¹Department of Hematology, National Kyushu Cancer Center Hospital, Fukuoka, Japan

²First Department of Pathology, School of Medicine, Fukuoka University, Fukuoka, Japan

Correspondence to: Dr Y Yufu, Department of Hematology, National Kyushu Cancer Center Hospital, Notame 3-1-1, Minami-ku, Fukuoka 811-1395, Japan

Post-transplant lymphoproliferative disorders (PTLD) of T cell type are a rare complication of solid organ and allogeneic hematopoietic cell transplantation (HCT), and usually are not associated with Epstein-Barr virus (EBV) infection. EBV-associated T cell PTLD has not been reported to occur after autologous HCT. We report an unusual case of T cell lymphoproliferation after autologous blood stem cell transplantation (ABSCT). A patient with relapsed Hodgkin's disease developed abdominal lymphadenopathy followed by atypical CD8⁺lymphocytosis in the peripheral blood 30 months following ABSCT. DNA studies of the atypical lymphocytes demonstrated rearrangements of the T cell receptor gene and a clonal proliferation of EBV. Bone Marrow Transplantation (2000) 26, 1339-1341.

post-transplant lymphoproliferative disorder; T cell; Epstein-Barr virus; autologous blood stem cell transplantation

Post-transplant lymphoproliferative disorders (PTLD) are a serious complication that occur in immunocompromised recipients of solid organ and allogeneic hematopoietic cell transplants (HCT).^1,2 PTLD are virtually always of B cell origin; PTLD of T cell origin is rare.^3,4,5 Also, while B cell PTLD are usually associated with Epstein-Barr virus (EBV) infection, most T cell PTLD are not associated with the EBV genome.^3,4,5

Three cases of T cell PTLD have been reported as occurring after allogeneic HCT, all unassociated with EBV infection;³ T cell PTLD after autologous HCT has not been reported. We describe an unusual case of T cell lymphoproliferation following autologous blood stem cell transplantation (ABSCT), in which DNA studies in the tumor cells demonstrated a monoclonal proliferation of T cells infected with EBV.

Case report

A 48-year-old male with bilateral cervical lymphadenopathy was diagnosed in 1992 with Hodgkin's disease (lymphocyte-rich classical type; clinical stage IIA). The patient achieved remission with doxorubicin-based chemotherapy, but the cervical lymphadenopathy recurred 2 years later. A repeat lymph node biopsy yielded a specimen with a histologic appearance similar to that at presentation. No rearrangements of the T cell receptor (TCR) or immunoglobulin heavy chain (IgH) genes were seen. After harvesting of blood stem cells mobilized by high-dose Ara-C and G-CSF (filgrastim), the patient underwent ABSCT and entered a second remission. The conditioning regimen included cyclophosphamide, 200 mg/kg; etoposide, 900 mg/m² and ranimustine, 400 mg/m². Unmanipulated blood stem cells were administered at a total CFU-GM count of 2.0 ´ 10⁵/kg. The patient experienced an uncomplicated recovery in the immediate post-transplantation period, with a neutrophil count >0.5 ´ 10⁹/l on day 8 and a platelet count >5 ´ 10¹⁰/l on day 9.

Thirty months after ABSCT, he was hospitalized for post-prandial vomiting, diarrhea and abdominal pain. Physical examination showed no lymphadenopathy. The serum contained elevated concentrations of soluble interleukin-2 receptor (1290 U/ml; normal <520). Multiple enlarged mesenteric lymph nodes were observed on computed tomography. Serum titers of antibodies against EBV were as follows: VCA IgG, 1:1280; VCA IgM, <1:10; VCA IgA, 1:20; EA-DR IgG, <1:10; EA-DR IgA, <1:10; and EBNA, 1:40. (Before ABSCT, the VCA IgG had been 1:80 and EBNA 1:40, with all the others negative.) The patient was seronegative for both human T cell lymphotropic virus type I (HTLV-I) and human immunodeficiency virus (HIV).

Histologic examination of mesenteric lymph nodes obtained at laparotomy showed lymphoid hyperplasia with mild sinus histiocytosis as well as proliferation of epithelioid cells. No Hodgkin's cells or Reed-Sternberg cells were observed, indicating that recurrence of the Hodgkin's disease was unlikely. Four months later, a leukocytosis (13.8 ´ 10⁹/l) with 58% atypical lymphocytes developed, accompanied by fever, sore throat and abdominal pain. Surface marker profile of the atypical lymphocytes was CD2⁺3⁺4^-5⁺8⁺19^-21^-25^-30^-56^-DR⁺TCR⁺. The karyotype of the blood cells was normal. EBV DNA (IR1) was detected in the serum by polymerase chain reaction. Many cells stained for EBER-1 (EBV-encoded small nuclear RNAs) by in situ hybridization performed on mesenteric lymph node tissue obtained 4 months earlier. Clonal rearrangement of the TCR gene was detected in DNA from peripheral blood mononuclear cells including atypical lymphocytes (Figure 1a). No rearrangement of the IgH gene was seen. Southern blot analysis of the EBV terminal repeat gene clearly showed that the atypical lymphocytes were clonally infected by an episomal form of EBV (Figure 1b). Despite administration of ganciclovir, aciclovir, -globulin, and vinblastine, abdominal lymphadenopathy and fever persisted. The patient died of septic shock 9 months after admission. Permission for autopsy was not granted.

Discussion

The presence of a clonal EBV strain infecting CD8⁺ T cells during the course of fatal infectious mononucleosis has been described.⁶ This probably was not the situation in our patient, since infectious mononucleosis is caused principally by a first infection with EBV, and the pattern of antibodies against EBV prior to ABSCT in this patient indicated that previous infection with EBV had occurred. Clonal lymphocytosis may occur after allogeneic and autologous HCT, possibly associated with known or unknown infections. Thus, the clonal expansion of CD8⁺ T cells in our patient may have been a reaction to agents other than EBV. From this point of view, the clonality pattern of EBV infection could have resulted simply from coincidental latent EBV infection of CD8⁺ T cells that clonally expanded in response to an exogenous stimulus. However, EBV was detected not only in clonal CD8⁺ T cells but also in lymph node tissue and serum. This indicates that the EBV had reactivated, and the reactivation caused clonal CD8⁺ T cell proliferation in our patient.

Although monoclonal lymphoproliferation was not evident in the patient's mesenteric lymph nodes, the atypical lymphocytes in the blood appeared to represent a monoclonal proliferation of EBV-infected CD8⁺ T cells. PTLD can occur as a polyclonal, oligoclonal, or monoclonal proliferation of lymphoid cells.⁷ An interval of 4 months separated the biopsy of the mesenteric lymph nodes and the appearance of atypical lymphocytes in the peripheral blood in our patient. Early in the course of PTLD, proliferation of EBV-infected cells in the lymph nodes may have been polyclonal, while subsequent genetic changes may have resulted in monoclonal proliferation.⁷ Alternatively, events in this patient may have been similar to those in patients with a severe chronic active EBV infection, where a monoclonal proliferation of EBV-infected T cells may be detected in the blood even when no evidence of lymphoma exists.⁸

The EBV-associated T cell lymphoproliferation seen in our patient may have been related to underlying Hodgkin's disease, which is known to cause immunodeficiency. However, no persistence or recurrence of Hodgkin's disease was demonstrated on histologic examination of lymph node biopsy specimens. Therefore, the disease was considered to be related to an immunodeficient state cause by ABSCT per se.

Several cases of EBV-associated PTLD following autologous HCT have been reported, but all involved B cells.^{9,10,11,12,13,14} These included cases where in vitro purging of the bone marrow with anti-T cell antibodies was performed,^9,12 or where a CD34-purified graft was infused;¹³ this suggests that T cell depletion of the graft increases the risk for developing PTLD after autologous HCT, as is the case after allogeneic HCT.² However, PTLD has occurred in cases where no purging was carried out,^11,14 or where only anti-B cell antibodies were used for purging.¹⁰ The present case of T cell PTLD developed after an ABSCT where the graft contained abundant T cells. The specific risk factors associated with PTLD following autologous HCT remain to be defined.

The prognosis of T cell PTLD, whether associated with EBV infection or not, appears to be generally poor.^3,4,5 Our patient did not respond to antiviral agents, -globulin, or a chemotherapeutic agent. Donor T cell infusion has been reported to be effective for treating EBV-associated B cell PTLD after allogeneic HCT.^1,2 Whether analogous approaches could be effective against EBV-associated T cell PTLD remains to be clarified.

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6 Mori M, Kurozumi H, Akagi K et al. Monoclonal proliferation of T cells containing Epstein-Barr virus in fatal mononucleosis. New Engl J Med 1992; 327: 58, MEDLINE

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9 Young L, Alfieri C, Hennessy K et al. Expression of Epstein-Barr virus transformation-associated genes in tissues of patients with EBV lymphoproliferative disease. New Engl J Med 1989; 321: 1080-1085, MEDLINE

10 Chao NJ, Berry GJ, Advani R et al. Epstein-Barr virus-associated lymphoproliferative disorder following autologous bone marrow transplantation for non-Hodgkin's lymphoma. Transplantation 1993; 55: 1425-1428, MEDLINE

11 Shepherd JD, Gascoyne RD, Barnett JD et al. Polyclonal Epstein-Barr virus-associated lymphoproliferative disorder following autografting for chronic myeloid leukemia. Bone Marrow Transplant 1995; 15: 639-641, MEDLINE

12 Briz, M, Forés R, Regidor C et al. Epstein-Barr virus associated B cell lymphoma after autologous bone marrow transplantation for T cell acute lymphoblastic leukaemia. Br J Haematol 1997; 98: 485-487, MEDLINE

13 Peniket AJ, Perry AR, Williams CD et al. A case of EBV-associated lymphoproliferative disease following high-dose therapy and CD34-purified autologous peripheral blood progenitor cell transplantation. Bone Marrow Transplant 1998; 22: 307-309, MEDLINE

14 Hauke RJ, Greiner TC, Smir BN et al. Epstein-Barr virus-associated lymphoproliferative disorder after autologous bone marrow transplantation: report of two cases. Bone Marrow Transplant 1998; 21: 1271-1274, MEDLINE

Figure 1 Southern blot analysis of the T cell receptor (TCR) gene and EBV terminal repeat (TR) gene. (a) TCR gene. DNA extracted from the peripheral blood mononuclear cells (PBMC) of a normal subject (left) and the patient (right) was digested with EcoRI(E), BamHI(B), and HindIII (H), then probed with a BglII-EcoRV fragment of YT35 (TCR C1). Germline and rearranged bands are indicated by arrows and asterisks, respectively. In the patient's atypical lymphocytes, a rearranged band is seen in each lane. (b) EBV TR gene. In cells with latent infection, the linear EBV genome is circularized by joining of its TR sequences to form an episome that subsequently is amplified in copy number, undergoing replication as the cell undergoes mitosis. Variable numbers of tandem-repeat sequences are present at each linear terminus. Therefore, the presence of EBV genomes and the clonal nature of the EBV-infected cells can be assessed with a DNA probe specific for one of the termini of the EBV genome. Southern analysis with this probe detects a single band in monoclonal tumors infected with EBV, while DNA representing polyclonal processes involving EBV shows multiple ladder bands. Lane 1, cervical lymph node at relapse of HD; lane 2, mesenteric lymph node; and lane 3, PBMC containing atypical lymphocytes. DNA was digested with EcoRI, and a 5.2-kb BamHI-EcoRI fragment containing TR was used as a probe (EBV Xho1a, kindly provided by Dr K Hirai, Department of Virology and Immunology, Tokyo Medical and Dental University). A single monoclonal band is detected in lane 3, as well as in a positive control (arrows). The position of the bands differ because it depends on the number of TR in the EBV genome that is present in each tumor. No clonal bands were seen in lanes 1 or 2. The numbers on the left denote DNA fragment sizes (in kilobases).