presence of active EBV tonsillar infection were analyzed to assess the relationship between timing of EBV seroconversion and evidence of active EBV infection in tonsillar tissue. We also expanded the subject age range to include patients up to 21 years, which is considered to be pediatric by our institution s and state s criteria, the U.S. Food 1719

3 and Drug Administration, and by the American Academy of Pediatrics Section on Adolescent Medicine. MATERIALS AND METHODS This study underwent institutional review board review and approval for human subjects. A retrospective analysis was performed on all patients ages 21 years and younger referred for tonsillectomy from the pediatric solid organ transplant (cardiac, liver, kidney) teams. There were no patients who had undergone lung or small bowel transplant referred for tonsillectomy during the study period. Patient records were reviewed for age at time of transplantation, type of organ transplanted, age at time of tonsillectomy, gender, and EBV serologic status before and after transplant and at the time of tonsillectomy. Tonsillectomy specimens were sent separately as fresh tissue specimens, and examined by hematoxylin and eosin staining at low and high magnification. Nodal architecture, cytology, differentiation, hyperplasia, and polarization of follicles were examined and recorded. Immunohistochemical analysis was performed using B cell, T cell, and Epstein-Barr viral markers, as previously described. 20 CD20 marker antibodies were used to label B cells, and kappa and lambda light-chain antibody probes were used to assess B-cell clonality. The presence of both light-chain types was considered to be indicative of a polyclonal B-cell population. T-cell antibody probes included CD3 and CD43. In situ hybridization and immunuhistochemical markers for EBV-EBER and EBV-latent membrane protein (EBV-LMP) were performed to determine presence and activity of EBV. Presence of EBV-EBER is seen during viral replication of active EBV infection. Following primary infection, the virus can remain dormant, whereby EBV-LMP is used to detect the virus. Tissue samples were classified based on the Society of Hematopathology Workshop 1997 recommendations for PTLD. 19 EBV serologic data were abstracted from electronic medical record data based on the following parameters for antibody titers at our institution: EBV-viral capsid antigen (VCA) immunoglobulin (Ig) M<1:20 ¼ negative; EBV-VCA IgM>1:20 ¼ positive; EBV-VCA IgG <1:10 ¼ negative; EBV-VCA IgG >1:10 and <1:640 ¼ equivocal; EBV-VCA IgG >1:640 ¼ positive or four-fold increase in antibody titer ¼ positive; Epstein-Barr nuclear antigen (EBNA) <1:5 ¼ negative; EBV >1:5 ¼ positive. Patients were divided into three major groups, with group 2 having two subgroups: group 1: EBV seronegative at the time of transplant and at the time of tonsillectomy; group 2a: EBV seronegative at the time of transplant with seroconversion to EBV positive in the early post-transplant period (6 months post-transplantation); group 2b: EBV seronegative at the time of transplantation with seroconversion to EBV positive in the late post-transplant period (6 months post-transplantation); and group 3: EBV seropositive pre- and post-transplantation. The criterion for delineating early (<6months) from late (>6 months) post-transplantation was based on prior studies that describe acute or early post-transplantation complications to occur in the 3- to 6-month period following organ transplant. 21,22 Analysis of histology and immunohistochemical data in relation to EBV status was performed. Statistical testing for relationships between EBV seroconversion status and histological markers of EBV infection was conducted with v 2 with significance set at P ¼.05. Fig. 1. Tonsillar tissue with immunohistochemical staining reveals Epstein-Barr virus-encoded small nuclear RNA (EBV/EBER) in large lymphoid cells (high-power magnification, 40x). RESULTS Sixty-one consecutive children who underwent tonsillectomy between 1999 and 2008 following solid organ transplantation were reviewed. Of these, 47 had sufficient data available for review regarding pre- and post-transplantation EBV serologies, and form the current study group. There were 23 female patients and 24 male patients. Mean age at time of organ transplantation was 3.1 years (range, 0 13 years), and mean age at tonsillectomy was 8.0 years (range, 2 19 years). Of the 47 children, 30 received liver transplants (63.8 %), 14 received renal transplants (29.8 %), and three received heart transplants (6.4%). All patients were referred for tonsillectomy based on signs and/or symptoms of adenotonsillar hypertrophy noted by the transplantation teams during post-transplantation clinic visits. The major reason for referral was to assess for PTLD and EBV activity in the tonsil specimens. Clinical signs and symptoms of adenotonsillar hypertrophy prompting referral for tonsillectomy included sleep-disordered breathing (41 patients), spontaneous tonsillar bleeding (one patient), recurrent tonsillitis (three patients), and otitis media (four patients), with two patients having more than one indication. Mean duration from organ transplantation to tonsillectomy was months (range, months; standard deviation [SD] 30.51). Of the patients who converted from pretransplantation EBV seronegative to post-transplantation EBV seropositive, mean number of months to seroconversion was (SD 21.32). Presence or absence of EBV seroconversion had no impact on the mean number of months from transplant to tonsillectomy (P ¼.294). Twenty-one of the tonsillectomy specimens (44.7%) demonstrated immunohistologic evidence of EBV-related lymphoid hyperplasia. None of the specimens had evidence of full fledged PTLD by histological criteria. Of the 47 specimens, 20 (42.6%) were positive for acute EBV infection (EBV-EBER), and three (6.3%) were positive for EBV-LMP (Fig. 1 and Fig. 2). On immunohistochemical staining, 10 (40%) of group 1 tonsil specimens were EBV- EBER positive, one (12.5%) of group 2a were EBV-EBER positive, seven (87.5%) of group 2b specimens were EBV- EBER positive, and two (33.3%) of group 3 specimens were EBV-EBER positive (Table II). These differences were statistically significant (P ¼.020, v 2 ). 1720

4 EBV-LMP activity did not differ between groups: group 1 had two (8.0%) specimens that stained positive for EBV-LMP, group 2a had zero (0.0%) specimens that stained positive for EBV-LMP, group 2b had one (6.4%) specimen that stained positive for EBV-LMP, and group 3 had zero (0%) positive specimens (P ¼.667). Fig. 2. Tonsillar tissue with immunohistochemical staining reveals Epstein-Barr virus-encoded latent membrane protein (EBV/LMP) in large lymphoid cells (high-power magnification, 40x). Of the eight patients who seroconverted to EBV positive in the early transplant period (group 2a), seven had undergone liver transplantation and one had undergone kidney transplantation. Mean age at transplantation for this group was 8.6 years, and mean number of months to seroconversion was 5.9. All had adenotonsillar hypertrophy due to follicular hyperplasia, with none having PTLD. All had polyclonal evidence of both kappa and lambda light chains. One specimen out of eight was EBV-EBER positive, and none were EBV- LMP positive. Of the eight patients who seroconverted from EBV seronegative to EBV seropositive in the late posttransplantation period (group 2b), six had received a liver transplant, one had received a second liver transplant, and one had received a kidney transplant. Mean age at transplantation was 7.6 years and the mean number of months to seroconversion in this group was All histological analyses of this group of specimens demonstrated follicular hyperplasia, and all were polyclonal for kappa and lambda light chains. Seven of the eight specimens were positive for EBV-EBER. In comparison of the group 2 subgroups, one out of eight tonsil specimens of group 2a were EBV-EBER positive (12.5%) versus seven out of eight EBV-EBER positive (87.5%) in group 2b tonsil specimens (P ¼.005). These results are summarized in Table II. DISCUSSION PTLD represents a spectrum of abnormal, unregulated lymphocyte cell proliferation, and has been linked to primary EBV infection and polyclonal immortalization of EBV-infected B lymphocytes, most often in EBV-naïve transplant recipients. 2,8,23 Although viruses other than EBV have been shown to be associated with post-transplantation illnesses and PTLD, the proclivity to the lymphoid tissue of Waldeyer s ring makes EBV the most likely virus to be associated with PTLD of the tonsils. 24 As a result of prolonged administration of immunosuppressants, a defect in T-cell regulation allows for uncontrolled proliferation of B cells (and rarely T cells) in the setting of EBV infection. These disorders differ from lymphoproliferative disorders in immunocompetent individuals, in that they manifest different histologic patterns, a more aggressive clinical course, poor response to chemotherapy, and poorer overall outcomes. 4 PTLD lies on a spectrum. In its mildest form it is classified as a polyclonal lymphocytic proliferation; at the other end of the spectrum it is an aggressive large cell lymphoma. 19 The head and neck region may be affected in 25% to 63% of cases, with abdominal and thoracic cavities also being common sites of involvement. 16,25 Head and neck involvement (including Waldeyer s ring or cervical lymph nodes), will often present as a mononucleosis-like illness, with acute adenotonsillitis, cervical lymphadenopathy, fever, and malaise. When PTLD presents acutely in the head and neck, computed tomography and magnetic resonance imaging may be useful adjuncts to physical examination to assess for orbital and sinonasal extension of disease. Progression of disease can be rapid in any organ system involved, leading to widespread dissemination and invariable mortality if left untreated. Therefore, a high index of suspicion for PTLD is necessary to establish early diagnosis and treatment. Definitive diagnosis is made by tissue analysis, either by tissue removal or core biopsy. 16 Histology will demonstrate diffuse effacement of normal lymphoid follicles, with sheets of small lymphocytes, immunoblasts, plasma cells, and plasmacytoid lymphocytes (Fig. 3). In situ TABLE II. Results of immunohistochemical analysls of tonsillectomy specimens after solid organ transplantation. Seroconversion group Group number N EBER Status LMP Status EBV-EBER ( ) N(%) EBV-EBER (þ)n(%) EBV LMP ( ) EBV LMP (þ) EBV seronegative throughout (60) 10 (40) 23 (92) 2 (8.0) EBV seroconversion (early) post-transplant 2a 8 7 (87.5) 1 (12.5) 8 (100) 0 (0) EBV seroconversion (late) post-transplant 2b 8 1 (12.5) 7 (87.5) 7 (87.5) 1 (12.5) EBV seropositive throughout (66.7) 2 (33.3) 6 (100) 0 (0) 1721

5 hybridization will show strong nuclear staining for EBV- EBER (Fig. 1). 20 Staining for EBV-LMP is also seen in premalignant forms of nasopharyngeal carcinoma, although this has not been commonly reported in the post-transplantation populations. 26 Treatment options vary as to degree of organ or multiorgan involvement, but may include immunosuppressant reduction, antiviral medications, intravenous immunoglobulin, or cytomegalovirus hyperimmune globulin. Other useful therapies include interferon and methotrexate. When possible, especially if the tonsils and adenoids are involved, surgical removal of the affected tissue is strongly recommended, for both diagnostic and therapeutic purposes. 4,8,27 To date, there are no standardized means of assessing early signs of PTLD as potentially manifested by adenotonsillar enlargement. Some institutions have recommended close follow-up of serum EBV polymerase chain reactions and/or EBV serum antibody titers. Our institution uses serum antibody titers to measure the level of EBV in the serum of transplant recipients. A rise in either will warrant active investigation to search for a potential source of indolent EBV infection or PTLD Clinical signs and/or symptoms of adenotonsillar enlargement may be an early manifestation of PTLD; monitoring for such clinical changes may enhance more directed serologic surveillance. 7 Adenotonsillectomy as an aggressive intervention in transplanted children with adenotonsillar hypertrophy has been shown to be the most accurate and definitive means of diagnosing PTLD in its earliest forms. 8,24,30 Known risk factors for PTLD in the pediatric population include young age at the time of transplantation and EBV seronegativity pretransplantation. As EBV seropositive status is more common in older patients, young age and EBV seronegativity are oftentimes coincident risks. Population studies have demonstrated that Fig. 3. Post-transplantation lymphoproliferative disorder of tonsillar tissue reveals diffuse effacement of normal tonsillar follicles, sheets of small lymphocytes, immunoblasts, few plasma cells, few plasmacytoid lymphocytes (hematoxylin and eosin stain; highpower magnification, 40x). intrinsic EBV seroconversion in the healthy population is occurring at older ages than in prior years. This may result in what is now the young at-risk population eventually including older children. 31,32 A previously published study comparing EBV-EBER in tonsils of healthy controls to EBV-EBER in tonsils of transplanted individuals demonstrated higher rates of EBV-EBER in transplanted patients overall, compared to healthy individuals. 2 However, this study did not identify timing of seroconversion in relation to active tonsillar EBV infection. Timing of EBV seroconversion is an area of question investigated in the present study. A large proportion of patients who are seronegative recipients will seroconvert in the first year following transplantation, with a peak incidence of PTLD in the first 6 months following transplantation. 4 However, delayed seroconversion (>6 months post-transplantation) may be manifested as low-grade EBV infection of the tonsils, as demonstrated by the higher number of EBV- EBERþ staining in tonsils of children with delayed seroconversion found in the current study. Conversion from EBV-seronegative to EBV-seropositive in the late posttransplant period may indicate acute EBV infection, albeit to a lesser degree than commonly seen with fulminant PTLD. The data in this report strongly suggest that patients who seroconvert close to the time of manifesting signs and symptoms of adenotonsillar hypertrophy are more likely to have tonsillar EBV-EBER markers. Patients who converted from EBV-seronegative to EBV-seropositive in the early post-transplant period did not have similar evidence of active EBV infection of the tonsils when presenting for adenotonsillectomy. EBV, a gamma double-stranded DNA herpesvirus, is present in more than 90% of adults, with most individuals exposed at some point during childhood, resulting in lifelong latent infection. It is associated with several lymphoid neoplasms including Burkitt s lymphoma, Hodgkin s disease, and post-transplantation lymphoproliferative disorders. 33 Primary EBV infection is rarely seen in the first year of life, likely due to presence of the protective effect of maternal antibodies. 34 Because of improved sanitation and industrialization, fewer children are exposed to EBV infection in early childhood, making more children susceptible to acute infectious exposure in late childhood and early adolescence. In Japan, seropositive rates in children ages 5 to 9 years dropped from 80% in 1990 to 59% from 1995 to A 2006 cohort study demonstrated that in the United States, 30% to 75% of college freshman were EBV seronegative. 32 Progressively higher incidence of EBV seronegativity in the healthy population has implications for the pediatric transplant population, whereby children are more susceptible to complications of EBV exposure if seronegative at the time of transplantation and onset of immunosuppressant therapy. EBV is spread via oropharyngeal droplet exposure, leading to viral replication in the oropharynx. Incubation periods may last from 30 to 50 days from exposure to symptom onset. 34 EBV colonization in tonsils and adenoids of healthy subjects may lead to EBV-related adenotonsillar enlargement. Endo et al. demonstrated 1722

6 that 72% of adenoid specimens of patients 2 to 13 years old had evidence of active EBV infection on immunohistochemical staining (EBV-EBER). 35,36 In the immunocompetent host, acute EBV may progress to infectious mononucleosis (IM), which is more commonly seen in young adults with no prior EBV exposure. 12,13,37 IM is primarily a disease of Waldeyer s ring, with cryptic adenotonsillitis, reactive posterior cervical lymphadenopathy, fever, malaise, and occasional hepatosplenomegaly. EBV primarily targets the tonsil tissue, gaining access to the tonsillar epithelium and high proportion of tonsillar B lymphocytes. Regulatory T lymphocytes (CD4þ) play a role in suppressing the cellular immune response to infection, providing balance to activation of immunopathologic disease such as IM. However, acute IM may result in florid activation of cytotoxic (CD8þ) T cells, which secrete cytokines inducing symptoms of IM. Despite this sporadic progression to an acute immune response, the immunocompetent individual will eventually recover from the acute event by regulatory T-cell activation, and the virus will then remain latent after the acute response subsides. Most acute symptoms resolve by 4 weeks; however, persistent fatigue may last for 6 months. 38 Diagnosis of IM in the healthy population includes clinical presentation, presence of atypical lymphocytes on peripheral blood smear, and positive heterophile antibody test (monospot). In the acute phase, IgM specific for EBV-VCA are present, which is diagnostic of an acute infection. IgM for EBV- VCA are not detected in chronic infection. EBV IgG VCA is detected at the time of or shortly after acute EBV infection, and will persist at low levels thereafter. 34 Although EBV serology is a standard means of monitoring EBV status, some studies have shown that EBV DNA quantification in the tonsils themselves is a more accurate means of long-term monitoring. 39 Treatment of acute IM is supportive in the healthy individual, with special care regarding minimizing physical activity in those with associated splenomegaly. Although EBV is a relatively benign, ubiquitous infection in the immunocompetent population, EBV infection in the immunocompromised host poses more concerning implications. These issues are unique to solid organ transplant recipients, who develop EBV sequelae from the recipient s B lymphocytes, as opposed to bone marrow transplant recipients, who develop EBV sequelae and PTLD from donor B lymphocytes. The highest risk recipient group is the pediatric population, who are most likely to be EBV seronegative at the time of transplantation. Transmission of EBV to this group most likely occurs via transplantation of an EBV seropositive donor organ. Less likely, recipients will incur community-acquired primary EBV infection via close contacts. In either case, EBV will undergo lytic replication due to lack of EBV-specific immune surveillance, leading to uncontrolled expansion of EBV-transformed B lymphocytes, potentially progressing to PTLD. 15 It is for this reason that some transplant centers institute prolonged antiviral therapy for EBV-seronegative patients receiving EBV-seropositive organs; nonetheless, EBV infection can still occur. 10 Young age and EBV seronegativity (which often go hand-in-hand) at the time of transplantation are significant risk factors for development of EBV infection and PTLD. 3,7,17 Recipient treatment with T-lymphocyte antibodies such as OKT3 and more intense T-lymphocyte immunosuppression with FK506 (tacrolimus) have also been shown to increase likelihood of EBV-related sequelae and PTLD, as the resultant lack of T cell-mediated response enables EBV proliferation to proceed unchecked. 15,27,40 Monitoring of patients at risk for EBV and PTLD includes analysis of EBV serum antibody levels and EBV-PCR levels, whereby rise in EBV genome copies may be a predictor of PTLD 41 and assessment of clinical signs and symptoms of acute or chronic EBV infection. 3 Adenotonsillar hypertrophy, even in the absence of symptoms of acute infection, may be a marker for EBV and PTLD. Adenotonsillectomy, followed by immunohistochemical analysis and in situ hybridization techniques to detect EBV viral proteins and RNA, is a diagnostic and therapeutic treatment modality used in this and prior studies. 2,27,42 Treatment for EBV disease and PTLD in this population is controversial, and may include surgical removal of the EBV-infected tissue, reduction of immunosuppressants, use of antiviral regimens such as acyclovir and ganciclovir, interferon, IVIG, or anti-b cell monoclonal antibodies. More recently, use of EBV-specific cellular therapies have been successful in bone marrow transplant (BMT) recipients. However, because BMT EBV infection originates from donor cells, it is possible to create EBV-specific cytotoxic t-lymphocytes (CTLs) from the donor marrow, which would recognize and destroy EBVinfected B cells in the BMT recipient. Because EBV in solid organ transplant recipients originates from recipient B cells, which were seronegative until the time of infection, there is no preexisting immunity whereby EBV-specific CTLs could be created. 1 Whereas some reports have demonstrated that adenotonsillar hypertrophy is not an indicator of EBV infection or early PTLD, 28,29 the majority of studies have recommended utilizing signs and symptoms of adenotonsillar enlargement as potential early markers for EBV infection and/or PTLD, and have recommended adenotonsillectomy in transplant recipients, even with asymptomatic adenotonsillar enlargement. The tissues of Waldeyer s ring are easily accessible, in that the physical examination does not require radiologic studies, and symptoms of airway obstruction due to adenotonsillar hypertrophy are easily elicited in pediatric patients. 3,7,8,18,27,30 Adenotonsillar enlargement in this population may present similarly to that seen in the healthy population, with snoring, sleep-disordered breathing, recurrent infections, or asymptomatic enlargement on routine follow-up examinations. Rarely, patients may develop acute life-threatening airway obstruction from fulminant EBV infection of the tissue of Waldeyer s ring. 8 Many children with adenotonsillar enlargement in this population will be asymptomatic, but will have evidence of active EBV infection by immunohistochemical markers in tonsil specimens. Thus, it is more important that physical examination and clinical symptoms of adenotonsillar 1723

7 enlargement be incorporated into close surveillance of EBV serology. 28,41 EBV-related hyperplasia, seen commonly in these patients, will have characteristic preservation of follicular architecture and polarization, with variable hyperplasia involving follicular and interfollicular areas. Most will test positive for both kappa and lambda light-chain markers, indicating polyclonality. Presence of EBV-EBER and EBV-LMP are variable. 20 Adenotonsillar hypertrophy in healthy children is often manifested as a spectrum of sleep-disordered breathing, ranging from primary snoring to obstructive sleep apnea syndrome. Adenotonsillectomy is curative in 85% to 95% of cases. 43,44 Histologic analysis of tonsil specimens of immunocompetent children demonstrates reactive follicular hyperplasia. Nodal architecture is preserved, without evidence of atypical lymphoid infiltrates (Fig. 4). On immunohistochemical staining, these tonsils will not likely have evidence of EBV-EBER, but may rarely have evidence of EBV-LMP (Fig. 5). All specimens will also have markers for T lymphocytes, as measured by CD3 activity. This latter finding is less likely in the tonsil specimens of children following organ transplantation. 2 To the authors knowledge, this is the first study to compare tonsillar EBV activity in transplanted children in relation to change in EBV serology over time. It supports prior studies, which demonstrate that transplant recipients have a higher rate of active EBV infection (EBV-EBER) of the tonsils compared with healthy counterparts, 2 but it also demonstrates that change in EBV status, and timing of that change, may have significance regarding development of active EBV infection. The finding that those children with the highest incidence of EBV-EBER were those who seroconverted from EBVseronegative to EBV-seropositive just prior to developing signs of adenotonsillar hypertrophy further strengthens the theory that seroconversion may reflect adenotonsillar involvement of EBV. There are several possible explanations for this: 1) EBV seronegative patients are Fig. 4. Lymphoid hyperplasia in normal tonsil. Nodal architecture and follicular polarization are well preserved. Hyperplasia is predominantly follicular (hematoxylin and eosin stain; low-power magnification, 4x). Fig. 5. Immunohistochemical staining in normal tonsil. Nodal architecture is preserved. Epstein-Barr virus-encoded small nuclear RNA and Epstein-Barr virus-encoded latent membrane protein stains are negative (low-power magnification, 4x). receiving antiviral protocols in the early post-transplant period, inhibiting potential development of acute EBV infection in the months following transplantation 10 ;2)in the later post-transplant period, when patients organ host status are stable and they are receiving relatively lower dose immunosuppressant regimens, they are more likely to be exposed to community-acquired EBV; and 3) in the early post-transplant period, the focus of the transplantation team tends to be on graft maintenance and minimization of severe post-transplantation complications such as organ rejection or life-threatening infections. Subtle changes in adenotonsillar size are less likely a concern during this critical period. CONCLUSIONS EBV infection in the setting of T lymphocyte suppression may be the earliest precursor of PTLD. Pediatric solid organ transplant recipients have high rates of active EBV infection, which in tonsils and adenoids may be manifested at higher rates of active EBV in the late post-transplant period. Conversion to EBV-seropositive >6 months following solid organ transplantation may be a marker for active EBV infection of Waldeyer s ring as a high incidence of EBV-EBER was found in these patients. Continued close surveillance for both EBV serologic status changes and evidence of adenotonsillar hypertrophy incorporate critical tools to enhance the early diagnosis and treatment regimens for these patients. Acknowledgments Dr. Shapiro would like to acknowledge Drs. Gerald Berke and Rinaldo Canalis, who have mentored and supported her work in preparation of her Triological Society thesis. She would like to thank Dr. Sunita Bhuta for her expertise in pathology and immunohistochemistry. She would also like to thank the Pediatric Liver and Kidney Transplant Teams at UCLA for their collegiality in patient 1724

8 care and support of this and other projects. Dr. Shapiro is indebted to her family, Dr. Elliot Abemayor, whose wise words and loving encouragement have helped her carry this to fruition, and to her children, Alessandra and Charles. BIBLIOGRAPHY 1. Green M, Michaels G, Webber SA, Rowe D, Reyes J. The management of Epstein-Barr virus associated post-transplant lymphoproliferative disorders in pediatric solid-organ transplant recipients. Pediatr Transplantation 1999;3: Mowry SE, Strocker AM, Chan J, et al. Immunohistochemical analysis and Epstein-Barr virus in the tonsils of transplant recipients and healthy controls. Arch Otolaryngol Head Neck Surg 2008;134: Shapiro NL, Strocker AM. Adenotonsillar hypertrophy and Epstein-Barr virus in pediatric organ transplant recipients. Laryngoscope 2001;111: Dror Y, Greenberg M, Taylor G, et al. Lymphoproliferative disorders after organ transplantation in children. Transplantation 1999;67: Gross TG. 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