Triple negative (CD3/CD4/CD8) adult T cell leukemia/lymphoma, histologically presenting as CD30 (Ki-1)-positive anaplastic large cell lymphoma with clonal Epstein–Barr virus genome

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The lymphotrophic viruses, Epstein–Barr virus (EBV) and human T cell leukemia virus type I (HTLV-I), play pathogenetic roles predominantly in lymphoid neoplasia. Adult T cell leukemia/lymphoma (ATLL) is associated with HTLV-I, which usually infects CD3+/CD4+/CD25+/HLA-DR+ mature T cells. Definitive diagnosis of ATL is made by documenting the presence of HTLV-I proviral DNA in the DNA of leukemic or lymphoma cells. On the other hand, EBV has been thought to be etiologically linked to human malignancies such as Burkitt's lymphoma and nasopharyngeal carcinoma. Recent studies have suggested that this virus plays a causative role in chronic active EBV infection syndrome, EBV-related hemophagocytic lymphohistiocytosis, EBV genome-positive T cell lymphoma, natural killer cell leukemia/lymphoma, Hodgkin's disease and gastric carcinoma. We describe here a patient with triple-negative (CD3/CD4/CD8) ATLL, histologically presenting as CD30 (Ki-1)-positive anaplastic large cell lymphoma (ALCL). Clonal EBV genome was also detected in lymphoma cells of this patient.

A 54-year-old man was admitted to Osaka City University Hospital because of left neck tumors. Based on histological findings for a biopsied lymph node, ALCL was diagnosed. Surface marker analysis revealed that malignant cells were positive for CD25 (77.3%) and CD30 (40.8%), but negative for CD2 (1.1%), CD3 (7.6%), CD4 (15.4%), CD5 (10%), and CD8 (1.3%). On immunohistochemical studies, lymphoma cells were CD45 (LCA) (±), CD20 (L-26) (−), CD45RO (UCHL-1) (+), CD30 (Ki-1) (+), and epithelial membrane antigen (EMA) (+). Chimeric ALK-NPM gene was not detected by polymerase chain reaction (PCR). No atypical cells were present in peripheral blood or bone marrow. Serum LDH level was high (919 IU/l) and serum Ca levels were within normal limits (4.5 mEq/l). Serum antibody against HTLV-I was positive by a routine particle agglutination method (titer of 4096) and Western blot analysis. Southern blot analysis was performed using DNA extracted from the biopsied lymph node. Monoclonal integration of HTLV-I proviral DNA was detected using ScaI fragment (8.25 kb) of HTLV-I proviral DNA as a probe (Figure 1). HTLV-I was also detected in the lymphoma cells by PCR in situ hybridization (ISH) using pX gene primers. Evaluation of T cell receptors by Southern blot analysis using human TCR-Cβ1 probe (Pr-1; Calbiochem, La Jolla, CA, USA) revealed rearrangement of the TCR-Cβ gene (Figure 1). The patient was finally diagnosed with triple-negative (CD3/CD4/CD8) ATLL, histologically presenting as CD30 (Ki-1)-positive ALCL. Furthermore, EBV-encoded RNA-1 (EBER-1) was detected in the lymphoma cells by ISH. We investigated the clonal nature of EBV in the patient by Southern blot analysis using BamHI–EcoRI fragment (4.6 kb) containing the tandem terminal repeated sequences of the EBV genome, which provides a virus-specific, clonotypic marker for the clonal pathogenesis of EBV-associated tumors. A single band was detected, which demonstrated clonal proliferation of EBV infected cells (Figure 1). Complete remission was obtained after chemotherapy with cyclophosphamide, doxorubicin hydrochloride, vincristine, and prednisolone (CHOP). However, the patient died of recurrence of lymphoma 8 months after admission.

Figure 1

Southern blot analysis of HTLV-I proviral DNA, T cell receptor gene, and EBV genome. Genomic DNA was extracted from biopsied lymph node and was digested with EcoRI (E), PstI (P), EcoRV (R), BamHI (B), and HindIII (H) restriction endonucleases. The digested DNA (5 μg) was electrophoresed through 0.8% agarose gels and transferred to nylon membranes. The blots were hybridized to probes (a) ScaI fragment of HTLV-I proviral DNA, (b) TCR Cβ1 DNA, (c) BamHI–EcoRI fragment of EBV terminal repeated sequences) labeled with 32P by random primer method. 1: ATL cell line, 2: placenta, 3: the presented case.

ALCL is essentially defined by features including proliferation of large lymphoid cells with strong expression of CD30 (Ki-1) antigen, which was reported by Schwab et al1 to be specific to Hodgkin cells and Reed–Sternberg cells. ALCL is now widely recognized to have a broad morphologic and phenotypic spectrum. It is closely associated with the expression of chimeric NPM-ALK protein activated by the (2;5)(p23;q35) chromosomal translocation. NPM/ALK-positive ALCL represents a distinct genetic entity that occurs in young patients, has a favorable prognosis, and should be differentiated from the NPM/ALK-negative tumors including ATLL, which have a relatively aggressive clinical course. It has been reported that monoclonal integration of HTLV-I proviral DNA was detected in 39.5% of cases of CD30 (Ki-1)-positive ALCL in an HTLV-I endemic area.2 Most of the ALCL patients with HTLV-I proviral DNA exhibited frequent lymphadenopathy and extranodal large tumor or nodules, but a few exhibited leukemic changes, bone marrow involvement, and hypercalcemia. The latter patients had a very poor prognosis despite immediate performance of chemotherapy. Some of these clinical features differ from those of ATLL. These results suggest that ALCL with HTLV-I proviral DNA may be a unique histologic subgroup of ATLL. The relationship between ALCL and HTLV-I infection has not been clarified. CD30 (Ki-1) antigen, an activated T cell antigen, is a member of the tumor necrosis factor/nerve cell growth factor receptor super-family. The pX region of HTLV-I, which has been shown to encode for the transacting regulator gene (tax and rex), might play an important role in expression of CD30 antigen. T cell markers of CD2, CD4, and/or CD3 were expressed in 88.2% of cases of ALCL with HTLV-I proviral DNA.2 In the present case, T cell markers (CD2, CD3, CD4, CD5, and CD8) were not expressed. The incidence of double-negative (CD4/CD8) ATL was 7%,3 while it has been reported that lack of CD2, CD3, and CD5 antigens worsens prognosis. Furthermore, the lymphoma cells of the presented case had a clonal EBV genome. Tokunaga et al4 reported that EBV gene (by PCR), EBER-1 (by ISH), EBNA-2, and LMP-1 (by immunohistochemistry) were detected within tumor cells in about 17% of patients with ATLL, while Herbst et al5 reported that LMP-1, EBNA-2, and ERER-1 were expressed in CD30 (Ki-1)-positive ALCL. HTLV-I is known to infect T cells early in life. Ohtsubo et al6 reported that HTLV-I Tax induces CD21/EBV receptor expression on T cells. EBV may also infect and proliferate in T cells in the usual pathway of initial infection. In fact, dual infection of HTLV-I and EBV has been observed in cultured lymphocytes.7 Both EBV and HTLV-I are powerful inducers of CD30 expression in lymphoid cells in vitro.8 These results suggest that both of these viruses may infect the same T cell in early life and play important roles in oncogenesis.


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Kumura, T., Hino, M., Yamane, T. et al. Triple negative (CD3/CD4/CD8) adult T cell leukemia/lymphoma, histologically presenting as CD30 (Ki-1)-positive anaplastic large cell lymphoma with clonal Epstein–Barr virus genome. Leukemia 15, 994–995 (2001) doi:10.1038/sj.leu.2402126

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