Epstein-Barr Virus and Posttransplant Lymphoproliferative Disorder in Solid Organ Transplantation

American Journal of TransplantationVolume 13, Issue s4 p. 107-120 Special ArticleFree Access Epstein-Barr Virus and Posttransplant Lymphoproliferative Disorder in Solid Organ Transplantation U. D. Allen, Corresponding Author U. D. Allen Departments of Pediatrics, and Health Policy, Management & Evaluation Research Institute, Hospital for Sick Children Division of Infectious Diseases, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto, Canada Corresponding author: Upton D. Allen, upton.allen@sickkids.caSearch for more papers by this authorJ. K. Preiksaitis, J. K. Preiksaitis Division of Infectious Diseases, Department of Medicine, University of Alberta, Alberta, CanadaSearch for more papers by this authorthe AST Infectious Diseases Community of Practice, the AST Infectious Diseases Community of PracticeSearch for more papers by this author U. D. Allen, Corresponding Author U. D. Allen Departments of Pediatrics, and Health Policy, Management & Evaluation Research Institute, Hospital for Sick Children Division of Infectious Diseases, Department of Pediatrics, Hospital for Sick Children, University of Toronto, Toronto, Canada Corresponding author: Upton D. Allen, upton.allen@sickkids.caSearch for more papers by this authorJ. K. Preiksaitis, J. K. Preiksaitis Division of Infectious Diseases, Department of Medicine, University of Alberta, Alberta, CanadaSearch for more papers by this authorthe AST Infectious Diseases Community of Practice, the AST Infectious Diseases Community of PracticeSearch for more papers by this author First published: 06 March 2013 https://doi.org/10.1111/ajt.12104Citations: 124AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abbreviations ACVBP chemotherapy (doxorubicin, cyclophosphamide, vindesine, bleomycin, prednisone); ANZDATA, Australia and New Zealand Dialysis and Transplant Registry; ATP, adenosine triphosphate; BAL, bronchoalveolar lavage; CHOP, Cyclophosphamide, Hydroxydaunorubicin (also called doxorubicin or Adriamycin), Oncovin (vincristine), Prednisone or prednisolone; CMV, cytomegalovirus; CNS, central nervous system; CT, computerized tomography; CTL, cytotoxic T lymphocyte; EBV, Epstein-Barr virus; ECOG, Eastern Cooperative Oncology Group; HHV6, human herpesvirus type 6; HIV, human immunodeficiency virus; IL6, interleukin 6; IVIG, intravenous immune globulin; LDH, lactate dehydrogenase; PET, positron emission tomography; PNCL, primary central nervous system lymphoma; PTLD, posttransplant lymphoproliferative disorder; RSST, risk stratified sequential treatment; SRTR, Scientific Registry of Transplant Recipients Introduction Posttransplant lymphoproliferative disorder (PTLD) is recognized as potentially one of the most devastating complications of organ transplantation. The Epstein–Barr virus (EBV) genome is found in the majority (>90%) of B cell PTLD occurring early (within the first year) after solid organ transplantation The entity referred to as EBV-associated PTLD encompasses a wide spectrum of clinical conditions characterized by lymphoproliferation after transplantation, which may or may not be symptomatic. These syndromes range from uncomplicated infectious mononucleosis to true malignancies 1-3. Disease may be nodal or extranodal, localized, often in the allograft, or widely disseminated. PTLD may resemble a self-limited infection or be indistinguishable from non-Hodgkin's lymphoma. Lesions may be localized and progress slowly or the patient may present with a fulminant multisystem sepsis-like syndrome. EBV is known to play a major role in the development of PTLD 4. The pathogenesis of these disorders is complex, and related to EBV's ability to transform and immortalize B lymphocytes, sometimes combined with secondary genetic or epigenetic events that occur during uncontrolled proliferation. Host and viral genomics affecting the response to EBV infection, local environmental factors including chronic antigenic stimulation, and the presence of other infections may impact outcome. Immunomodulation caused directly by EBV viral proteins, the coordinated effects of viral and cellular miRNAs 5 and exogenous immunosuppressive drugs alter the proliferative response and survival of infected cells 6, 7 and the innate and adaptive immune responses, particularly the EBV-specific cytotoxic T lymphocyte (CTL) responses critical for controlling EBV infection. Although B cell transformation and PTLD are a result of latent EBV infection, lytic EBV infection appears to be extremely important during primary EBV infection prior to the development of the CTL response 8. For a patient experiencing EBV infection for the first time in the early posttransplant period, delay in development of the immune response theoretically would prolong the one-way self-amplifying circuit of naïve B cell infection, latency in memory cells and reactivation with infectious virus production. The resulting high virion peak results in massive infection of the B cell pool and perhaps other cells not normally infected (T cells, NK cells, memory B cells), thereby setting the stage for secondary events that lead to malignancy. Although the role of EBV in EBV-negative PTLD is uncertain, recent data support the hypothesis that over time, immune escape occurs in initially EBV-driven lymphoproliferation, with cellular mutations replacing the functions of EBV oncogenes 9. This document summarizes current recommendations and supporting data that guide the prevention, diagnosis and treatment of PTLD in the solid organ transplant recipient. The recent literature was reviewed, including recommendations for the diagnosis and management of PTLD that were published by notable groups (e.g. the British Transplantation Society [10, 11]). Although the focus is largely on PTLD, relevant aspects of non-PTLD EBV syndromes are addressed, as appropriate. Epidemiology Humans are the only known hosts of EBV. In immunocompetent individuals, this virus is transmitted in the community by exposure to infected body fluids such as saliva. Although infection may also be acquired in the community by the traditional routes of transmission seen in immunocompetent patients, for solid organ transplant recipients, EBV that is transmitted from the seropositive donor organ is an important source of infection. Transmission is also possible when nonleukoreduced blood products are used. In the least affluent nations, greater than 90% of individuals are EBV-seropositive before the age of 5 years 12. However, in more affluent developed nations, this level of seropositivity is not attained until the fourth decade of life. The diagnosis of PTLD requires tissue examination. In many settings tissue is not available or accessible. When laboratory evidence of EBV infection is present and other causes have been ruled out, investigators have used the term EBV “disease” to describe a number of clinical syndromes where EBV is believed to play a causative role. Although the highest rate of PTLD in the solid organ transplant setting is seen in the first year after transplant, recent analyses suggest that the incidence of early PTLD is decreasing 13, 14. However, cases occurring in the first year after transplant represent only one-fifth of the total cumulative 10-year post transplant PTLD burden 15. Analyses of both French and ANZDATA renal PTLD registries suggest a biphasic pattern of disease with a second peak occurring in years 7–10 after transplant after a period of reduced incidence in years 2–7. A significant proportion of late B cell PTLD is monomorphic and may be EBV-negative (∼20%), with the relative proportion of EBV-negative lesions increasing over time after transplant; NK or T cell PLTD (approximately 37% are EBV positive) may also occur late after transplant 16. As transplant patient survival improves, late and EBV-negative PTLD will represent an increasing proportion of cases seen in adult populations. Although historically the median time of onset of primary EBV infection after solid organ transplantation is 6 weeks and reactivation/infection events were most often observed in the 2–3-month period after transplantation, recent studies in patients monitored serially using EBV viral load, note later initial detection of EBV DNAemia at a median of 110 days 17 and a mean of 276 days 18. PTLD incidence is also dependent on the type of organ transplanted, which may reflect immunosuppressive regimens, lymphoid load in the allograft and chronic antigenic exposure when organs directly communicate with the environment 8. Small intestine transplant recipients are at the highest risk for development of PTLD (up to 32%), while recipients of pancreas, heart, lung and liver transplants are at moderate risk (3–12%). Renal transplant recipients are at relatively low risk (1–2%). Recently, Caillard also described a temporal sequence of sites of PTLD involvement in adult renal allograft recipients, with disease localized to the graft occurring within the first two years, CNS disease occurring between years 2 and 7 and gastrointestinal disease occurring between years 6 and 10 and becoming the predominant site of late disease 13. Although PTLD in solid organ transplant recipients is most often of recipient origin 19, PTLD limited to the graft occurring early after transplant is predominantly donor in origin 20. Risk Factors The risk factors for the development of early (<12 months after transplant) and late PTLD (>12 months after transplant) in solid organ transplant recipients are shown in Table 1 21-24. Analyses of risk factors for PTLD have used both smaller single center and larger registry datasets. Both approaches have limitations and often involve specific subsets of patients, adults versus children or specific allograft types. Many of the risk factors are interrelated and multivariate analysis is required to identify independent risk factors. Even using this approach, results are not always consistent 25. An overwhelming risk factor in most analyses is primary EBV infection, placing pediatric populations at higher risk of developing PTLD than their adult counterparts 14, 26. Surprisingly, in a recent Collaborative Transplant Study database analysis, pretransplant EBV seronegativity in liver transplant recipients, unlike other allograft types, was not associated with an increased risk of developing non-Hodgkin's lymphoma. However, a subsequent analysis of the SRTR data in the United States confirmed that being EBV seronegative was a risk factor for PTLD development even in liver transplant recipients (but less so than in kidney and heart transplant recipients) because of a higher baseline risk in seropositive liver transplant recipients 27. Individuals who are R+ are not devoid of PTLD risk, and account for up to 25% of PTLD cases in children 28. Intestinal transplant recipients who are EBV-seropositive remain at a high risk of PTLD. Although, PTLD rates increased after calcineurin inhibitors became the backbone of most immunosuppressive regimens in the 1990s, it is likely that the net state of immunosuppression, an entity difficult to measure, is a major risk factor. Attempts to quantify the risk associated with specific immunosuppressive agents used for induction or maintenance therapy have often led to inconsistent results 25, 29. Antilymphocyte globulins that result in selective T cell depletion, particularly when used in high dose or repetitive courses, have historically been associated with increased PTLD risk. Among the newer biologic agents, alemtuzumab does not seem to be associated with an increased PTLD risk. Very high rates of PTLD presenting predominantly as primary CNS lymphoma were observed in renal transplant patients who received belatacept and were EBV seronegative prior to transplant, leading to prohibition of the use of this agent in this subset of patients 30-32. The duration of immunosuppression and older recipient age are risk factors for late PTLD development. This highlights the need for studies to optimize minimization of long term immunosuppression in individual patients including the accommodation of immunosenescence associated with aging in patients surviving for long periods after transplant. Cytomegalovirus infection may contribute to the net state of immunosuppression and is known to be a risk factor for PTLD. Table 1. Risk Factors for PTLD in solid organ transplant recipients Early PTLD Primary EBV infection Type of organ transplanted OKT3 and polyclonal antilymphocyte antibodies Young recipient age (i.e. infants and young children) CMV mismatch or CMV disease Late PTLD Duration of immunosuppression Type of organ transplanted Older recipient age (i.e. adults) Contradictory/controversial evidence exists for the role of the following as risk factors for primary disease: Tacrolimus in pediatric recipients; HLA matching; certain cytokine gene polymorphisms; preexisting chronic immune stimulation; Hepatitis C infection; viral strain virulence (EBV1 vs. EBV-2 and LMP1 deletion mutants). Manifestations of Non-PTLD EBV Syndromes Although the most feared EBV-associated disease after transplantation is PTLD, patients may experience non-PTLD-related disease. The features of this might include the manifestations of infectious mononucleosis (fever, malaise, exudative pharyngitis, lymphadenopathy, hepatosplenomegaly and atypical lymphocytosis), specific organ diseases such as hepatitis, pneumonitis, gastrointestinal symptoms and hematological manifestations such as leucopenia, thrombocytopenia, hemolytic anemia and hemophagocytosis. Some of these manifestations may be identical to the features of PTLD (Table 2). EBV-associated posttransplant smooth muscle tumors can occur de novo or after PTLD at a median interval of 48 months after transplant and develop earlier in children than adults. They can be of donor or recipient origin, and appear in atypical sites such as solid organs. When involving multiple sites, disease is multifocal rather than metastatic in origin 33. HHV6 reactivation may theoretically be an indirect cofactor for PTLD due to the potential for interaction with CMV 34. Table 2. Presenting symptoms and signs in patients with lymphoproliferative disorder Symptoms/complaints Signs Swollen lymph glands Lymphadenopathy Weight loss Hepatosplenomegaly Fever or night sweats Subcutaneous nodules Sore throat Tonsillar enlargement Malaise and lethargy Tonsillar inflammation Chronic sinus congestion and discomfort Signs of bowel perforation Anorexia, nausea and vomiting Focal neurologic signs Abdominal pain Mass lesions Gastrointestinal bleeding Symptoms of bowel perforation Manifestations and Diagnosis of PTLD Clinical assessment Relevant clinical information includes, but is not limited to the following: EBV serostatus of transplant recipient and donor. CMV donor/recipient serostatus. Time from transplantation to PTLD diagnosis. Type of allograft. An adequate physical examination is required to detect the manifestations of PTLD, which may be quite nonspecific (Table 2). Given the predilection for the reticuloendothelial system to be involved, this clinical examination should include a meticulous assessment for lymphadenopathy and adenotonsillar hypertrophy. The general physical examination might elicit signs referable to the site(s) of organs affected by PTLD. Laboratory tests Blood tests (Non-EBV) Initial tests include a complete blood count with white blood cell differential. In the case of the latter, lymphopenia might suggest less overall CTL activity, which is essential in containing EBV-driven lymphoproliferation. In some patients with PTLD, there may be evidence of anemia, which is usually normochromic, normocytic, but may be hemolytic. In patients with gastrointestinal tract PTLD and occult bleeding over a prolonged period of time, there may be evidence of iron-deficiency anemia with hypochromia and microcytosis. The source of bleeding can be determined by performing additional testing, such as examination of the stools for occult blood. Thrombocytopenia has also been observed in non-PTLD EBV disease. Depending on the location of PTLD lesions, there may be evidence of disturbances in serum electrolytes, liver and renal function tests. Elevations in serum uric acid and lactate dehydrogenase may occur. Serum immunoglobulin levels may be elevated as part of an acute phase reaction. CMV infection status should be determined using CMV pp65 antigenemia assays, plasma or whole blood quantitative nucleic acid testing for CMV DNA as well as the examination of biopsy tissue for viral inclusions, CMV DNA or CMV antigens by immunohistochemistry. Other adjunctive tests that might predict PTLD risk have been investigated. Promising initial results have been obtained for biomarkers that include serum 1L-6 35, serum/plasma free light chains 36, serum sCD30 37, serum CXCL13 38 and host genetic polymorphisms particularly in cytokine genes 25 but require further validation. How these markers relate to each other and to EBV viral load in predicting PTLD risk should be the subject of future research. Blood tests (EBV-related) EBV serology In immunocompetent patients, primary EBV infection can be determined by measuring EBV antiviral capsid antigen IgM and IgG antibodies, antibodies to early antigen (EA) and Epstein–Barr nuclear antigen. Persistence of anti-EA antibodies has been shown to be more likely in PTLD patients 39 and patients who are known to be seropositive before transplantation may have falling anti-EBNA-1 titers in the setting of elevated EBV loads and the presence of PTLD 40. Serology is unreliable as a diagnostic tool for either PTLD or primary EBV infection in immunocompromised patients, due to delayed or absent humoral responses. Another important drawback is that if these patients are receiving blood products, the passive transfer of antibodies may render EBV IgG antibody assays difficult to interpret. The most important role of EBV serology in the setting of transplantation is the determination of pretransplant donor and recipient EBV serostatus for PTLD risk assessment. Detection of EBV nucleic acids or protein in tissue Documenting the presence of EBV-specific nucleic acids in tissues is of value in the diagnosis of EBV-associated PTLD. RNA in situ hybridization targeting EBV-encoded small nuclear RNA (EBER; Refs. 41, 42) is the preferred approach and is more sensitive for detecting EBV-infected cells than in situ hybridization directly targeting viral DNA because EBERs are expressed at levels several orders of magnitude higher in infected cells. EBV latent or lytic antigens can also be detected in fixed tissues by immunohistochemistry using commercial antibodies directed against EBNA-1, EBNA-2 and LMP-1 or BZLF1, respectively 41, 43 and used to document the presence of EBV although these techniques are less sensitive than in situ hybridization. Direct EBV DNA amplification from tissue is less useful as it does not allow cellular localization or differentiation of EBV in lesions from that present in passenger lymphocytes. Viral load determination The optimal way to perform, interpret and utilize quantitative EBV viral load assays for surveillance, diagnostic and disease monitoring purposes remains uncertain 44. In October 2011, the World Health Organization approved the 1st International Standard for EBV created by the National Institute for Biological Standards and Controls for calibration of the wide array of commercial and in house developed assays currently being used for EBV nucleic acid testing. This international reference standard should reduce the significant and extreme interlaboratory variability in both qualitative and quantitative viral load results previously documented 45, 46. Until the impact of the standard on result harmonization among assays is validated, interinstitutional result comparison requires formal crossreferencing of assays between institutions. Data suggest that in most laboratories intralaboratory result reproducibility and result linearity over the dynamic range of the assay is reasonable. Therefore trends in patients over time within individual institutions using a single assay are valid and more useful than single values 45, 46. Optimal extraction methods, gene targets and instrument platforms for EBV viral load assessments have not been determined. Although EBV viral load in whole blood and lymphocytes appears comparable and normalization of reporting units to cellular DNA does not change dynamic trending in individual patients (reporting IU/mL of whole blood is adequate), controversy with respect to preferred sample type (whole blood vs. plasma) remains and should be the focus of future research studies 47-49. Whole blood or lymphocyte EBV viral load monitoring is more sensitive than plasma for detection of early EBV reactivation. Although, generally, EBV DNA becomes detectable in plasma as EBV viral load rises in matched whole blood samples, the quantitative correlation between EBV viral load measured in whole blood or lymphocytes versus plasma is suboptimal. Studies of the sensitivity and specificity of quantitative EBV viral load for the diagnosis of early PTLD and symptomatic EBV infection are limited 50-53. Pediatric populations have been the focus of many of these studies. Data from prospective studies targeting adult patients are limited 54, 55. In high-risk asymptomatic solid organ transplant recipients being serially monitored, the use of EBV viral load as a diagnostic test (i.e. levels above a specific quantitative threshold being diagnostic of PTLD) has good sensitivity for detecting EBV-positive PTLD but misses EBV-negative, some cases of localized and donor-derived PTLD. However, it has poor specificity, resulting in good negative (greater than 90%) but poor positive predictive value (as low as 28% and not greater than 65%) in these populations. When used in the diagnostic context, this would result in significant unnecessary investigation of patients for PTLD. Formal evaluation of EBV viral load assessments as a diagnostic tool using a single evaluation in patients presenting with symptoms and/or signs (usually mass lesions) with no history of recent or previous monitoring have not been carried out in populations at high risk for PTLD. In low-risk seropositive adult transplant recipients presenting for investigation with signs and symptoms compatible with PTLD, high EBV viral load lacked sensitivity, understandably missing all cases of EBV-negative PTLD and some cases of localized EBV-positive PTLD, but was highly specific for EBV-positive PTLD 52. EBV viral load measured in plasma appears to improve the specificity of the test as a diagnostic tool for EBV-positive PTLD while not significantly lowering its sensitivity relative to assessments in cellular blood compartments 50-53, 56. Preliminary data suggest that EBV viral load testing in samples other than peripheral blood, that is, broncoalveolar lavage (BAL) fluid or CSF may be useful. Among pediatric lung and heart lung transplant patients in whom the lung is often the primary site of PTLD, high quantitative levels of EBV load in BAL fluid may be a more sensitive predictor of PTLD than peripheral viral load assays 57. However, EBV DNA, often at high levels were detected in BAL fluid of adult lung transplant recipients in the absence of PTLD 58. Similarly, extrapolating from experience in HIV-infected patients, qualitative and quantitative EBV testing in CSF is performed to assist in the diagnosis of CNS lymphoma 59. However, further data regarding the sensitivity and specificity of testing in BAL and CSF are required in order to meaningfully interpret testing at these sites. Adjunctive laboratory testing may improve the specificity of high viral load as a predictor of PTLD. The best studied and most promising are assays measuring T cell restoration or EBV-specific T cell responses 60. Although data suggest that the specificity and positive predictive value of EBV viral load can be significantly improved by using concomitant EBV-specific T cell ELISPOT and tetramer assays, these assays are complex, costly and difficult to implement in a routine diagnostic laboratory 10. Simpler rapid assays to measure global and EBV-specific T cell immunity using commercial ATP release assays (Cylex Immuknow and T Cell Memory) have undergone preliminary evaluation as adjunct markers of PTLD risk when combined with viral load testing in pediatric thoracic transplant recipients but require further validation 61. Viral gene expression profiling in peripheral blood as an adjunctive test of PTLD risk has been studied (62) and is still the subject of research. To date no distinctive pattern that is indicative of PTLD or PTLD risk has been demonstrated. Radiographic imaging Most centers employ a total body CT scan (head to pelvis) as part of the initial assessment of PTLD. Beyond this, the choice of tests depends largely on the location of suspected lesions and the historical sequence of prior radiographic testing. Many experts recommend that a head CT or MRI be included as part of the initial work-up, as the presence of central nervous system lesions will significantly influence treatment and outcome. CT scanning of the neck may help to define the extent of involvement or detect subtle early changes that necessitate biopsy to rule out PTLD. Depending on the location (e.g. CNS lesions), MRI may be a more suitable modality than CT scanning due to radiation concerns with CT scans and more precise lesion delineation with MRI. Pulmonary lesions that are visible on chest radiographs may require high-resolution CT scanning for better delineation prior to biopsy. Furthermore, CT of the chest may reveal mediastinal adenopathy and small pulmonary nodules that are not visible on the plain chest radiograph. Suspected intra-abdominal lesions may be evaluated with ultrasonography and CT scanning. This is in addition to other modalities of assessment, including GI endoscopy in the case of intestinal hemorrhage, persistent diarrhea and unexplained weight loss, where necessary. Positron emission tomography–computerized tomography (PET–CT) is emerging to be a useful test in the evaluation of PTLD 63, 64, although additional data are needed on its utility across the known heterogenous spectrum of PTLD lesions. It may be more useful for monitoring response to therapy than for initial diagnosis. A major disadvantage is that the amount of radiation exposure is significantly greater than that associated with regular CT scans. Histopathology Pathology remains the gold standard for PTLD diagnosis 2, 65. Although excisional biopsy is preferred, needle biopsy is acceptable when larger biopsies are impractical as in the case of allograft organ biopsy. The tissue specimen should be interpreted by a hematopathologist or pathologist familiar with histopathologic features of PTLD. Institutional protocols should be put in place to ensure that tissue is handled appropriately for ancillary diagnostic tests. It is essential that reactive conditions such as plasma cell hyperplasia and infectious mononucleosis be clearly segregated in the classification process from potentially neoplastic lesions, which contain monoclonal elements. The Society for Hematopathology has published a working categorization of PTLD under the auspices of the World Health Organization 65 and is recommended for use (III). Table 3 summarizes the key features of this classification system. Intrinsic weaknesses are present in the purely histologic classification of PTLD. Additional pathologic tools have provided a better understanding of the pathogenesis of PTLD with the goal of developing more effective and more targeted therapy. Use of ancillary diagnostic tests identified as essential is strongly recommended if available (AIII). In addition to EBER and the detection of latent antigens as outlined previously, these tests are as follows: Immunophenotyping to determine lineage and therapy dependent markers (i.e. CD20) (essential). EBV clonality studies (rarely required/research). Molecular genetic markers of antigen receptor genes to assess clonality (useful). Donor versus recipient origin (useful). Fluorescent in situ hybridization or gene profiling by microarray to detect alterations in oncogenes, tumor suppressor genes or chromosomes (rarely required/research). Table 3. Categories of posttransplant lymphoproliferative disorder (PTLD) Early lesions11Some mass-like lesions in the posttransplant setting may have the morphologic appearance of florid follicular hyperplasia or other marked but non-IM-like lymphoid hyperplasias. Plasmacytic hyperplasia Infectious mononucleosis-like lesion Polymorphic PTLD Monomorphic PTLD (classify according to the lymphoma they resemble) B cell neoplasms Diffuse large B cell lymphoma Burkitt lymphoma Plasma cell myeloma Plasmacytoma-like lesion Other22Indolent small B cell lymphomas arising in transplant recipients are not included among the PTLD. T cell neoplasms Peripheral T cell lymphoma, NOS Hepatosplenic T cell lymphoma Other22Indolent small B cell lymphomas arising in transplant recipients are not included among the PTLD. Classical Hod