Tetsuyuki Kiyokawa

Hypercalcemia and osteoclast proliferation in adult T-cell leukemia

Eighteen autopsy cases of adult T‐cell leukemia (ATL) were investigated clinicopathologically. Thirteen of the patients had hypercalcemia during their clinical course. Nine of the thirteen had a high level of serum calcium at the terminal stage, even after extensive chemotherapy. Microscopic examination of the bone revealed proliferation of osteoclasts and bone resorption in eight patients. No osteoclast proliferation or bone resorption was found in the other nine normocalcemic patients. The infiltration of ATL cells was observed in only two patients—one was hypercalcemic and the other, normocalcemic. The factors affecting the serum calcium level were examined in two hypercalcemic patients. Hypercalcemia could not be accounted for by parathyroid hormone or prostaglandins E levels, which were in the normal range, or by 25‐hydroxyvitamin D and 1,25‐dihydroxyvitamin D, which were low. Our findings are consistent with the mechanism proposed by several investigators, that the malignant T‐lymphocytes produced an osteoclastactivating‐factor‐like substance that caused osteoclast proliferation and hypercalcemia. Cancer 59:1187‐1191, 1987.

Polyclonal integration of HTLV-I proviral DNA in lymphocytes from HTLV-I seropositive individuals: an intermediate state between the healthy carrier state and smouldering ATL

We have studied 15 individuals (aged 14‐74 years) with antibodies to HTLV‐I in their serum and random integration of HTLV‐I proviral DNA in their peripheral blood lymphocytes. All but one of these patients suffered from a variety of non‐specific complaints which did not correspond to those of adult T‐cell leukaemia (ATL). All of them were born in Kyushu and Okinawa which are endemic areas for HTLV‐I infection; 25% of their family members were also seropositive for HTLV‐I. The only haematological abnormality in these patients was the presence of few atypical lymphoid cells in the peripheral blood. The CD4/CD8 ratios were normal but the proportion of Tac positive cells was slightly higher than normal.

Analysis of a chronic myelogenous leukemia patient vaccinated with leukemic dendritic cells following autologous peripheral blood stem cell transplantation.

Dendritic cells (DCs) are believed to be the most potent antigen‐presenting cells and may be important in the induction of anti‐leukemia specific T cell responses. In this preliminary clinical study, a patient with chronic phase chronic myelogenous leukemia (CML) was vaccinated with autologous leukemic DCs following autologous peripheral blood stem cell transplantation (PBSCT). In an in vitro study, leukemic DCs were generated using granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), tumor necrosis factor‐α, and interleukin‐4 from granulocyte colony‐stimulating factor (G‐CSF)‐mobilized PBSC fraction of this patient, and were found to be Phl+, and to possess the morphologic and phenotypic characteristics of mature DCs. These cells could also elicit antigen‐specific immune responses, including a vigorous cytotoxicity specific to CML cells. In the clinical experiment, we obtained evidence that infused leukemic DCs could induce T cell clones expressing the same T cell receptor usage as a cytotoxic T cell line, suggesting that the immune repertoire includes tumor‐reactive T cells. These cytotoxic T lymphocytes are activated in vivo. The vaccination of leukemic DC caused a decrease in the number of Phl+ cells in the peripheral blood and bone marrow. These results indicate that the activity is an immunologically mediated phenomenon and vaccination therapy with leukemic DC following autologous PBSCT may be effective in treating CML.

The absence of the p15INK4B gene alterations in adult patients with precursor B-cell acute lymphoblastic leukaemia is a favourable prognostic factor.

Summary. We examined deletion and methylation of the p15INK4B(p15) and p16INK4A(p16) genes, using Southern blotting and methylation‐specific polymerase chain reaction (PCR), in 70 untreated adult patients with precursor B‐cell acute lymphoblastic leukaemia (PBC‐ALL) and analysed the relationship between their genetic changes and clinical outcome. Methylation and homozygous deletion of the p15 gene were detected in 30 (43%) and 18 (26%) patients, while those of the p16 gene were found in 16 (23%) and 11 (16%) patients respectively. Thirteen out of 17 patients with wild‐type p15 gene showed expression of p15 mRNA, whereas 31 out of 39 patients with alteration (deletion and methylation) of the p15 gene showed no p15 mRNA expression by reverse transcription–PCR, suggesting that alterations of the p15 gene are highly associated with loss of␣p15 mRNA expression. Disease‐free survival (DFS) at 4 years in patients with wild‐type p15 gene is 33%, compared with 4% of those with p15 gene alterations (P = 0·049). Multivariate analysis showed that the absence of p15 gene alterations was an independent significant favourable prognostic factor for longer DFS (P = 0·0001). These results suggest that alterations in the p15 but not p16␣gene can be used as a genetic prognostic indicator in PBC‐ALL.

Treatment of post-transplanted, relapsed patients with hematological malignancies by infusion of HLA-matched, allogeneic-dendritic cells (DCs) pulsed with irradiated tumor cells and primed T cells.

Patients with hematological malignancies who relapse after bone marrow transplantation (BMT) are often treated with donor lymphocyte infusion. However, this procedure often results in graft-versus-host disease (GVHD). While, Dendritic cells (DCs), which present antigens to naive T cells, have been used in the immunotherapy of cancer, this approach has been logistically difficult due to limiting numbers of DCs. We have now developed a method for obtaining a large number of DCs by treating the granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSCs) from healthy donors with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor-α (TNF-α). The resulting cells possess the morphologic, phenotypic, and functional characteristics of mature DCs. In in vitro studies, culture of these HLA-matched donor derived-DCs with irradiated each patients tumor cells as an antigen source, followed by incubation with T cells from the patient, induced the production of highly cytotoxic T lymphocytes (CTLs) specific for the respective tumor cells in the semi-allogeneic setting. A transient, but objective clinical response was obtained in the absence of GVHD when we injected the DCs which had been pulsed with irradiated tumor cells as well as primed T cells from the same original donor of related-allogeneic stem cell transplantation into the relapsed patients. Our findings suggest that treatment of relapsed patients with such donor-derived DCs, and primed T cells may be effective as an adjunctive immunotherapy.

Association of the pX gene product of human T-cell leukemia virus type-I with nucleus

Human T-cell leukemia virus type I (HTLV-I) contains a unique gene pX coding for p40 chi, and this protein was suggested to activate the transcription from the LTR of HTLV. By a similar mechanism, this viral function might be involved in immortalization of T-cells and leukemogenesis in adult T-cell leukemia induced by HTLV-I. In this communication, a part of the p40 chi was found to be tightly associated with nuclei in infected cell lines by subcellular fractionation and immunofluorescence staining.

Comparison of immunoperoxidase staining with indirect immunofluorescence, ELISA, and Western blotting assays for detecting anti-HTLV-I antibodies in systemic lupus erythematosus.

Serum antibodies against human T cell leukaemia virus type I (HTLV-I) were investigated in 12 patients by four methods: indirect immunoperoxidase staining, indirect immunofluorescence, enzyme linked immunosorbent assay (ELISA), and strip radioimmunoassay based on the Western blotting assay. Seven patients had systemic lupus erythematosus (SLE) and five various autoimmune diseases with one or more circulating autoantibodies. Serum samples from three patients were found to be HTLV-I-positive by the ELISA assay and sera from five patients showed a non-specific reaction by indirect immunofluorescence. These sera were negative when tested by indirect immunoperoxidase staining and Western blotting assay. All four methods gave positive results when tested with samples from 19 HTLV-I carriers and 16 patients with adult T cell leukaemia. Indirect immunoperoxidase staining and Western blotting assay are probably useful and more specific assays for the detection of anti-HTLV-I antibodies in samples from patients with autoimmune diseases.

Follow-up of asymptomatic HTLV-I carriers among blood donors in Kyushu, Japan

We examined mortality from adult T-cell leukemia/lymphoma (ATL/ATLL) and other diseases alleged to be associated with human T-lymphotropic virus type I (HTLV-I) among anti-HTLV-I antibody-positive blood donots in Kyushu, Japan. During 1984–87, a total of 3,991 blood donors aged 40 years or over were followed from the date of donation to the date of death or the end of the study. Crude mortality rates from ATL (with 95 percent confidence intervals) were 68 per 100,000 (13–202) for males and 36 per 100,000 (3–132) for females. The rates were underestimated by approximately 50 percent because of self-selection and short observation periods. Neither death rates from other cancers not death rates from all cancers were elevated.

Treatment of Adult T Cell Leukaemia by Extracorporeal Photochemotherapy.

Four patients with chronic or smouldering type adult T cell leukaemia (ATL) were treated by extracorporeal photochemotherapy (photopheresis). From 4 to 8 months after starting photo-pheresis, skin lesions with ATL cell infiltration began to disappear. Cell surface markers in three patients showed improvement. In one patient, a serial decrease of soluble interleukin-2 receptor levels (950 U/ml to 620) in the serum was observed after 4 months. This pilot study suggests that photopheresis may be successfully applied in ATL. It is still necessary to continue follow-up observations in these patients and to treat a larger number of cases, before definite conclusions can be drawn in the future.

Elderly ATL patients in ageing society of Japan

Results More than 70% of newly diagnosed ATL patients were elderly persons aged 65 or older. Twenty-three of 66 patients at age from 41 to 61-year-old underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) in our hospital. On the other hand, 38 patients were not suitable for allo-HSCT therapy. Their ages were from 51 to 89-year-old and elderly aged 65-yearold and over occupied 73.3% of them (22 of 30, who we confirmed their outcome). Seven patients of them died before treatment or during the primary therapy. Then, we focused patients cause acute transformation from chronic type to figure out the timing of primary intervention and the efficiency of the therapy. Elevating sCD30 was observed earlier than other markers (sIL-2R, LDH) due to acute transformation and reduced by the treatment regimen changed even during treatment of relapse (recurrence).