Shigehiko Ishihara

Clonal lymphoproliferation following chronic active Epstein-Barr virus infection and hypersensitivity to mosquito bites.

In order to elucidate the possibility of lymphoproliferation in cases of chronic active Epstein‐Barr virus infection (CAEBV), to clarify the clonality and genotype of proliferating lymphocytes, and to search for the factors that induce lymphoproliferation, we studied 11 cases of CAEBV, using genetical and immunological techniques.

Hypersensitivity to Mosquito Bites Conceals Clonal Lymphoproliferation of Epstein-Barr Viral DNA-positive Natural Killer Cells

In order to clarify the relationship between Epstein‐Barr (EB) virus and hypersensitivity to mosquito bites (HMB), and to search for the mechanism which induces EB virus‐associated lymphoproliferative diseases, we investigated patients with HMB, using hematological, immunological and virological techniques. Among 5 cases of HMB, CD56+ cells had proliferated and CD3+ cells were diminished in 4 cases. Although anti‐EB virus antibody titers were not consistent with chronic active EB virus infection, EB viral DNA was detected in the peripheral blood mononuclear cells in all 5 cases. Moreover, EB viral DNA‐positive cells had proliferated monoclonally in 4 cases, and hiclonally in 1 case. It was proved that most of the EB viral DNA existed in natural killer (NK) cells through polymerase chain reaction analysis. These findings suggest that the basis of HMB may be clonal lymphoproliferation of EB viral DNA‐positive NK cells and this hematological abnormality may induce the characteristic symptoms of HMB. In some cases, the proliferating NK cells can metamorphose into leukemic cells, and hemophagocytic syndrome, which has been assumed to be a complication of HMB, may then occur.

Clinical significance of CD7-positive stem cell leukemia : a distinct subtype of mixed lineage leukemia

Ten leukemia cases with mixed phenotype were investigated in terms of clinical characteristics and cellular origin. Three patients were infants and six patients were older children. Six of them had a high leukocyte count and a mediastinal mass was found in three cases. All but one showed hepatosplenomegaly and/or lymphoadenopathy. In spite of intensive chemotherapy, most of them responded poorly. Cytochemical analysis of their leukemic cells revealed a low percentage of positivity for myeloperoxidase reactivity (< 25%) in two cases and electron microscopic platelet peroxidase reactivity was found in one of three analyzed cases. Phenotypically, these cells all expressed CD7, and other T‐lineage‐associated, B‐lineage‐associated, and/or myeloid‐associated antigens were also detected to some extent. In addition, three cases expressed CD41 and one case expressed CD56. The T‐cell receptor (TCR) genes and immunoglobulin gene were in the germline configuration in seven cases. In three rearranged cases two showed only the TCR‐δ gene rearrangement, and one had both TCR‐γ and δ gene rearrangements. Cell culture studies with 12–0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) revealed differentiation to the T‐lineage in two cases and to a myeloid lineage in one case. Megakaryocytic differentiation was detected in two cases in culture without TPA. These results suggest that the cells from these cases arose from stem cells capable of both lymphoid and nonlymphoid differentiation. Although the cells were heterogeneous with regard to their potency of differentiation, they have similar clinical characteristics. Because of poor prognosis, it is important to identify this type of leukemia, and allogenic or autologous bone marrow transplantation should be considered. Cancer 68:2273–2280, 1991.

Elevated serum soluble Fas ligand in natural killer cell proliferative disorders

We evaluated the serum level of soluble Fas ligand (sFasL) in patients with natural killer lymphocyte proliferative disorders (NK‐ LPD). The serum sFasL level was elevated in neoplastic groups of aggressive NK leukaemia, indolent NK leukaemia and NK lymphoma, all of which contained clonal EBV‐DNA. In NK leukaemia the serum sFasL level was significantly higher than that found in others. However, it was not elevated in the patients with reactive NK‐LPD and in one patient with NK leukaemia in remission. These findings indicate that the serum sFasL level is a useful indicator in evaluating disease activity.

Analysis of chromosome 6q deletion in EBV-associated NK cell leukaemia/lymphoma.

Deletions involving chromosome 6q have been reported in a number of human cancers such as ovarian and breast tumours as well as haematopoietic malignancies. It seems that this region might contain tumour-suppressor genes. Putative natural killer cell lymphomas/leukaemias (NKLL) represent a group of recently characterized haematolymphoid malignancies sharing an immunophenotype of CD3/Leu4 m CD3epsilon+ CD56+, a genotype of germline T-cell receptor genes, and have a close association with Epstein-Barr virus (EBV). Deletion at 6q21-q25 was demonstrated in three recently reported cases of NKLL. Here we investigated the possible involvement of 6q deletions in the pathogenesis, and especially the tumorigenesis of NKLL. The regions of D6S1574 (6p25), DS276 (6p12), D6S257 (6q11), D6S434 (6q14), D6S287 (6q15), D6S292 (6q21), D6S308 (6q22), D6S264 (6q25), and D6S446 (6q26) were analysed by PCR in 25 cases of NKLL, including seven cases with chronic NK leukaemia, six with acute NK leukaemia and 12 with NK lymphoma. 6q deletions, especially 6q15-25, were frequently detected, but 6p deletions were not detected in any cases. Analysis of 6q21 showed possible deletion in two of seven cases (29%) with chronic NK leukaemia, three of six (50%) with acute leukaemia, and 12 of 12 (100%) with NK lymphoma. The frequency of deletion increased in clinical phases. In three cases with lymphoma, fluorescence in situ hybridisation was performed, which confirmed 6q21 deletion in two cases, although 6q telomeric and centromeric regions were preserved. The other case failed to show deletion. Our results suggest that 6q deletion, especially 6q21-25, might be involved in NKLL tumorigenesis, and support the presence of the tumour suppressor genes associated with the development of NKLL.

Remarkable Depression of CD4+2H4+ T Cells in Severe Chronic Active Epstein‐Barr Virus Infection

In order to better understand the features of chronic active Epstein‐Barr (EB) vims infection, we employed two‐colour immunofluorescence staining with monoclonal antibodies and flow cytometry analysis to study the lymphocyte phenotypes of two patients with severe symptoms of this disorder as well as four patients with mild symptoms. We found an increased number of activated T cells, as characterized by CD4+ la+, CD8+Ia+, or CD4+Tac+ phenotypes, and a markedly decreased CD4+2H4+ T cell subpopulation, previously characterized as a suppressor‐inducer subset, in the patients with severe symptoms. In contrast, the four patients with mild symptoms showed only a slightly elevated number of activated T cells and a normal CD4+2H4+/CD4+ ratio. These phenotypic differences may suggesst heterogeneity in this disorder. Also, a failure in ihe suppressor‐inducer population could contribute to changes in the host‐virus relationship and the degree of the decrease in this population may correlate directly with ihe severity of the disease.

Increased expression of the multidrug-resistance gene in undifferentiated sarcoma

We analyzed multidrug‐resistance gene (mdr1 gene) expression in a patient with undifferentiated sarcoma of the liver using the cloned cDNA for the mdr1 gene. Tissue samples were available at the time of initial diagnosis and of two intracranial relapses after chemotherapy with a regimen including doxorubicin and teniposide. The level of mdr1 gene expression was increased sevenfold in the intracranial tumor at the time of first relapse and 11‐fold at the second relapse. This case may be an example of acquired multidrug resistance associated with overexpression of the mdr1 gene.

Developmental process of the T-cell receptor α and δ gene assembly in B-cell precursor acute lymphoblastic leukaemia

Summary We analysed the organization of Vδ genes and δ recombining element (δRec) in 27 children with B‐cell precursor acute lymphoblastic leukaemia. Twenty‐two of 54 alleles showed rearrangements of the T‐cell receptor (TCR) δ locus. These rearrangements resulted either from D2Dδ3 (2 alleles) or Vδ2(Dn)Dδ3 (20 alleles) recombinations, and the other Vδ and δRec were not rearranged. Of 23 alleles with deletion of Cδ and rearrangements of Jα. Vδ2, Vδ4 and Vδ5 appeared to rearrange to Jα on five alleles. With regard to the relationship between the rearranged Vα/δ and Jα genes, gene segments 5’to Vδ2 frequently rearranged to Jα more proximal to Cα, whereas Vδ2 and gene segments 3’to Vδ2 showed a tendency to rearrange to Jα distal to Cα. Based on these findings, we suggest that the initial recombination event of the TCR‐α/δ gene may be D2Dδ3 joining, followed by Vδ2 recombination with the D2Dδ3 complex. It was also suggested that use of Vα/δ and Jα/δ may depend on the distance between the involved Vα/δ and Jα/δ at least in B‐lineage cells. These rearrangements in B‐precursor cells appear to be aberrant. However, this recombinational process may be one of the normal differentiation pathways in T‐lineage cells, because cells with a Vδ2(Dn)Dδ3 rearrangement were detected in 0·1–0·01% of normal peripheral mononuclear cells by the polymerase chain reaction.

Poor prognosis of mediastinal non-Hodgkin's lymphoma with an immature phenotype of CD2+, CD7 (or CD5)+, CD3−, CD4−, and CD8−

Nine children with mediastinal non‐Hodgkins lymphoma (NHL) were treated according to our new regimen which is characterized by intensified therapy with high‐dose cytosine arabinoside (HDCA). After induction therapy with a combination of five drugs, such as vincristine, doxorubicin, cyclophosphamide, 1‐asparaginase, and prednisolone, intermediate dosages of methotrexate (MTX) (1 g/m2) and HDCA (1.5 g/m2 × 12 doses) were administered. All but one patient (88.9%) achieved complete remission and then received this intensified therapy. With a median follow‐up period of 25.5 months, five patients are still in complete remission, but three patients have relapsed. From the phenotypic point of view, these relapsed patients showed only very immature T‐cell differentiation antigens such as CD2 and CD7 (or CD5). These results suggest that HDCA as intensified therapy for children with mediastinal NHL seems to be effective. However, for patients with an immature phenotype of T‐lineage cells, more sophisticated regimens should be prepared.

Concomitant rearrangements of T-cell β- and γ-chain genes in childhood T-lineage leukemia/lymphoma☆

Abstract Similar to the immunoglobulin (Ig) gene rearrangements in B-lineage cells, identification of T-cell receptor (TCR) gene rearrangements is a novel clonal marker and necessary to establish a T-cell lineage. The function of T-cell γ-chain (Tγ) gene is still unknown, but because of its shared properties with T-cell α-chain (Tα) and Tβ genes, we analysed Tγ gene organization in 10 patients with T-lineage leukemia/lymphoma as well as in non-T-lineage leukemias. All 10 cases of T-lineage leukemia/lymphoma, whose phenotypes were different, demonstrated Tγ gene rearrangements as well as Tβ gene rearrangements. In contrast, among the non-T-lineage leukemias, the emergence of Tβ and/or Tγ gene rearrangements was varied. Based on these findings, concomitant rearrangements of Tβ and Tγ genes are characteristic in childhood T-lineage leukemia/lymphoma regardless of their phenotypic differences. Furthermore, no obvious developmental hierarchy was observed between Tβ and Tγ gene arrangements in these leukemia/lymphoma cells.