Immune responses and serum levels of cytokines in adult T-cell leukemia patients and human T-cell leukemia virus type-I carriers.

To find predictive parameters for development and progression of adult T-cell leukemia (ATL) in human T-cell leukemia virus type-I (HTLV-I) carriers, we investigated cellular immune responses such as mitogenic responses and natural killer activity of the peripheral blood mononuclear cells (PBMC). And serum or plasma levels of cytokines, including tumor-necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and immunosuppressive acidic protein (IAP), were also measured in patients with ATL, healthy HTLV-I carriers and healthy HTLV-I non-carriers as controls. Results are as follows: (1) increased spontaneous proliferation and decreased mitogenic responses of PBMC already existed in HTLV-I carriers; (2) IAP was significantly higher in patients with acute/lymphoma type ATL than in those with chronic/smoldering type, HTLV-I carriers and HTLV-I non-carriers. These results suggest that spontaneous proliferation or mitogenic responses and IAP may be useful parameters for the development and progression of ATL from the carriers. Since HTLV-I carriers already have various grades of immunosuppression, we should seriously try to prevent further HTLV-I transmission.     

Urinary excretion of parathyroid hormone-related protein in patients with adult T-cell leukemia and other hematologic disorders]

Urinary excretion of parathyroid hormone-related protein (PTH-rP) was measured by radioimmunoassay in 25 patients with adult T-cell leukemia (ATL), in 68 patients with other hematologic disorders and in 13 asymptomatic individuals seropositive for human T-cell leukemia virus type I (HTLV-I). The mean levels of urinary PTH-rP in ATL patients with hypercalcemia (11.01 micrograms/g.Cr) were higher than in ATL patients with normocalcemia (5.16 micrograms/g.Cr). The mean levels in patients with acute type (8.84 micrograms/g.Cr), lymphoma type (4.18 micrograms/g.Cr) and crisis ATL (18.20 micrograms/g.Cr) were significantly higher than in urine of healthy controls. However, all asymptomatic carriers of HTLV-I and patients with chronic and smoldering ATL had normal urinary PTH-rP levels. In 7 patients with acute myelogenous leukemia, 1 patient with blastic crisis of chronic myelogenous leukemia and 3 patients with malignant lymphoma, the urinary levels of PTH-rP were above the normal range. Urinary levels of PTH-rP of the ATL patients with hypercalcemia correlated with the serum calcium levels. Urinary levels of PTH-rP of the all ATL correlated with serum lactic dehydrogenase level. These findings suggest that the measurement of urinary levels of PTH-rP is useful for evaluation of ATL and that some tumor cells of other hematologic diseases may produce PTH-rP.     

Natural antibodies in sera from Japanese individuals infected with HTLV- I do not recognize HTLV-III

Seventy-one sera from Japanese individuals infected with human T cell leukemia virus type I (HTLV-I) were examined for the presence of antibodies to HTLV-III by an enzyme-linked immunosorbent assay (ELISA) and by a strip radioimmunoassay based on the Western blot technique. The sera were from 23 healthy carriers and from 48 patients, including 18 with smoldering adult T cell leukemia (ATL), 13 with chronic ATL, and 17 with acute ATL. All people tested lived in the southwestern part of Japan, a known endemic area for HTLV-I infection. Antibodies against HTLV-I were detected in all sera both by indirect immunofluorescent methods and strip radioimmunoassay using cell lysates. Six sera were reactive in the ELISA assay for HTLV-III. But these sera did not react specifically to HTLV-III-related proteins (p15, p24, gp41) when analyzed by strip radioimmunoassay. Our data suggest that coincidental infection of HTLV-I and HTLV-III is quite rare in Japan.     

Immunological studies on opportunistic infection and the development of adult T-cell leukemia.

Three patients with opportunistic infection preceding adult T-cell leukemia (ATL) are presented: a 66-year-old woman with cryptococcosis and Pneumocystis carinii pneumonia, a 46-year-old man with Pneumocystis carinii pneumonia, and a 55-year-old woman with cryptococcosis. Although, at the first examination, the first two had smoldering type ATL and the third case was an HTLV-I carrier, all three developed overt ATL 14-16 months after the onset of opportunistic infection. It is considered that there is immune suppression already present in HTLV-I carriers and in patients with smoldering ATL, and opportunistic infection is predictive of the development of ATL.     

Clonal integration and expression of human T-cell lymphotropic virus type I in carriers detected by polymerase chain reaction and inverse PCR

Adult T-cell leukemia (ATL) is a neoplasm of mature helper (CD4) T lymphocytes, and human T-cell lymphotropic virus type-I (HTLV-I) has been suggested to be the causative virus of ATL. HTLV-I integrates its proviruses into random sites in host chromosomal DNA. Clonal integration has been observed in patients with ATL, including smoldering, chronic, and acute states. However, random and/or polyclonal integration has only been reported in a few asymptomatic HTLV-I carriers. To clarify the clonality of HTLV-I-infected cells in carriers, we used an inverse polymerase chain reaction (IPCR), which is more sensitive than Southern blot analysis. We used the peripheral blood momonuclear cells (PBMC) from 16 asymptomatic carriers and the separated CD4-positive cells. No cases showed either a monoclonal or polyclonal integration of the HTLV-I provirus by Southern blot. But, using IPCR, 7 of 16 cases showed either mono- or oligoclonal integration. In addition, the populations of clonal provirus in the total PBMC were frequently different from those in the CD4-positive cells. Three cases showed expression of HTLV-I tax/rex mRNA in the total PBMC, but no such expression was found in CD4-positive cells. In this study, an unexpected frequency of clonal HTLV-I provirus DNA was observed in HTLV-I carriers. These findings indicate that the clonal but nonmalignant proliferation of HTLV-I-infected cells already occurs even in HTLV-I carriers, and therefore that some other step is necessary to induce malignant proliferation. Am. J. Hemato. 54:306–312, 1997. © 1997 Wiley-Liss, Inc.     

Human T lymphotropic virus type-I and adult T-cell leukemia in Japan

HTLV-I is the first retrovirus to be associated directly with human malignancy. In ATL-endemic areas, the rate of HTLV-I carriers is high. Both HTLV-I and ATL have been shown to be endemic in some regions of the world, especially in southwest Japan, the Caribbean islands, South Americas, and parts of Central Africa. Antibodies against HTLV-I have been found in over one million individuals, and more than 700 cases of ATL have been diagnosed each year in Japan alone. The cumulative incidence of ATL among HTLV-I carriers in Japan is estimated at 2.5% (3-5% in males, 1-2% in females). In endemic areas, HTLV-I Ab were found in the sera of 6 to 37 percent of healthy adults over 40 years of age. This clustering is thought to be due to the limited transmission of virus between socially isolated populations. The diagnostic criteria for HTLV-I associated ATL have been defined as follows. 1) Histologically and/or cytologically proven lymphoid malignancy with T cell antigens. 2) Abnormal T-lymphocytes present in the peripheral blood, except in the lymphoma type. 3) Serum specimens for all patients with ATL have HTLV-I Ab. 4) Demonstration of clonality of HTLV-I proviral DNA is a definite diagnosis of ATL. ATL shows diverse clinical features but can be divided into four subtypes: acute, chronic, smoldering, and lymphoma type. The pattern of HTLV-I transmission is through one of three different modes. Infected mothers can transmit the virus to newborns mainly via breast milk. The virus also can be transmitted from male to female by sexual intercourse, and through blood transfusion. Chemotherapy is not effective; the acute and lymphoma types have a poor prognosis. ATL is generally treated with curative intent using combination chemotherapy, although long-term success has been very limited. Unfortunately that advance did not translate into an improvement in the overall survival; the median remain 10 months. In contrast, smoldering ATL, or some cases of chronic ATL, may have a more protracted natural course, which may be compromised by aggressive chemotherapy. Alternative strategies for both acute and chronic forms are clearly needed. After infection of HTLV-I, there is a long latent period before onset of ATL. Analyses by PCR showed that clearly proliferation occurred in intermediate state or even carriers with high virus load. Such clonal proliferation might be preleukemic stage, which suggested that carriers with high virus load should be risk group to have ATL.     

Clinical study on the serum levels of immunosuppressive acidic protein in patients with adult T-cell leukemia

Serum levels of immunosuppressive acidic protein (IAP) were measured in patients with adult T-cell leukemia (ATL) in order to clarify its significance in this disease. The mean levels in patients with both acute (854 +/- 404 micrograms/ml) and chronic ATL (439 +/- 103 micrograms/ml) were significantly higher than in sera of healthy controls (367 +/- 104 micrograms/ml). However, mean levels in patients with smoldering ATL, healthy T-cell lymphotropic virus type 1 (HTLV-1) carriers and healthy controls showed no differences. Levels in crisis in chronic and smoldering ATL were similar to those in patients with acute ATL. Serial measurements of serum IAP in a number of patients revealed that the levels reflected each patient's clinical course, suggesting a potential for use in evaluating the effects of chemotherapy.     

Studies on association between the ATL and the development of multiple malignant neoplasms--analysis of 1171 cases of hematological malignancies during the past 24 years

A high incidence of multiple primary neoplasms has been observed in our patients with ATL in comparison to persons with other forms of hematologic malignancy who we have observed during the past 24 years (1963-1985). Five of 15 patients with ATL (33.3%) have had at least one other associated neoplasm in comparison to only 44 of 1156 patients with other forms of hematological malignancy (3.8%). The incidence figures for secondary neoplasms associated with the other hematologic malignancies were 4.3% (16/370) for acute non-lymphocytic leukemia (ANLL), 2.2% (2/90) for acute lymphocytic leukemia (ALL), 4.8% (1/21) for acute unclassifiable leukemia, 2.2% (5/225) for chronic myelogenous leukemia, 4.7% (2/43) for chronic lymphocytic leukemia, 5.9% (8/136) for malignant monoclonal gammopathy and 3.7% (10/271) for malignant lymphoma. The incidence of multiple neoplasms in patients with ATL in comparison to those with other hematological malignancies was significant (p less than 0.01 or p less than 0.001). The neoplasms associated with ATL have been adenocarcinoma of the thyroid or lung, and squamous cell carcinoma of the larynx, lip or lung. We identified ATL-derived factor (ADF) in the cytoplasm of the secondary neoplasms of the ATL patients by means of indirect immunofluoroscopy and immunohistochemical techniques utilizing anti-ADF antibody. We also identified ras p21 products in these neoplasms by means of p21 ras monoclonal antibody studies. The possibility that HTLV-I was the cause of the secondary neoplasms thus was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infection of human T-cell leukemia virus type I and development of human T-cell leukemia lymphoma in patients with hematologic neoplasms: a possible linkage to blood transfusion

Among 354 adult patients with either hematological malignancy or aplastic anemia, eight were positive for anti-HTLV-I antibodies; six of eight had received multiple transfusions. There was an approximately 3.5-fold increase (P less than .001) of HTLV-I seropositivity in the patients with hematologic disease (8 of 354, 2.23%) compared to the healthy adults older than 20 years (34 of 5252, .65%). Two hematological patients, one with Hodgkin's disease and one with acute promyelocytic leukemia, were found to be positive for HTLV-I, and developed and died of adult T-cell leukemia/lymphoma (ATL) subsequently. Both were long-term survivors of the primary disease and had received multiple transfusions. The latent period from blood transfusion to onset of ATL was 6 months and 11 years, respectively. Immunocompromised patients, who were seropositive for HTLV-I, may be at increased risk for ATL compared to healthy carriers of HTLV-I, and the latent period may be shorter.     

Detection of HTLV-ImRNA by in situ hybridization technique using biotinylated oligonucleotide probe]

The expressions of human T-lymphotropic virus type I (HTLV-I) mRNA in the HTLV-I-infected lymphocytes were studied in the peripheral blood of two HTLV-I carriers and of three patients with adult T-cell leukemia (ATL) (acute, chronic and lymphoma types) by in situ hybridization technique using two biotinylated single-stranded oligodeoxynucleotide probes complementary to different nucleotide sequences in the mRNA for the HTLV-IpX region. A low percentage of leukocytes (1.5-7.4%) reacted with the probes in ATL patients, while less than 1% of leukocytes was reactive in HTLV-I carriers. These results indicate that a part of ATL cells express the HTLV-IpX, which is thought to be involved in the leukemogenesis of ATL in the peripheral blood of the patients.     

Natural history of HTLV-I infection]

Adult T-cell leukemia (ATL), a disease entity first described by Takatsuki et al., is endemic in southwestern Japan, the Caribbean Islands, and in some parts of Africa. ATL patients are classified into four subtypes according to the clinical picture: acute, chronic, smoldering, and lymphoma type. The diagnosis of ATL is made from the characteristic clinical findings, the detection of serum antibodies to HTLV-I, and when necessary, the confirmation of monoclonal integration of HTLV-I proviral DNA in cellular DNA of ATL cells. Recently, diagnostic criteria for clinical subtypes of ATL were proposed by the Lymphoma Study Group in Japan: 1) smoldering type, normal lymphocyte level, no hypercalcemia, lactate dehydrogenase (LDH) value 1.5 times the upper limit of normal or lower, no lymphadenopathy, no involvement of liver, spleen, central nervous system (CNS), bone or gastrointestinal tract, and no ascites or pleural effusion: 2) chronic type, absolute lymphocytosis with T-lymphocytosis of greater than 3 x 10(9)/1, LDH value twice the upper limit of normal or lower, no hypercalcemia, no involvement of CNS, bone, or gastrointestinal tract, and no ascites or pleural effusion: 3) lymphoma type, no lymphocytosis, 1% or less abnormal lymphocytes, and histologically-proven lymphadenopathy: 4) acute type, remaining ATL patients who are not classified as any of the above types. Infection with HTLV-I is a direct cause of ATL. Furthermore, infection with this virus can indirectly cause many other diseases via the induction of immunodeficiency, such as chronic lung diseases, opportunistic lung infections, cancer of other organs, monoclonal gammopathy, chronic renal failure, strongyloidiasis, non-specific dermatomycosis, non-specific lymph node swelling, HTLV-I associated myelopathy (HAM/TSP), and HTLV-I uveitis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Soluble interleukin 2 receptors in sera of Japanese patients with adult T cell leukemia mark activity of disease

Serum concentrations of soluble interleukin 2 receptors (sIL 2R) were measured by an enzyme-linked immunosorbent assay (ELISA) in 30 patients with adult T cell leukemia (ATL), in 9 patients with other hematopoietic malignancies, and in 17 asymptomatic individuals seropositive for human T cell leukemia virus type I (HTLV-I). Sixty HTLV-I seronegative, age-matched controls showed a normal range of form 63.2 to 480.8 U/mL. All asymptomatic carriers of HTLV-I had sIL 2R in their sera within the normal range. sIL 2R in sera was not related to the anti-HTLV-I antibody titer. Eleven patients with acute ATL, a clinical phenotype with median survival rate of 4.4 months, had markedly elevated sIL 2R (11,100 to 99,000 U/mL), but eight patients with smoldering ATL had low sIL 2R values (less than 480.8 U/mL) comparable to controls. Eleven patients with chronic ATL had intermediate elevated levels of sIL 2R (480.8 to 37,300.0 U/mL). Serum levels of sIL 2R correlated with the number of ATL cells (r = 0.812) and CD25-positive cells (r = 0.725) circulating in the peripheral blood. Longitudinal studies performed in four patients with ATL showed significant correlation between serum concentration of sIL 2R and activity of the malignancy. These findings suggest that the level of sIL 2R in serum indicated tumor load and, possibly, prognosis.     

Cytogenetic studies of healthy HTLV-I carriers (Report 1): Cytogenetic studies of an ATL family

The chromosome 14q11 anomaly has been reported to be specific to adult T-cell leukemia (ATL) and this anomaly has also been confirmed in preleukemic state of ATL (pre-ATL) patients though the frequency is low. In an attempt to clarify if the same chromosome aberrations could be found also at the stage of HTLV-I carrier and if there is any cytogenetic difference from non-HTLV-I carriers, a cytogenetic study of lymphocytes stimulated with phytohemagglutinin in three HTLV-I healthy carriers and three non-HTLV-I carriers in an ATL family was performed. The results were as follows. 1. In three HTLV-I carriers, 7 of 311 cells examined (2.3%) showed chromosome aberrations, and 4 cells (1.3%) had 14q11 anomaly. 2. In three non-HTLV-I carriers, 4 of 260 cells examined (1.5%) showed chromosome aberrations, whereas no cells had 14q11 anomaly. These findings suggest that 14q11 anomaly is already present at the stage of HTLV-I carrier and seems to be an important cytogenetic clue to the pathogenesis of ATL.     

Serum dehydroepiandrosterone and DHEA-sulfate in patients with adult T-cell leukemia and human T-lymphotropic virus type I carriers

The serum levels of dehydroeplandrosterone (DHEA) and DHEA-sulfate (DHEA-S) were determined by radioimmunoassay in 38 patients with adult T-cell leukemia (ATL). Levels of serum DHEA and DHEA-S were also measured in 60 human T-lymphotropic virus type I (HTLV-I) carriers, and did not differ from those in 60 healthy control subjects. Serum levels in patients with ATL were lower than those in the age- and sex-matched healthy controls and in HTLV-I carriers with statistical significance. Serum DHEA and DHEA-S in male patients with acute and lymphoma-type ATL were 1.06 ± 0.77 ng/ml and 245.8 ± 192.9 ng/ml, respectively. Levels in male patients with chronic and smoldering-type ATL were 1.69 ± 0.68 ng/ml and 477.6 ± 251.5 ng/ml, respectively. Serum levels of DHEA and DHEA-S in patients with acute and lymphoma-type ATL were significantly lower than those in patients with chronic and smoldering-type ATL (P < 0.05). These data suggest that a decrease in serum levels of DHEA and DHEA-S may be associated with patients who have some clinical subtypes of ATL. Moreover, androgens may have a therapeutic role in patients with ATL, as administered in patients with hairy-cell leukemia. Because there is at present no curative chemotherapy for ATL, a trial combination of androgens and standard chemotherapy may be a reasonable therapeutic option in such patients. © 1996 Wiley-Liss, Inc.     

Existence of escape mutant in HTLV-I tax during the development of adult T-cell leukemia

Although Tax protein is the main target of cytotoxic T lymphocyte (CTL) on human T-cell lymphotropic virus type I (HTLV-I)-infected cells, and Tax peptide 11 through 19 binding to HLA-A*02 has been shown to elicit a strong CTL response, there are patients with adult T-cell leukemia (ATL) bearing HLA-A*02. To explore whether there is genetic variation in HTLV-I tax that can escape CTL recognition during the development of ATL, the HTLV-I tax gene was sequenced in 55 patients with ATL, 61 patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and 62 healthy carriers, and it was correlated with the presence of HLA-A*02. First, a premature stop codon in the 5' half of the tax gene that looses transactivation activity on the viral enhancer was observed in 3 patients with acute and 1 patient with chronic ATL. This stop codon was revealed to emerge after the viral transmission to the patient from sequence analysis in family members with ATL. Second, amino acid change in Tax peptide 11-19 was observed in 3 patients with ATL. CTL assays demonstrated that this altered Tax 11-19 peptide, observed in ATL patients with HLA-A*02, was not recognized by Tax 11-19-specific CTL. Two patients with ATL had large deletions in tax by sequencing, and 5 patients with ATL had deletions in HTLV-I by Southern blotting. These findings suggest that at some stage of ATL development, HTLV-I-infected cells that can escape the host immune system are selected and have a chance to accumulate genetic alterations for further malignant transformation, leading to acute ATL.     

A monoclonal antibody detecting a novel antigen expressed in the HTLV-I- infected cells

A monoclonal antibody, FTF 148, was prepared by hybridizing murine myelomal cells (NS-1) and spleen cells of BALB/c mice immunized with cultured cells derived from an adult T cell leukemia (ATL) patient (KUT- 2 cells). This monoclonal antibody reacted with all of the human T cell leukemia virus I (HTLV-I)-infected cell lines tested but did not react with other T cell lines derived from acute lymphocytic leukemia, Epstein-Barr virus-transformed B cell lines, or an erythroleukemic cell line. This monoclonal antibody was not directed to viral antigens because it reacted equally well with almost all KUT-2 and MT-1 cells, only 1% to 3% of which were ATL-associated antigen-positive. In contrast to interleukin 2 receptors expressed on both ATL cells and normal phytohemagglutinin-stimulated blasts, this antigen was not expressed on the latter cells. The antigen, mainly expressed on the cell membrane, was analyzed by metabolic labeling with 3H-leucine and surface labeling with 125I followed by cell lysis and immunoprecipitation with the FTF 148 antibody. The findings obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that p50 and p74 proteins were specifically precipitated and the antigen was also different from the product of the Xs gene of HTLV-I.     

Expression of fos proto-oncogene product by monoclonal antibody FO-120 in smouldering adult T-cell leukaemia (ATL)

We investigated the expression of c-fos gene product in peripheral blood mononuclear cells of patients with smouldering adult T-cell leukaemia (ATL) and healthy human T-lymphotropic virus type-I (HTLV-I) carriers by an immunofluorescence assay, using a mouse monoclonal antibody (FO-120) specific for fos gene product. Peripheral blood mononuclear cells derived from healthy HTLV-I carriers were rarely positive for FO-120, less than 2% of the cells weakly reacted with FO-120, whereas positive cells were detected in more than about 10% of cells from patients with smouldering ATL.
FO-120 appears to be a useful tool for detecting smouldering ATL in asymptomatic HTLV-I infected people without using molecular techniques.     

Heterogeneity in clonal nature in the smoldering subtype of adult T-cell leukemia: continuity from carrier status to smoldering ATL

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.     

Diagnosis and management of adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell malignancy caused by human T-cell leukemia virus type I (HTLV-1). Between 3% and 5% of HTLV-1-infected individuals develop ATL after a long latency. Confirmation of seropositivity of anti-HTLV-1 antibody, and clonal proliferation of CD4 and CD25 positive lymphocytes with nuclear pleomorphism in patients suspicious of malignant lymphoma or chronic lymphocytic leukemia is crucial for the diagnosis of ATL. The clinical course of ATL is very heterogeneous, and divided into acute, lymphoma, chronic, and smoldering types. The chronic type is further subclassified into the favorable and unfavorable subtypes. Acute, lymphoma, and unfavorable chronic type ATL, and favorable chronic and smoldering type ATL are defined as aggressive and indolent ATL, respectively. Recently identified prognostic indices based on clinical parameters and/or genetic predictors of outcomes need to be confirmed and incorporated for more stratified therapeutic interventions. The standard of care for aggressive ATL is multiagent chemotherapy followed by allogeneic hematopoietic stem cell transplantation if possible, while that for indolent ATL is watchful waiting until progression to aggressive ATL. The combination of interferon-α and zidovudine is also standard for leukemic type ATL. In addition, mogamulizumab, lenalidomide, and brentuximab vedotin have been incorporated into clinical practices in Japan. Furthermore, several novel drugs are currently undergoing clinical trials.