Torahiko Makino

Expression of the multidrug transporter, P-glycoprotein, in acute leukemia cells and correlation to clinical drug resistance

The overexpression of a cell‐surface glycoprotein termed P‐glycoprotein (P‐gp) is frequently associated with multi‐drug resistance (MDR) in cell lines in vitro. To evaluate the implications of P‐gp expression in clinical drug resistance, the authors examined the expression of P‐gp in leukemia cells from patients with acute myelogenous leukemia (AML) and those with acute lymphoblastic leukemia (ALL) at initial presentation and relapse, using immunoblotting with a monoclonal antibody against P‐gp, C219. Nine of 17 patients with AML and four of 11 patients with ALL had P‐gp‐positive results at the initial presentation, and most P‐gp‐positive patients did not respond to chemotherapy. Four of seven patients at the relapsed stage and all three patients with preceding myelodysplastic syndrome had P‐gp‐positive results. The expression of P‐gp and clinical refractoriness to chemotherapy were highly correlated. These data indicate that the expression of P‐gp is closely related to clinical drug resistance in acute leukemia.

Arsenic trioxide inhibits growth of human T-cell leukaemia virus type I infected T-cell lines more effectively than retinoic acids.

Adult T‐cell leukaemia (ATL) is difficult to cure using conventional therapies. Recently the therapeutic possibility of retinoic acids (RA) has been reported. In this study, suppression of in vitro growth of human T‐cell leukaemia virus type I (HTLV‐I) infected T‐cell lines and fresh ATL cells by arsenic trioxide (As2O3) were evaluated by comparison with a series of RA derivatives. Proliferation of four HTLV‐I‐infected T‐cell lines was significantly reduced within 72 h by 1.0 μmol/l As2O3. Growth of two out of four HTLV‐I‐infected T‐cell lines was also inhibited by 1.0 μmol/l RA, but to a lesser extent than by As2O3. The mechanism of this growth inhibition was due to the induction of apoptosis. Apoptosis was also induced in fresh ATL cells from patients by As2O3, but far less by RA. As described in patients with acute promyelocytic leukaemia, 1.0 μmol/l of As2O3 can be safely achieved in the serum of patients; however, it is difficult to maintain this concentration of RA. In conclusion, As2O3 has therapeutic potential for the treatment of ATL and may be far more clinically beneficial than RA.

The prevalence of human T-cell leukemia virus type I infection in patients with hematologic and nonhematologic diseases in an adult T-cell leukemia-endemic area of Japan

In order to clarify the prevalence of human T‐cell leukemia virus type I (HTLV‐I) infection in the Kagoshima district, Japan, a highly endemic area for HTLV‐I, antibodies for HTLV‐I (anti‐HTLV‐I) were examined in the sera of 6167 from healthy residents and patients with various hematologic and nonhematologic diseases. In healthy residents, including blood donors, the prevalence of anti‐HTLV‐I was 11.9% (562/4741 persons). The prevalence increased with age, and was significantly higher in females than in males (P < 0.01). The prevalence of anti‐HTLV‐I in blood donors was 8.5%. In hematologic diseases, the prevalence of anti‐HTLV‐I was 98.3% in ATL, 28.9% in lymphoproliferative disorders except ATL, and 10.6% in myeloproliferative disorders. In nonhematologic diseases, the prevalence of anti‐HTLV‐I was shown to be 29.5% in pulmonary tuberculosis, 25.8% in leprosy, 33.8% in chronic renal failure (CRF), 21.9% in autoimmune diseases, and 47.8% in strongyloidiasis. The various diseases except myeloproliferative disorders had significantly higher prevalence of anti‐HTLV‐I than healthy residents (P < 0.01 or 0.05). For autoimmune diseases, the prevalence of anti‐HTLV‐I in patients with blood transfusion (55.6%) was higher than in those without blood transfusion (8.7%), and healthy residents. In hemodialysis patients with CRF who had received blood transfusions the prevalence of anti‐HTLV‐I increased with the number of blood transfusions. Therefore, HTLV‐I transmission via blood transfusion would partially explain these high prevalence of anti‐HTLV‐I. However, the prevalence of anti‐HTLV‐I in hemodialysis patients with CRF was statistically higher than that in healthy residents, regardless of blood transfusion (P < 0.01). Furthermore, hemodialysis patients showed significantly higher prevalence of anti‐HTLV‐I than healthy residents, even at a younger age. Patients with pulmonary tuberculosis and leprosy showed the same results as hemodialysis patients. These results suggest the possibility that HTLV‐I infection has some relation not only to ATL but also to other diseases. Therefore, it seems very important to halt the spread of HTLV‐I transmission as soon as possible.

Phase II study of cladribine (2-chlorodeoxyadenosine) in relapsed or refractory adult T-cell leukemia-lymphoma

Adult T-cell leukemia-lymphoma (ATL) is a retrovirus-associated T-cell malignancy with an extremely poor prognosis; the median survival time of ATL patients with the acute or lymphoma type is less than 1 year with various combination chemotherapies. Cladribine (2-chlorodeoxyadenosine; 2-CdA), a purine analog resistant to degradation by adenosine deaminase, has shown definitive clinical activity against various lymphoid malignancies, including hairy cell leukemia, indolent lymphoma, and cutaneous T-cell lymphoma. An in vitro study showed the sensitivity of T-lymphoblastoid cell lines to cladribine, and a preceding Japanese phase I study of cladribine showed that 1 refractory patient with ATL achieved an objective response. To evaluate the therapeutic efficacy of cladribine in treating ATL, we conducted a multicenter phase II study. The plan was to administer cladribine to 30 ATL patients as 0.09 mg/kg per day by 7-day continuous intravenous infusion every 4 weeks for up to 3 courses. Before the planned interim analysis, 16 patients with relapsed or refractory ATL were enrolled, 15 of whom were eligible. Only 1 of the 15 eligible patients showed an objective response (overall response rate, 7%; 90% confidence interval, 0% to 28%), and 11 patients (73%) showed progressive disease, mostly during the first course of treatment. Because the upper limit of the 90% confidence interval of the overall response rate did not reach 30% in the interim analysis, the Independent Monitoring Committee advised us to discontinue patient enrollment. In conclusion, cladribine is not worthy of further investigation for the treatment of ATL.

Combination chemotherapy (RCM protocol: response-oriented cyclic multidrug protocol) for the acute or lymphoma type adult T-cell leukemia.

43 patients with the acute or lymphoma type ATL were treated with the new combination chemotherapy (RCM protocol: response-oriented cyclic multidrug protocol) between January 1989 and December 1991. Complete response (CR) and partial response (PR) were achieved in 20.9% and 65.1% of all treated patients respectively. The median duration of survival was 6.0 months. The survival duration of patients with a high serum lactate dehydrogenase (LDH) value (> or = 1,000 unit) and/or a poor performance status (PS) (PS 3 or 4) were also improved but not in patients with a severe leukocytosis (> or = 35,000/microliters). Toxicity was mild (grade 1 or 2) except hematologic toxicity in 4 patients (9.3%) and alopecia in one patient (2.3%). In spite of many patients with a poor PS (PS 3 or 4), our chemotherapeutic results are equal or superior to other previous reports. It seems that response-oriented chemotherapy is suitable for the ATL patients with poor prognostic factors. These results indicate that the RCM protocol is very useful as the first choice chemotherapy for the acute or lymphoma type ATL.

Random integration of HTLV-I provirus; increasing chromosomal instability

Adult T-cell leukemia/lymphoma (ATLL) is a neoplasm of mature helper (CD4) T-lymphocytes. Human T-cell lymphotropic virus type-I (HTLV-I) is etiologically considered to cause ATLL. It has been suggested that HTLV-I integrates its provirus into random sites in host chromosomal DNA after infection. Clonal integration has been observed in patients with ATLL, including smoldering, chronic and acute leukemia states. Almost all cases with ATLL demonstrate clonal chromosome abnormalities, with karyotypes being very complicated in both number and structure. However, there are no specific karyotype abnormalities in ATLL. In order to examine the role of HTLV-I in the pathogenesis of ATLL, we investigated whether or not HTLV-I randomly integrates and whether the integration site in the human genome is associated with any chromosomal abnormality. We analyzed 18 cases with ATLL, which included 15 cases with acute states, two cases with chronic states and one case with a smoldering state. In four of the 18 cases, the HTLV-I provirus integrated into the 9th chromosome, while in three cases, it integrated into the 1st or 10th chromosome. However, the integrated site in the chromosome varied in each case and the random integration was considered to be true. All 15 cases with acute ATLL had complicated chromosomal abnormalities and two cases with chronic and smoldering ATLL showed simple abnormal karyotypes, while one case with chronic ATLL showed a normal karyotype. In 15 of the 18 cases, the chromosomes with HTLV-I integration showed abnormalities. In particular, in two cases with simple chromosome abnormalities, HTLV-I integrated into the abnormal chromosome, but not into the normal chromosome. The HTLV-I proviral integration thus seems to be associated with chromosome abnormalities. In the multistage leukemogenesis of ATLL, these findings indicate that HTLV-I integration might play an important role in the induction of chromosomal instability.

Treatment for adult T-cell leukemia

Abstract The purpose of this study was to clarify the clinical efficacy of multidrug chemotherapy for aggressive adult T-cell leukemia (ATL). We report the therapeutic results of treatment of patients with aggressive ATL undertaken between 1986 and 1995. A total of 120 newly diagnosed patients with a performance status of 0–3 and aged <70 years at diagnosis were entered into the study. Clinical features, including clinical subtypes, serum levels of lactate dehydrogenase and blood urea nitrogen, the response to chemotherapy, and doses of individual chemotherapeutic agents, were evaluated. Of the 120 patients enrolled, 97 had acute-type and 23 lymphoma-type ATL. The complete response rate and median survival of these patients were 25.3% and 9 months, respectively. The 2- and 5-year survival rates were 18.4% and 8%, respectively, and five patients have been alive for >5 years and are disease-free. These long-term survivors had good prognostic factors at diagnosis. There was no correlation between the doses of the various chemotherapeutic agents and the survival duration. These results indicate that ordinary combined chemotherapy has limited ability to improve the prognosis of aggressive ATL. Our previous study indicated that expression of P-glycoprotein in ATL cells might be involved in resistance to chemotherapeutic agents, particularly doxorubicin, vincristine, and etoposide. Therefore, new therapeutic strategies will be necessary to improve the prognosis of ATL patients.

GRANULOCYTE COLONY-STIMULATING FACTOR IN THE COMBINATION CHEMOTHERAPY FOR ADULT T-CELL LEUKEMIA (ATL)

We evaluated the effect of granulocyte colony-stimulating factor (G-CSF) on the median survival of 17 patients with Adult T-cell leukemia (ATL). Standard-dose combination chemotherapy using the response-oriented cyclic multidrug (RCM) protocol with G-CSF (lenograstim 2 microg/kg/day or filgrastim 50 microg/m2/day) was administered between October 1990 and December 1994. Complete responses (CR) were achieved in 11 (64.7%) patients, and partial responses (PR) in 4 (23.5%) patients. The median duration of survival was 7.4 months, compared with 6.0 months in ATL patients treated with the RCM protocol alone (historical controls) (n.s.). Infectious complications were the cause of death in 4 (26.7%) of the 15 patients who died. The median duration of neutropenia (absolute neutrophil count < 1.0 x 10(9)/L) was 6 days. G-CSF, in the doses and schedules used here, may have shortened the duration of neutropenia and reduced the incidence of fatal infectious complications. However, concomitant use of G-CSF did not prolong the median duration of survival in patients with ATL treated according to the RCM protocol.

Aggressive NK cell leukaemia after splenectomy: association with CD95‐resistant memory T‐cell proliferation and recalcitrant clinical course of haemophagocytic syndrome

We describe a 44‐yr‐old Japanese woman with persistent polyclonal T‐cell proliferation and recalcitrant clinical course of haemophagocytic syndrome (HPS). T cells bearing αβ T‐cell receptors (TCR) expressed increased amounts of CD95 and of CD45RO, which are phenotypically memory T cells. The TCR repertoire was broad and diverse. Regardless of CD95 expression, these cells were resistant to CD95‐mediated apoptosis. Aggressive natural killer cell leukaemia (ANKL) without an association with Epstein–Barr virus was detected 1 month after therapeutic splenectomy that followed 3 yr of immunosuppressive therapy against HPS. The immunophenotype of these leukaemia cells was CD56, CD16dim, CD7, CD45RA and they expressed some CD2, CD8 and HLA‐DR. Moreover, hyperdiploid clones with complex chromosomal abnormalities were also detected. Latent NK‐cell malignancy seemed to cause the CD95‐resistant memory T‐cell proliferation and splenectomy resulted in overt ANKL progression. There should be careful consideration of the risks versus benefits of splenectomy in HPS, in light of the possibility of fatal leukaemia/lymphoma progression.

Identification of interleukin-6 producing fibroblastoid cells in cerebrospinal fluid from patients with leukemic meningitis

Cytokine producing native cells in cerebrospinal fluid (CSF) have not been identified. So, we investigated the cytokine producing ability of floating cells in CSF from patients with leukemic meningitis. Morphologic study revealed that established cell lines were polygonal or elongated in shape and had an abundant and irregular branched cytoplasm. Immunocytochemical analysis demonstrated positive reactivity with monoclonal anti-fibroblast antibody only. Interleukin-6 (IL-6) was constitutively produced in vitro by these cell lines; both interleukin-1 and lipopolysaccharides significantly increased its synthesis. These findings imply that these fibroblastoid cells are floating in CSF of patients with leukemic meningitis and produce IL-6 in response to various inflammatory stimulations in vivo.