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To better understand indeterminate HTLV-1 carriers and smoldering (SM) subtype of adult T-cell leukemia (ATL), HTLV-1 proviral integrated status, proviral load (PVL) and ATL-related biomarkers were examined in 57 smoldering cases, including unusual carriers with a percentage of ATL-like cells. We found that according to Southern blot hybridization analytic features, 28 patients with SM ATL could be divided into 3 groups consisting of 16 (57.4%) patients with a monoclonal band, 6 (21.4%) with oligoclonal bands and the remaining 6 with smears. Although no clinical differences were observed among the 3 SM subtypes, HTLV-1-infected CD4 T-cell counts increased in order of poly-, oligo- and monoclonal subtypes. This trend began in the carrier stage and also was observed in PVL, CD25 and CCR4, indicating that a clone consisting of leukemic phenotypic cells was continuously growing. Moreover, the antigen modulation rates of CD26 and CD7 and the increasing rate of CD25 and CCR4 cells were closely correlated to growing clonal size, indicating that these markers had the possibility to predict a monoclonal band. In particular, CD26 or the ratio of CD26/CD25 had a validity differential for leukemic nature and predictive detection of clonal band. Conclusively, the present study shows that smoldering ATL is heterogeneous in the leukemogenic process, and the behavior of CD26 plays a central role in the evolution from early occult to overt smoldering ATL.

The p53 gene is one of the most important genes involved in carcinogenesis and its role in part has been clarified by research using cell lines. To know the comprehensive characteristics of 22 hematopoietic cell lines (T, 13 and non-T, nine lines), the relationship between p53 mutational status, its altered functioning, and its mRNA and protein levels were examined. p53 mutations were less frequent in T-cell lines (38% vs. 78%) with mainly single nucleotide substitutions generating missense codons. Of 22 different p53 mutations, 12 (54.5%) resulted in mutated proteins, with the mutations clustering mainly in the sequence-specific DNA-binding site region located from amino acid residues 102 to 292. p53 mRNA and protein assays determined that wild-type cell lines expressed constant levels of both mRNA and protein, but mutated cell lines demonstrated two expression patterns: protein over-expression with reduced mRNA levels, because of missense mutations; and protein under-expression with little mRNA expression, because of other mutations. The resistance to Nutlin (MDM2 inhibitor)-induced apoptosis was associated with p53 mutations independently of MDM2 expression levels. This clarification of the unique associations in cell lines useful for bio-medical studies will contribute to a better understanding of p53-associated carcinogenesis.

Primary effusion lymphoma (PEL) was initially designated as a body-cavity-based lymphoma and recognized as a distinct clinical entity without a contiguous tumor mass. PEL was first reported in patients with acquired immunodeficiency syndrome (AIDS) and the distinctive feature of PEL originally reported as a B-cell neoplasm characterized by infection of the tumor cells by human herpes virus 8 (HHV-8). However, there have recently been several reports of PEL in patients without human immunodeficiency virus (HIV) or HHV-8 infection.

Human T-cell leukemia virus type I (HTLV-1) causes adult T-cell leukemia (ATL), a fatal T-cell leukemia resistant to chemotherapy, after more than 50 years of clinical latency from transmission through breast-feeding. Polyclonal expansion of virus-infected T cells predisposes them to transformation. Constitutive activation of nuclear factor-kappaB (NF-kappaB) in the leukemic cells is essential for their growth and survival. Blocking NF-kappaB has been shown to be a potential strategy to treat ATL. We tested this approach using a novel NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), and also examined its application to chemoprevention by selective purging of the HTLV-1-infected cells. DHMEQ inhibited NF-kappaB activation in primary ATL cells and cell lines derived from them and induced apoptotic cell death. NF-kappaB inhibition down-regulated expression of genes involved in antiapoptosis or cell-cycle progression. DHMEQ protected severe combined immunodeficiency (SCID) mice inoculated with HTLV-1-transformed cells from death. In addition, DHMEQ selectively targeted HTLV-1-infected cells in the peripheral blood of virus carriers in vitro, resulting in a decreased number of infected cells. We conclude that NF-kappaB is a potential molecular target for treatment and prevention of ATL. As a potent NF-kappaB inhibitor, DHMEQ is a promising compound allowing the translation of this strategy into clinical medicine.

Adult T-cell leukemia/lymphoma (ATLL) is a lymphoproliferative disorder caused by human T lymphotropic virus type 1 (HTLV-I). Although ATLL cells display an activated helper/inducer T-cell phenotype, CD4+ and CD25+, they are known to exhibit strong immunosuppressive activity. As regulatory T cells (Treg cells) express CD4+ and CD25+ molecules and possess potent immune response suppressive activity, we investigated a possible link between ATLL cells and Treg cells. In primary ATLL cells, the expression levels of the Treg cell marker molecules Foxp3 and glucocorticoid-induced tumor necrosis factor receptor family related protein (GITR) were significantly higher than in those from healthy adults. Furthermore, ATLL cells are unresponsive in vitro to concanavalin A stimulation and suppress the proliferation of normal T cells. GITR mRNA expression was induced by the HTLV-I transactivator Tax, and GITR promoter analyses revealed that this induction depends on the kappaB site from -431 bp to -444 bp upstream of the putative transcription site. Taken together, ATLL cells may originate from HTLV-I-infected Treg cells, and GITR seems to be involved in the progression to ATLL.

Constitutive expression of the IL-2 receptor (IL-2R) on adult T-cell leukemia (ATL) cells and the presence of permanent IL-2-dependent ATL cell lines indicate that the signal transduction system via IL-2R is a key element for the development of this disease. IL-2R is a member of the common gamma-chain (gammac)-receptor family and shares gamma with IL-4R, IL-7R, IL-9R, and IL-15R. In addition to IL-2R, ATL cells express IL-15R and respond to IL-15. In the present study, we examined other members of this receptor family. ATL cells showed various levels of IL-4Ralpha (CD124) and IL-7Ralpha (CD127) expression, and responded to these cytokines. In contrast, ATL cells hardly responded to IL-9. As primary samples were a mixed population and the results may have been modified by contaminating normal cells, we used ATL cell lines as pure ATL cell populations. Here, we report that IL-2-dependent ATL cell lines also express IL-4Ralpha and respond to IL-4, which was verified by the activation of cytoplasmic transcriptional activator Stat6 protein. Moreover, a novel ATL cell line that grows stably in an IL-7-dependent manner was established from one of the cell lines, and IL-7 induced Stat5 activation in this cell line. These results indicated that ATL cells have the potential to express all gammac-receptors except IL-9R. Overlapping and switching of cytokine receptors supported the idea that ATL cells can rapidly select the appropriate gammac-receptor according to conditions.

Derangement of either apoptosis or cell division is known to play an important role in tumorigenesis. Fas-mediated apoptosis on normal and leukaemic T cells is finely tuned by inhibitory proteins, such as FAP-1, FLIP and survivin, and defective caspase isoform which can attenuate the function of its intact caspase as a decoy molecule. However, complex involvement of such inhibitors in tumour biology relating to apoptotic pathology remains unclear in the neoplasms. We report the aberrant expression of FAP-1, FLIP and survivin mRNAs on leukaemic T cells from adult T-cell leukaemia (ATL) patients. Among these inhibitors, only survivin was aberrantly expressed in all ATL cases, but not in any normal peripheral blood mononuclear cells (PBMCs). Furthermore, survivin mRNA expression level was characteristic in each subtype of ATL and represented an important determinant for ATL prognosis. However, the apoptotic effector of casp-8, which is essential in Fas-mediated signal transduction, was dominant in defective casp-8 rather than intact casp-8 in ATL cells, suggesting a favourable biological situation for escape from apoptosis. Taken together, ATL cells probably possess many different regulatory mechanisms in order to attenuate Fas-mediated signalling and subsequently expand their populations under escape from apoptosis. Among these inhibitors, survivin is a useful bio-marker to assess tumour biology and may be a potential new target for apoptosis-based selective therapy in neoplasms as the expression is a general feature of neoplasia, but not normal tissues.

To characterize the prodromal phase of adult T-cell leukemia (ATL), a prospective follow-up study was conducted on 50 carriers in a putative pre-ATL state. This state was defined by the presence of molecularly-detectable monoclonal proliferation of human T-lymphotropic virus type I (HTLV-I)-infected T lymphocytes, and the absence of clinical symptoms of leukemia. The median observation time was 50 months. The pre-ATL subjects were divided into two groups according to initial white blood cell (WBC) counts: group A, those with a normal WBC count (9,000/microL) (n = 30), and group B, those with an increased WBC count (9,000 to 15,000) (n = 20). Comparisons were made between the two groups and with a group of 25 patients with chronic ATL (group C) who had WBC counts of more than 15,000. Significant differences in survival rate were found between groups A and B (10-year survival 65.7%) and group C (32.8%) (P < .01), and between group A (10-year survival 90.0%) and group B (52.1%) (P < .05). The incidence of transformation to overt ATL was 10% (3 of 30) in group A and 50% (10 of 20) in group B (P < .01). In six transformed cases (one in A and five in B) we found exactly the same integration sites in pre-ATL and overt ATL phases, confirming the multistep leukemogenesis hypothesized for this disease. However, the pre-ATL subjects could be divided into two distinct prognostic groups based on the initial WBC count; those with good and those with poor prognosis. Although the 10% transformation rate (2.5% annually) in group A seemed to be extremely high compared with that in the general population of HTLV-I carriers (around 0.06% to 0.4% annually), the majority of group A subjects and some in group B showed stable clinical courses without transformation. Further, development of ATL was not observed in four group A subjects with HTLV-I-associated myelopathy (HAM), which is rarely associated with ATL. We propose to call this group of rather benign HTLV-I carriers "HTLV-I carriers with monoclonal proliferation of T lymphocytes (HCMPT)." Thus far we have been unable to identify reliable parameters other than WBC counts that prospectively distinguish HCMPT from the true pre-ATL state, in which there is a high probability of developing ATL. Further clinical and biologic approaches should elucidate the natural history of the HTLV-I carrier state and early events in ATL leukemogenesis.

Surface phenotypes of leukemic cells from six patients with adult T cell leukemia (ATL) were analyzed by the use of monoclonal antibodies, both at the time of initial diagnosis and at either relapse or exacerbation phase after chemotherapy. Changes of cell surface antigens were observed in four of the six cases. The majority of the leukemic cells of these patients were reactive with anti-Leu-1 and anti-Leu-3a, but unreactive with anti-Leu-2a and MAS 036c monoclonal antibodies at the time of initial diagnosis, indicating that ATL cells are of peripheral inducer/helper T cell origin. In three cases, the Leu-1 antigen disappeared at relapse or at exacerbation phase, and in one of these cases, a small percentage of ATL cells became reactive with MAS 036c, which identifies cortical thymocyte antigen. ATL cells of one other case did not have Leu-1 antigen from the start, but gained Leu-2a antigen at exacerbation phase and became double-labeled cells (Leu-2a+, 3a+), which is known to be a feature of thymocytes. Thus, it appeared that ATL cells sometimes change their surface phenotype to that of an earlier stage of T cell differentiation at relapse or at exacerbation phase. Chronic myelocytic leukemia (CML) cells also usually change to immature cells at blastic crisis involving morphological change. However, this morphological change was not so prominent in the ATL cases studied, except one, in which typical ATL cells with nuclear indentation changed to large immature cells with basophilic cytoplasm at relapse.

The leukemic cells of 57 patients with adult T cell leukemia (ATL) were analyzed for their immunologic surface markers. Forty-four cases showed normal mature inducer/helper T cell phenotype (typical group: E-RFC+, Leu-1+, 2a-, 3a+ MASO36c-), but the other 13 cases showed unusual surface phenotypes (variant group) and could be subdivided into several groups (V1 to V5). Four cases had absent or low Leu-1 positivity (V1: E-RFC+, Leu-1-, 2a-, 3a+, MASO36c-), while two other cases with low Leu-1 positivity had both Leu-2a and 3a, a characteristic of cortical thymocytes, but were unreactive with MASO36c (V2: E-RFC+, Leu-1-, 2a+, 3a+, MASO36c-). Three cases lacked both Leu-2a and 3a despite having other T cell markers (V3: E-RFC+, Leu-1+, 2a-, 3a-, MASO36c-). The next three cases had low rosette-forming ability with sheep RBCs (V4: E-RFC-, Leu-1- approximately +, 2a- approximately +, 3a+, MASO36c-). Interestingly, one other case showed high reactivity against anti-Leu-7, which is believed to be one of the monoclonal antibodies directed against natural killer cells (V5: E-RFC+, Leu-1+, 2a-, 3a+, 7+, MASO36c-). Clinical and hematologic differences between the typical group and variant group were investigated, and it was found that the variant group (excluding V5) have statistically significant (P less than .002) higher serum lactic dehydrogenase (LDH) activity. The overall survival in the variant group was worse than in the typical group, but it was not quite statistically significant (P = .072). The median survival time was eight months for typical cases and only four months for variant cases; six cases died within two months. The V5 case was unusual not only because the patient's leukemic cells have Leu-7 antigen but also because she survived more than nine years after initial diagnosis. There seems to be some correlation between phenotypic diversity of ATL cells and prognosis.

We report the clinical, hematologic, and immunologic features of 18 preleukemic adult T cell leukemia (pre-ATL) cases with abnormal T lymphocytosis induced by human adult T cell leukemia-lymphoma virus (HTLV/ATLV). The patients were from the Nagasaki district, which is one of the most endemic areas of ATL in Japan. Pre-ATL is a subclinical T cell abnormality differing from ATL. It is characterized by an insidious onset and appearance of abnormal T lymphocytes (10% to 40%) in the peripheral blood without clinical symptoms except for a few cases transiently presenting fever, skin eruptions, and slight lymphadenopathies. Most abnormal T lymphocytes were small and mature with incised or lobulated nuclei and formed E rosettes with sheep RBCs. Virologic and biomolecular analysis revealed that all cases were infected with HTLV, and proviral DNA was integrated in host lymphocytes from 12 of the 14 cases examined. Furthermore, the lymphocyte populations, including abnormal T lymphocytes, were monoclonal with respect to the site of the provirus integration. Abnormal T lymphocytosis persisted from one to more than seven years in six cases, three of which developed ATL after a one- to five-year pre-ATL stage, whereas abnormal T lymphocytes spontaneously decreased in the other seven patients. However, HTLV-infected monoclonal lymphocytes were detected in four cases examined, even after most of the abnormal T lymphocytes had disappeared. Moreover, the same clonally provirus-integrated lymphocytes persisted in two of four cases not only during the course of abnormal lymphocytosis, but also in the subsequent almost-normal blood. These results indicate that the majority of the cases were in a pre-ATL state with a potential to develop ATL.

We report phenotypic and functional characterization of lymph node cells from 27 patients with T lymphoma from the Nagasaki district of Japan. Nagasaki is one of several areas where adult T-cell leukemia is endemic, and sera from 18 of 23 patients examined (78.3%) were positive for antibody to adult T-cell leukemia-associated antigen. The majority of cases (24 of 27) showed the Leu-2a-, 3a+, MASO36c- phenotype similar to adult T-cell leukemia, but seemed to be subdivided into four groups according to the presence or absence of Leu-1 antigen and the receptor for sheep erythrocytes (SRBC). Eleven cases had both Leu-1 antigen and the receptor for SRBC (E-RFC+, Leu-1+, 2a-, 3a+), but 6 cases lacked Leu-1 antigen (E-RFC+, Leu-1-, 2a-, 3a+), 4 cases lacked receptors for SRBC (E-RFC-, Leu-1+, 2a-, 3a+), and 3 cases lacked both of these markers (E-RFC-, Leu-1-, 2a-, 3a+). The effects of lymph node T cells on pokeweed mitogen (PWM)-induced normal B-cell differentiation was studied in 10 cases. Lymph node T cells of 5 cases showed helper activity without any suppressor activity, and 2 cases showed suppressor activity with almost no detectable helper activity, similar to adult T-cell leukemia cells. Two other cases lacked particular functional effects. These results suggest the possibility that Leu-3a+ T-cell lymphoma may be functionally subdivided into at least two types, a helper type and a suppressor (adult T-cell leukemia) type.

By using human T-cell growth factor (TCGF), 10 cell lines were established from tissue samples of 10 patients with adult T-cell leukemia (ATL). Three cell lines were adapted to growth in medium lacking TCGF. The surface markers of all cell lines were characteristic of inducer/helper T cells, i.e., OKT3+, OKT4+, OKT6-, OKT8-, OKIa1+, and human Lyt2+ and Lyt3+, except that one cell line was OKT3-. The expression of the viral antigen was examined during establishment of 8 of the 10 cell lines. The viral antigen was not expressed in leukemic cells before cultivation. In 5 lines, the viral antigen was detected by immunofluorescent staining after a short period of cultivation. However, 3 cell lines, ATL-6A, ATL-9Y, and ATL-1K did not express the viral antigen during short-term culture: the ATL-6A and ATL-9Y cell lines became positive for the viral antigen after 5 and 2 months of cultivation, respectively; the ATL-1K cell line remained antigen-negative throughout a culture period of 13 months. Southern blot hybridization assay showed that all of the cell lines, including the viral antigen-negative ATL-1K cell line, contained the viral genome. Thus, the retrovirus was associated with all 10 cell lines established from ATL patients, but there was a heterogeneity in the expression time of the retroviral antigen in leukemic cells maintained in vitro. Our findings suggested that the expression of the viral antigen was not required for maintenance of the leukemic state in vivo and for growth of leukemic cells in vitro.