Downregulation of an AIM-1 kinase couples with megakaryocytic polyploidization of human hematopoietic cells.

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3-dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.

Predominance of a distinct subtype of hairy cell leukemia in Japan.

Forty Japanese patients with hairy cell leukemia (HCL) were reviewed. Nine cases were diagnosed as typical HCL, and two cases had the features of HCL variant (prolymphocytic variant). The remaining 29 cases (72.5%) differed morphologically and hematologically from the other two groups in that they usually had a moderately high leukocyte count (average 27.9 x 10(3)/microliters), and abnormal cells showing a densely stained round nucleus and an inconspicuous nucleolus. Tartrate-resistant acid phosphatase reaction was weak, and their cells exhibited generally smooth or slightly irregular, cellular outlines in smears. The cells showed weak expression of surface immunoglobulin G (IgG) with kappa-chain predominance. CD25 antigen was not detected. Some of these findings resemble those of B-cell chronic lymphocytic leukemia, but the patients also had several important features of HCL. They had splenomegaly without significant lymphadenopathy. The abnormal cells were CD20+, CD11c+ and showed typical 'hairy morphology' under phase-contrast and scanning electron microscopy. Furthermore, spleen sections revealed diffuse infiltration by the abnormal cells in the red pulp. From these findings, we speculated that this group of patients constitute a distinct subtype of HCL which is commonly seen in Japan. We propose to term the disease as HCL Japanese variant.

Association of protein tyrosine phosphorylation with B cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA).

We have assessed whether tyrosine protein kinase (TPK) is involved in B cell differentiation. In vitro phosphorylation of an endogenous substrate in B cell leukemias showed that leukemic B cells at different stages of differentiation had specific endogenous substrates in tyrosine phosphorylation as well as distinct TPK activity. To clarify the relationship between TPK and the process of B cell differentiation, we studied protein tyrosine phosphorylation in two kinds of leukemic B cells, which showed distinct responses to TPA (12-O-tetradecanoylphorbol-13-acetate) in B cell differentiation. TPA-treated leukemic B cells from patients with B cell chronic lymphocytic leukemia (B-CLL) differentiated into cytoplasmic immunoglobulin (clg)+ plasmacytoid cells, while TPA-treated leukemic B cells from patients with hairy cell leukemia (HCL) did not differentiate into clg+ cells, but showed a peculiar morphological change, spreading. Untreated B-CLL cells and HCL cells showed similar TPK activities and tyrosine protein phosphorylation. When treated with TPA, enhanced phosphorylation was seen in B-CLL cells, while a clear reduction in phosphorylation was found in HCL cells. However, using 4-hydroxycinnamide derivatives which reduce TPK activity, we found that only the reduction of TPK activity did not lead HCL cells to spreading. These data suggest that protein tyrosine phosphorylation and/or dephosphorylation might be involved in B cell differentiation, but only the change of TPK activity in HCL cells is not sufficient to induce effects.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on leukemic cells from chronic B cell leukemia.

It is not clear whether cells from various chronic B cell leukemias including B-chronic lymphocytic leukemia (CLL), CLL in prolymphocytoid transformation (CLL-PLT), B-prolymphocytic leukemia (PLL), and hairy cell leukemia (HCL) simply represent different stages of a single B cell differentiation pathway. Furthermore, it is not known whether cells from any given B cell leukemia are characterized by the same population during the differentiation process. Differentiation of various B cell leukemic cells was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and the resulting changes in their morphology, cytoplasmic immunoglobulin (clg), and cytochemistry were evaluated. With respect to peculiar morphological change, i.e. extending long thin processes (spreading) and the appearance of clg, each sample showed different responses. According to these two indices samples were classified into three groups; spread+ clg- samples (one case of CLL-PLT, all HCL), spread+ clg+ samples (one of CLL, one of CLL-PLT), and spread- clg+ samples (a majority of CLL, one of CLL-PLT, and all PLL). Unexpectedly, both CLL and CLL-PLT consisted of heterogenous populations as to the reactivity to TPA. In the process of TPA-induced differentiation in CLL cells, features similar to those of HCL cells were not found. Since three different TPA-induced response patterns were observed in each chronic B cell leukemia type, it was not possible to sequentially assign each of these leukemias along a single B cell differentiation pathway. In order to explain this result, we introduced the hypothesis that these groups might be divided into different lineages in B cell differentiation. Since TPA-induced spreading cells were present in the B cell fraction of normal peripheral blood mononuclear cells, this morphological change should not be associated with malignant transformation.

Immunophenotypic features and configuration of immunoglobulin genes in hairy cell leukemia-Japanese variant.

Immunophenotypes and Ig gene rearrangements were investigated in 12 patients with a variant form of hairy cell leukemia (HCL) termed HCL-Japanese variant (HCL-J), and in an HCL-J-derived cell line. The leukemic cells of HCL-J characteristically showed the phenotype of CD20+, CD5-, CD10-, CD11c+, CD22+, CD24- and CD25-. Ig light (L) chain was undetected in nine cases, and the remaining four cases expressed kappa chain. Expression of Ig heavy (H) chain was studied in nine cases. In addition to Igkappa+ cases showing expression of predominantly gamma H chain isotype, alpha chain was detected in one case without expression of L chain. Rearranged bands in Ig heavy chain (JH) genes were recognized in all 12 cases tested. Rearranged bands in kappa chain genes and germline configuration in chi chain genes were seen in all three Igkappa+ cases tested. Four of nine cases without expression of L chain had a rearranged chi chain gene. The other three cases had chi chain genes in the germline configuration and rearranged and/or deleted kappa chain genes. In the remaining two cases, no rearrangement in either kappa or chi chain genes was detected. The Ig gene configuration and expression in HCL-J, partially overlapping with those described for immature B cell leukemia, were dissociated from the cytological features and CD20+, membrane CD22+ phenotype characteristic of mature B cells.

Effects of interferon-alpha on L-BCGF- and TNF-alpha-induced proliferation of hairy cell leukemia in Japan.

Interferon-alpha (IFN-alpha) is very effective in patients with hairy cell leukemia (HCL), although its mechanism of action is still unknown. To investigate this issue, we studied the in vitro response to IFN-alpha of a variant type of HCL, recently reported by us as the Japanese variant. Their clinical response to IFN-alpha (remission rate 35.7% in the multicenter study in Japan) was inferior to that of typical HCL (remission rate 80%; mean of previous reports). We found that both low molecular weight B-cell growth factor (L-BCGF) and tumor necrosis factor-alpha (TNF-alpha) induced the proliferation of HC from patients with the Japanese variant, as well as those with typical HCL. While, in typical HCL, IFN-alpha strongly inhibited the in vitro proliferation of HC induced by L-BCGF and TNF-alpha, the inhibitory effect of IFN-alpha on L-BCGF and TNF-alpha-induced proliferation was low in most Japanese variant patients. These in vitro findings may be related to the extent of clinical efficacy of IFN-alpha in the Japanese variant, obtained in the multicenter study. Since the degree of inhibition was parallel in L-BCGF- and TNF-alpha-induced proliferation in three patients examined simultaneously, it appeared that the antiproliferative effect of IFN-alpha is not specific to individual growth factors. Rather, IFN-alpha might affect fundamental growth mechanisms triggered by these factors.

Phenotypical heterogeneity of CD4+CD8+ double-positive chronic T lymphoid leukemia.

Chronic T lymphoid leukemias are defined as leukemias of post-thymic T cells. The CD4+CD8+ double-positive (DP) phenotype is seen in a few cases. Since DP generally occurs in thymic T cells, whether the DP T leukemia cells represent thymic or peripheral T cells has been a matter of controversy. To address this issue, we studied phenotypical features in eight cases of DP T cell leukemia. Thymic DP T cells and peripheral CD8+ T cells have CD8 of alphabeta subunit, while CD8alphaalpha is induced in CD4+ T cells on activation with IL-4. We found that two patients with DP T large granular lymphocyte leukemia (LGLL) showed dim expression of CD8alphaalpha, identical to the phenotype on IL-4-activated DP-T cells. The leukemic cells of these patients expressed IL-4 mRNA and produced high levels of IL-4. These findings suggest that they may be derived from peripheral CD4+ T cells. Three patients with adult T cell leukemia/lymphoma (ATLL) showed CD8alphaalpha, suggestive of an activated peripheral T cell origin. One case expressed CD8alphaalpha dim and IL-4 mRNA, while the other two cases expressed no IL-4 mRNA and showed CD8alphaalpha bright phenotype, features not found in normal T cell populations. Three patients with T-prolymphocytic leukemia (T-PLL) expressed CD8alphabeta. The DP phenotype is relatively common in T-PLL, and CD4+CD8alphabeta+ is characteristic of thymic T cells. The DP T-PLL cells did not express TdT,CD1 or recombination activating gene-1 (RAG-1), which is down-regulated at the late stage of thymic T cell development. On the basis of these findings, we propose a late thymic origin for DP T-PLL. The phenotype of DP T cells differed for each entity and appeared to correlate with minor normal DP T cell population.

Platelet activation induced by combined effects of anticardiolipin and lupus anticoagulant IgG antibodies in patients with systemic lupus erythematosus--possible association with thrombotic and thrombocytopenic complications.

Antiphospholipid antibodies (aPL) are well known to be associated with arterial and venous thrombosis. In a series of 180 patients with systemic lupus erythematosus (SLE), the prevalence of arterial thrombosis was obviously higher in the patients who had both anticardiolipin antibodies (aCL) and lupus anticoagulant (LA) (17/35, 48.6%, p<0.05) (Table 1) than in the other patients bearing aCL or LA alone or neither of them (2/145, 1.4%). Since a substantial fraction of the former group of patients with arterial thrombosis also had thrombocytopenia (12/17, 70.6%), there was a possibility that aCL and LA might have enhanced platelet activation and aggregation. To test this possibility, we studied the in vitro effects of aCL and LA on the enhancement of platelet activation by flow cytometric analysis using anti-CD62P and anti-CD41 monoclonal antibodies directed against platelet activation-dependent granule-external membrane (PADGEM) protein and platelet glycoprotein IIb (GPIIb), respectively. Platelet activation defined by the surface expression of CD62P was not induced by aCL+ x LA+ plasma only, but was significantly augmented by aCL+ x LA+ plasma in combination with adenosine diphosphate (ADP) at a low concentration that had only a modest effect on platelet activation. In contrast, aCL+ x LA-, aCL- x LA+ and aCL- x LA- plasma samples were incapable of enhancing platelet activation in the presence or absence of ADP stimulation. In addition to plasma samples, the purified IgG from aCL+ x LA+ plasma (aCL+ x LA+-IgG) also yielded apparent enhancement of platelet activation induced by ADP. Furthermore, platelet activation was generated by the mixture of aCL+ x LA--IgG and aCL- x LA+-IgG fractions prepared from individual patients, but not by each fraction alone. These results suggest that aCL and LA may cooperate to promote platelet activation, and may be involved, at least partially, in the pathogenesis of arterial thrombosis and thrombocytopenia in patients with SLE.

HTLV-I associated myelopathy (HAM) after blood transfusion in a patient with CD2+ hairy cell leukemia.

Hairy cell leukemia complicating hemolytic anemia developed in a 46-year-old woman. Morphologically and cytochemically typical hairy cells were found to express both CD20 and CD2 antigens. Expression of surface IgG of kappa-chain type and the rearrangement of Ig but not T-cell receptor beta genes confirmed a B-cell origin of the leukemia. Blood transfusion was followed by disappearance of the hemolysis and a marked improvement of the leukemia. However, the patient developed progressive spastic spinal paraplegia about seven months after transfusion and was diagnosed as having HTLV-I associated myelopathy (HAM) by the demonstration of HTLV-I antibodies in serum and cerebrospinal fluid. HTLV-I infection via the transfusion may have been involved in the hematologic improvement seen in this patient. Autopsy showed demyelination, vacuolar degeneration, gliosis, and perivascular cuffing in the white matter of spinal cord without evidence of leukemic infiltration.

Immunoreactivity of neoplastic and non-neoplastic monocytoid B lymphocytes for DBA.44 and other antibodies.

AIMS--To evaluate the immunoreactivities of neoplastic and non-neoplastic monocytoid B cells (MBC) and compare them with hairy cell leukemia (HCL) and mantle cell lymphoma (MCL). METHODS--An immunohistochemical study of paraffin wax embedded sections was done on surgically resected specimens of spleens with MBC clusters from patients with gastric cancer (14 cases), tonsils (five cases), and lymph node (two cases) showing lymphoid follicular hyperplasia (LFH), submandibular lymph nodes containing MBC in Sjögren's syndrome (one case). Extranodal organs affected by MCL (three cases) and monocytoid B cell lymphoma (MBCL) (seven cases), and spleens from HCL (four cases) were also studied. These specimens were fixed in 10% formalin and routinely processed for paraffin wax embedding. Fresh spleen specimens from patients with liver cirrhosis (one case) and gastric cancer (seven cases) were snap frozen. RESULTS--Mantle zone lymphocytes were DBA.44, CD74 positive and showed a weaker reaction for CDw75 than marginal zone lymphocytes and MBC, which were almost DBA negative. In neoplastic diseases tumour cells in MCL were DBA.44, CD74, and CDw75 positive. MBCL showed a positive reaction for CD74 and CDw75, but positivity for DBA.44 was observed in only one of seven cases. The HCL specimens, all positive for DBA.44, showed a weaker reaction for CD74 and a stronger reaction for CDw75 than either MCL and MBCL specimens. CONCLUSION--These results show that mantle zone lymphocytes and MCL more closely matched HCL for reactivity to DBA.44 than MBC and MBCL. Reactivities for DBA.44 and CDw75 were greater in MBCL compared with its non-neoplastic counterpart, MBC.

Early age onset of hairy cell leukemia presenting with leukocytosis.

Hairy cell leukemia (HCL) is an uncommon type of chronic B cell leukemia mainly affecting middle-aged adults. HCL presenting with pancytopenia is rare in Japan and a distinct subtype of HCL termed HCL-Japanese variant is predominantly seen. We describe a HCL patient with unusual presentation. The patient was a 26-year-old male, such early onset of HCL being quite rare. The patient showed leukocytosis with many circulating hairy cells and cellular bone marrow. These findings were preferentially seen in HCL-Japanese variant, but, cytomorphologic, cytochemical and immunophenotypical studies on the pathologic cells were consistent with those of typical HCL seen in Western countries. Interferon-alpha therapy was very effective in this case. Differentiation of the subtype of HCL appears to be important for the choice of the treatment. The cytological findings were useful for the differential diagnosis of HCL presenting with leukocytosis.

Blastic transformation of splenic lymphoma with villous lymphocytes after a well-controlled chronic phase of more than 10 years.

A 30-year-old Japanese man with splenomegaly and lymphocytosis was examined in 1985. Blood analysis revealed that some of the lymphocytes had short-surface villi with polar distribution. The cells showed Ig lambda+, CD5+, CD11c+, CD19+, CD22+, CD23+, CD24+, FMC7+ phenotype. A small M peak was detected in the serum. Splenic lymphoma with villous lymphocytes (SLVL) was diagnosed on the basis of these findings. Remission was induced and was maintained with low-dose chlorambucil for more than 10 years. In 1996, the patient developed splenomegaly and lymphadenopathy with "B" symptoms and a high serum lactase dehydrogenase (LDH) level. Large blastoid cells with prominent nucleoli were observed in the bone marrow; later, a small number appeared in the peripheral blood. The bone marrow cells showed a complex chromosomal abnormality involving del(7)(q32). Southern blot analysis of immunoglobulin gene rearrangements in SLVL cells that had been cryopreserved in 1986 and of bone marrow cells in 1996 showed 2 rearranged bands in each cell sample; 1 band showed identical sizes in the 2 samples, and the other showed different sizes. These findings suggest that the blastoid cells were derived from SLVL cells through transformation. After this transformation, the disease followed a highly aggressive course. Various chemotherapeutic agents had little effect, and the patient died 3 months later.

Overexpression of the PRAD1 oncogene in a patient with prolymphocytic leukemia with t(11;14)(q13;q32).

Prolymphocytic leukemia (PLL) was diagnosed by morphologic and immunophenotypical studies in a 72-year-old Japanese man. Massive splenomegaly was present but lymphadenopathy was minimal in this case. Chromosomal analysis of peripheral mononuclear cells showed t(11;14)(q13;q32) in all metaphases examined, except for one normal karyotype. Northern blot analysis of RNA prepared from leukemic cells obtained from the patient revealed overexpression of the PRAD1/cyclin D1 proto-oncogene, which has not been described previously in patients with PLL.

Establishment of a new cell line from a patient with hairy cell leukemia-Japanese variant.

A cell line, JHC-2, was established from the peripheral blood of a patient with hairy cell leukemia (HCL)-Japanese variant. The JHC-2 cells have cytologic features similar to those of the original tumor cells. They displayed hairy cytoplasmic projections by phase contrast and scanning electron microscopy. The tartrate-resistant acid phosphatase reaction was weakly positive. The immunophenotype of the JHC-2 cells was CD5-, CD10-, CD11c+/-, CD19+, CD21+, CD23+, CD24-, CD25+/-, CD38- and FMC-7+. The expression of surface immunoglobulin (IgG, kappa) and the configuration of Ig gene rearrangements in the JHC-2 cells were identical to those in the original leukemic cells, and the JHC-2 cells displayed trisomy 9 on cytogenetic examination. Southern blot analysis for the Epstein-Barr virus (EBV) genome showed that the JHC-2 cells contained the EBV genome, although the freshly isolated leukemic cells did not. These results indicate that the JHC-2 cell line is an EBV spontaneously transformed B cell line originating from HCL cells.

Dehydroepiandrosterone sulfate deficiency in chronic fatigue syndrome.

The chronic fatigue syndrome (CFS) is a condition of unknown etiology, characterized by a persistent debilitating fatigue, the muscle-related symptoms and the neuropsychiatric symptoms. Recently, it has been reported that the patients with CFS might have impaired activation of the hypothalamic-pituitary-adrenal axis, and suggested that a part of the patho-genesis of CFS might be associated with abnormalities of the endocrine system. Herein, we show that the majority of Japanese patients with CFS had a serum dehydroepiandrosterone sulfate (DHEA-S) deficiency. Serum DHEA-S is one of the most abundantly produced hormones which is secreted from the adrenal glands, and its physiological function is thought to be a precursor of sex steroids. DHEA-S has recently been shown to have physiological properties, such as neurosteroids, which are associated with such psychophysiological phenomena as memory, stress, anxiety, sleep and depression. Therefore, the deficiency of DHEA-S might be related to the neuropsychiatric symptoms in patients with CFS.

Low levels of serum acylcarnitine in chronic fatigue syndrome and chronic hepatitis type C, but not seen in other diseases.

Recently, we found a serum acylcarnitine (ACR) deficiency in Japanese patients with chronic fatigue syndrome (CFS). To clarify whether this ACR abnormality is a characteristic of CFS or not, we also studied the levels of serum carnitine in Swedish subjects. Both serum ACR and free carnitine (FCR) levels in normal healthy subjects were quite different between Japanese (n=131) and Swedish people (n=46) (p<0.001). However, it is confirmed that Swedish patients with CFS (n=57) also had serum ACR deficiency (p<0.001). When we studied the levels of serum ACR and FCR in Japanese patients with various kinds of diseases (CFS, hematological malignancies, chronic pancreatitis, hypertension, diabetes mellitus, chronic hepatitis type C, psychiatric diseases), a significant decrease in the levels of serum ACR was only found in patients with CFS and chronic hepatitis type C (p<0.001). Therefore, we concluded that ACR deficiency in serum might be a characteristic abnormality in only certain types of diseases.

Splenic lymphoma with villous lymphocytes with CD5+, CD11c+ B-cell phenotype.

A 64-year-old male suffered from splenomegaly without lymphadenopathy. His WBC count on admission was 6.1 x 10(9)/l with 55% abnormal lymphocytes. No monoclonal gammopathy was detected. Abnormal cells shown in films usually had relatively abundant cytoplasm with serrated edges. Under phase-contrast microscopy, the cells displayed short, needle-like processes. The immunophenotype of peripheral blood mononuclear cells were CD19+, CD20+, CD11c+, FMC7+, CD5+, CD10-, CD25- and SIg+. The spleen histology showed a distinctive pattern of white pulp infiltration by abnormal lymphocytes with features of plasma cell differentiation. These findings were compatible with the features of splenic lymphoma with villous lymphocytes.

[Large granular lymphocyte leukemia].

Large granular lymphocyte leukemia (LGLL) is defined as clonal proliferation of LGLs in peripheral blood. The following studies were conducted to address some issues in chronic LGLL. (1) Chronic LGLL is characterized by the indolent course, and the diagnosis of leukemia is difficult in such patients as those without distinct organomegaly and/or any evidence of monoclonality. We performed immunohistological studies in a patient with persistent NK lymphocytosis. No organomegaly had been seen in the patient during a three-year-observation, who died from cerebrovascular accident. The autopsy findings revealed multi-organ infiltration including spleen, liver, bone marrow, lymph nodes and lung. These findings suggest that the cells of chronic LGLL have infiltrative capacity characteristic of malignant cells. (2) Lymphocytosis in chronic LGLL is usually stable for a long period. We found that both T- and NK-LGLL cells strongly expressed CD95, an apoptosis related protein. Anit-CD95 did not induce apoptosis, but suppressed proliferation induced by IL-2 or anti-CD3. These results suggest that CD95-CD95 ligand system is involved in the slow cell growth characteristic of chronic LGLL. (3) CD4+CD8+ double positive (DP) cases are rarely seen in LGLL, and the physiologic counterpart of the leukemic cells has not been determined yet. We found that the DP-LGLL had alpha alpha type in the CD8 subunit and did not express RAG-1, these findings being characteristic of peripheral T cells. We also found that they expressed IL-4 mRNA and secreted IL-4 on activation. These results strongly suggest that DP-T-LGLL represents an expansion of a rare subset of peripheral DP-T cells, possibly derived from IL-4 activated CD4 single positive T cells.

[LGL lymphoma].

We reported in this article two patients with large granular lymphocytic lymphoma (abbreviated as LGL lymphoma). One was the patient with LGL leukemia/lymphoma (patient 1) and other was the patient with NK-LGL lymphoma (patient 2). Because the gene of TCR delta was rearranged in the patient 1, the clonality of the LGL leukemia/lymphoma was confirmed. However, it is not determined yet whether the lineage of tumor cells is T cells or NK cells. The cytochemical features of the lymphoma cells of the patient 2 were studied. It was found that NK cell-derived lymphoma cells of the patient were positively stained with these two monoclonal antibodies that are reactive with T cell; one is anti-CD45RO (UCHL-1) and other is anti-CD3. Judging from the result, malignant NK-LGL cells in some patients are cytoplasmic CD3+ and UCHl-1+. It is emphasized that May-Grünwald-Giemsa stain of biopsied specimen of the lymphoma is required for making the diagnosis of LGL lymphoma.

[A case of T-cell prolymphocytic leukemia].

A 54-year-old man who had been known to have a high prolymphocyte count for four years was admitted to our hospital because of dyspnea in September, 1990. Physical examination revealed skin eruption, lymphadenopathy and hepatosplenomegaly. Chest X-ray demonstrated bilateral pleural effusions. The leukocyte count was 232,900/microliter with 99% lymphoid cells possessing single nucleoli. The cells expressed the phenotype CD2+, CD3-, CD4+, CD7+, and CD8-. Southern blot analysis of DNA from these cells revealed monoclonal rearrangement of T-cell receptor beta-chain genes. Anti-human T-cell lymphotropic virus type 1 (HTLV-1) antibody and HTLV-1 proviral DNA were not detected. A biopsy specimen from the skin lesions showed infiltration of the leukemic cells which were positive for anti-MT1 antibody. Histological finding of the axillary lymph node was malignant lymphoma, diffuse, medium-sized, T-cell type. Combination chemotherapy resulted in the improvement of skin eruption, lymphadenopathy, hepatosplenomegaly and pleural effusions, although his prolymphocyte count increased to 910,000/microliters. He died of cerebral bleeding in July, 1991. We diagnosed this case as T-cell prolymphocytic leukemia, observed for five years.