* Posttransplant lymphoproliferative disorders are often accompanied by >500 Epstein-Barr virus (EBV) genome copies/10^sup 5^ lymphocytes, and they occur shortly after transplantation. Hodgkin lymphoma occurs rarely after transplantation, appearing a mean of 4.2 years posttransplant, and although Hodgkin lymphoma has strong associations with EBV, no quantitative analysis of peripheral blood EBV genome copies has been reported. A mixed cellularity Hodgkin lymphoma developed in a 17-year-old boy 4 years after a renal transplant. Serial EBV genome copy numbers from blood by competitive polymerase chain reaction had been obtained to assess for lymphoproliferative disease. Epstein-Barr virus genome copy numbers peaked at 500 copies/10^sup 5^ lymphocytes 8 months prior to Hodgkin lymphoma diagnosis but fell to 8 copies/10^sup 5^ lymphocytes at diagnosis. Reliance on EBV levels greater than 500 copies may result in delay of biopsy and diagnosis of Hodgkin disease in the posttransplant setting.
(Arch Pathol Lab Med. 2001;125:1480-1482)
Hodgkin lymphoma, although one of the more frequent lymphomas encountered is rare in the spectrum of lymphoproliferative disorders that follow renal transplantation. Epstein-Barr virus (EBV) plays a role in both posttransplant lymphoproliferative disorders and Hodgkin lymphoma. Epstein-Barr virus posttransplant lymphoproliferative disorders are usually accompanied by greater than 500 EBV genome copies/10^sup 5^ lymphocytes.1 Although EBV has been detected in the blood of patients with EBVassociated Hodgkin lymphoma,2 there are no reports quantitating genome copy in Hodgkin lymphoma as there have been in the posttransplant lymphoproliferative disorders.
A 17-year-old boy received an allograft kidney at age 13. Multiple episodes of EBV reactivation led to decreased immunosuppressive therapy and serial quantitations of blood EBV genome copies. Four years after transplantation, he developed Hodgkin lymphoma despite decreased immunosuppressive therapy and decreasing blood EBV genome copies.
REPORT OF A CASE
At 13 years of age, a white male child presented with chronic renal failure and was diagnosed with end-stage renal disease. Diagnoses of right renal agenesis and left renal hypoplasia were based on renal ultrasound. Epstein-Barr virus, hepatitis A, and cytomegalovirus serologies indicated prior exposure. Herpes simplex I and II and hepatitis B and C serologies were negative. After 3 months on hemodialysis, he underwent a 3-antigen-match, living related donor renal transplant. His induction of immunosuppression therapy included antithymocyte globulin, which was discontinued after 3 doses because of a skin reaction. Other initial immunosuppressive agents consisted of prednisone, azathioprine, and cyclosporin microemulsion. Three weeks after transplantation the patient experienced an acute rejection episode, which was treated with a 7-day course of monoclonal CD3 antibodies (OKT3) with good results. Two months after transplantation, his immunosuppression regime was changed to prednisone and tacrolimus.
Eighteen months after transplantation, a febrile illness was diagnosed as reactivation of latent EBV, based on an elevated immunoglobulin (Ig) M titer. Over the ensuing 2 years, he had recurrent fevers presumed to be caused by EBV disease, and he was treated with ganciclovir and decreased immunosuppressive drugs. During this time, he had multiple abdominal, chest, and head computed tomographic scans; the only abnormality was a slightly enlarged spleen and small para-aortic abdominal lymph nodes, which were not biopsied.
At 4 years posttransplantation, the patient again developed recurrent fevers, this time with axillary and pulmonary hilar lymphadenopathy. The spleen was enlarged, but imaging scans showed no discrete masses. Bone marrow and axillary lymph node biopsies both contained mixed cellularity Hodgkin lymphoma. Treatment for stage IVB Hodgkin lymphoma was begun, consisting of cytoxan, oncovin, prednisone, and procarbazine alternating with adriamycin, bleomycin, and vinblastine. The patient achieved complete remission status after 3 courses of chemotherapy and is in remission 17 months after diagnosis. Renal function has remained stable, with no acute rejection episodes.
MATERIALS AND METHODS
All tissues for histology were initially fixed in B-5 (Columbia Diagnostics, Springfield, Va) and were then transferred to 10% neutral buffered formalin, processed routinely, and embedded in paraffin. Antibodies for immunoperoxidase studies included anti-CD30, clone 1612, prediluted (Novocastra Laboratories, Newcastle-Upon-Tyne, UK); anti-CD15, clone MMA; anti-LCA, clone Rp2/18; anti-CD3, polyclonal; and anti-CD20, clone L26 (Ventana, Tucson, Ariz), all prediluted. CD30, CD15, and CD3 required heat epitope enhancement (citrate buffer, pH 6.8). Immunoperoxidase techniques used avidin-biotin reaction and were performed on the Ventana NexES automated immunostainer. For demonstration of the Epstein-Barr virus-encoded small RNAs (EBER), an in situ hybridization kit (K5201, Dako Corporation, Carpinteria, Calif) was used. Prior to hybridization the sections were incubated with a 1:10 dilution of proteinase K (Dako). All immunoperoxidase stains and in situ hybridizations were performed on lymph node and bone marrow.
Nodal tissue for ploidy status was studied by image analysis. Fifty-micrometer sections of paraffin-embedded, 10% neutral buffered formalin (not B-5)-fixed tissues were dewaxed and rehydrated, and a whole nuclei prep was made by digesting with trypsin. The suspension was placed on a superfrost-plus slide, treated with RNase and propidium iodide, and analyzed on the laser scanning cytometer (CompuCyte Corporation, Cambridge, Mass) using WinCyte Version 3.3 software. The first peak in the gated histogram of red fluorescence value is defined as the diploid GO/Gl population. An interpretation of aneuploidy requires a separate and distinct peak to the right of the diploid GO/Gl peak population. The methodology allows discrimination of DNA subpopulations differing by as little as 5% in total DNA (ie, it can distinguish aneuploid peaks greater than 1.05).
For the year prior to the diagnosis of Hodgkin lymphoma, monthly EBV genome copies by quantitative competitive polymerase chain reaction assay were obtained commercially from the Department of Infectious Disease and Microbiology at the University of Pittsburgh.'
Three months after transplantation, EBV titers became elevated (IgM, 1:160; IgG, 1:1280), peaking at 1.5 years posttransplant (IgM, 1:80; IgG, 1:10240). Allograft biopsy at 18 months posttransplant demonstrated mild acute rejection (moderate lymphocytic tubulitis). The interstitial lymphoid infiltrate was a mixture of T and B cells, with approximately 20% of the lymphocytic nuclei positive for EBER. There was no nuclear enlargement, nuclear atypism, immunoblasts, or serpiginous necrosis.
Epstein-Barr virus genome quantitation by polymerase chain reaction in blood became available 3 years posttransplant. Levels peaked at 500 genomic copies/10^sup 5^ lymphocytes, then decreased over the next 8 months as immunosuppressive drugs were tapered (Table). At 4 years posttransplantation, the bone marrow biopsy and axillary lymph node both contained an abnormal infiltrate composed of a mixture of inflammatory cells with frequent Reed-Sternberg (RS) cells and their variants (Figure 1). In both the lymph nodes and bone marrow, the RS cells stained with CD30 (prominent golgi zone pattern), CD15 (prominent golgi zone staining with occasional delicate membrane staining), and EBER (nuclear pattern) (Figure 2), but they did not stain with LCA, CD20, or CD3. Based on the histology, along with the supportive immunoperoxidase reactions, stage IV mixed cellularity Hodgkin lymphoma was diagnosed.
On image analysis, RS cells made up 1.3% of the total cells analyzed. Gating on this subpopulation demonstrated a DNA index of 1.1 (aneuploid), but the low numbers precluded estimation of the proliferative activity.
The renal biopsy at this time showed mild interstitial fibrosis without acute rejection. The few lymphocytes present in the kidney did not stain with EBER. Epstein-Barr virus genome copies in peripheral blood were reduced to 8 copies/105 lymphocytes.
Epstein-Barr virus has long been associated with lymphoproliferative diseases in transplant recipients. Evidence supports that this proliferation of B cells is driven by the infecting EBV and escape T-cell control.3 Many of the lymphoproliferative disorders will develop within the first year after transplantation, decreasing in occurrence after this time. Epstein-Barr virus is also associated with Hodgkin lymphoma, being demonstrated in 50% to 60% of sporadic cases, but it contributes a much higher percentage in the Hodgkin lymphoma associated with immunosuppression, especially human immunodeficiency virus.4 Hodgkin lymphoma can arise after transplantation, but it is much rarer than the other lymphoproliferative disorders, comprising only 2% of posttransplant lymphomas, as compared to 18% of lymphomas in the general population.5 Since the introduction of cyclosporin, an immunosuppressant for transplantation, this figure may even be lower.6
Hodgkin lymphoma has been reported in 19 patients after renal transplantation, and the disease has followed the transplantation by 4.2 years on average.5-9 There is a marked male predominance (17 of 19). The frequency of the subtypes were 9 mixed cellularity, 4 lymphocyte depletion, 2 nodular sclerosis, and 1 lymphocyte predominant, with no subclass provided in 3 reports. This contrasts with the general population, in which nodular sclerosis is the most frequent subtype. It has been suggested that these transplant patients present more often with disseminated disease and do not do as well as individuals with sporadic Hodgkin lymphoma, but the coexistent renal disease and small number of patients are confounding issues. EBER and/or EBV latent membrane protein have been demonstrated in the RS cells in 11 of the 13 postrenal transplant Hodgkin lymphomas (7 have demonstrated both, 3 latent membrane protein, and 1 EBER) in which EBV was assessed histologically.
Immunohistochemical stains facilitated identification of the RS cells as true RS cells rather than RS-like cells, which are frequently encountered in lymphoproliferative disorders. Reed-Sternberg cells show golgi staining with CD15 and CD30 but are negative for the T- and B-cell markers. The RS-like cells in posttransplant lymphoproliferative disorders are most often CD20 positive, with no staining by CD15 or CD30.
The 20% EBV-positive lymphoid cells in the biopsy at 18 months posttransplant is higher than the up to 10% positive cells described in a variety of inflammatory conditions. This could represent a very early lymphoproliferative disorder, like that described by Bierman et al.9 Four years following a liver transplant, that child developed a polymorphous immunoblastic proliferation that responded to ganciclovir. Eighteen months later Hodgkin lymphoma developed. However, EBV is usually detected in 50% to 80% of the lymphoid cells (if detected at all) that compose a lymphoproliferative infiltrate.10 In the child discussed here, there was no other histologic feature of a lymphoproliferative disorder, and we interpreted this 20% EBV lymphoid positivity as a reflection of an EBV infection in an immunocompromised host.
Although no specific cytogenetic abnormality has been described with Hodgkin lymphoma, aneuploid populations have been detected by both flow cytometry and image analysis. Although flow cytometry has been more sensitive in detecting aneuploidy, because of the larger number of cells evaluated, image analysis has allowed better resolution of the type of cell analyzed (ie, reactive lymphocyte vs RS cell). This has allowed detection of diploid, hyperdiploid, and aneuploid peaks, with aneuploid peaks maintaining their identity on multiple biopsies, supporting a hypothesis that Hodgkin lymphoma may contain multiple or serial subpopulations of clonal Reed-Sternberg cells simultaneously.11
Usually, increasing or high numbers of copies of EBV genome determined by polymerase chain reaction in blood is associated with an increased chance of developing a lymphoproliferative disease.12 In this young man, EBV genome copies were decreasing when he developed Hodgkin lymphoma. Falling numbers of EBV genome copies reflected improving immunocompetence, as he was off most immunosuppressive drugs. Based on this case report, one cannot rely on EBV levels to suggest the presence of Hodgkin lymphoma, at least in the posttransplant setting.
The possibility of Hodgkin lymphoma arising de novo in this child must be considered. Epstein-Barr virus can be detected in the RS cell in about half of the de novo cases. The temporal distance from transplantation of 4 years may also support the theory that the Hodgkin lymphoma developed de novo. However, the predominance of mixed cellularity Hodgkin lymphoma instead of nodular sclerosing Hodgkin lymphoma in the posttransplant setting suggests some influence exerted by the transplantation process.
1. Rowe DT, Qu L, Reyes J, et al. Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders. J Clin Microbiol. 1997;35: 1612-1615.
2. Gallagher A, Armstrong AA, MacKenzie J, et al. Detection of Epstein-Barr virus (EBV) genomes in the serum of patients with EBV-associated Hodgkin's disease. Intl Cancer. 1999;20:442-448.
3. Opelz G, Henderson R. Incidence of non-Hodgkin's lymphoma in kidney and heart transplant recipients. Lancet. 1993;342:1514-1516.
4. Jarrett RF, MacKenzie J. Epstein-Barr virus and other candidate viruses in the pathogenesis of Hodgkin's disease. Semin Hematol. 1999;36:260-269.
5. Oldhafer KJ, Bunzendahl H, Frei U, Kemnitz J, Vogt P, Pichlmayr R. Primary Hodgkin's lymphoma: an unusual cause of graft dysfunction after kidney transplantation. Am] Med. 1989;87:218-220.
6. Gamier JL, Lebranchu Y, Dantal J, et al. Hodgkin's disease after transplantation. Transplantation. 1996;61:71-76.
7. Goyal RK, McEvoy L, Wilson DB. Hodgkin's disease after renal transplantation in childhood. J Pediatr Hematol Oncol. 1996;18:392-395.
8. Birkeland SA, Andersen HK, Hamilton-Dutoit SJ. Preventing acute rejection, Epstein-Barr virus infection, and post transplant lymphoproliferative disorders after kidney transplantation: use of aciclovir and mycophenolate mofetil in a steroid-free immunosuppressive protocol. Transplantation. 1999;67:1209-1214.
9. Bierman PJ, Vose JM, Langnas AN, et al. Hodgkin's disease following solid organ transplantation. Ann Oncol. 1996;7:265-270.
10. Randhawa PS, Magnone M, Jordan M, Shapiro R, Demetris AJ, Nalesnik M. Renal allograft involvement by Epstein-Barr virus associated post-transplant lymphoproliferative disease. Am J Surg Pathol. 1996;20:563-571.
11. Haber MM, Liu J, Knowles DM, Inghirami G. Determination of the DNA content of the Reed-Sternberg cell of Hodgkin's disease by image analysis. Blood. 1992;80:2851-2857.
12. Riddler SA, Breinig MC, McKnight JLC. Increased levels of circulating Epstein-Barr virus (EBV)-infected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of post-transplant lymphoproliferative disease in solid-organ transplant recipients. Blood. 1994;84:972984.
Randall D. Craver, MD; Wm. Douglas Scheer, PhD; Hernan Correa, MD; V. Matti Vehaskari, MD; Lolie C. Yu, MD
Accepted for publication April 24, 2001.
From the Departments of Pathology (Drs Craven Scheer, and Correa) and Pediatrics (Drs Craven Vehaskari, and Yu), Louisiana State University Medical Center, and Children's Hospital (Drs Craven Scheer, Correa, Vehaskari, and Yu), New Orleans, La.
Reprints: Randall D. Craven MD, Laboratory, Children's Hospital, 200 Henry Clay Ave, New Orleans, LA 70118 (e-mail: rcrave@ Isuhsc.edu).
Copyright College of American Pathologists Nov 2001
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