Shiroh Hidaka

NF-κB involvement in the activation of primary adult T-cell leukemia cells and its clinical implications

The HTLV-I provirus-encoded Tax protein induces NF-kappaB in Tax-transfected Jurkat T cells or HTLVL-I- infected T cells in vitro. Tax induction of NF-kappaB is presumed to be involved in proliferation and activation of primary leukemia cells in vivo. Recent studies have demonstrated that NF-kappaB activities in human T cells are mediated by at least four c-Rel-related DNA binding proteins - p50, p55, p75 and p85. We examined the significance of NF-kappaB induction in primary adult T cell leukemia cells and the induction kinetics of each of the four NF-kappaB species. Marked NF-kappaB activity was detected using an electrophoretic mobility shift assay (EMSA) in the primary cells of patients with acute disease, but little activity was noted in the cells of chronic patients. NF-kappaB activity was enhanced in a time-dependent manner in acute type cells cultured with mitogen-free medium; there was no induction of activity in chronic type cells. UV crosslinking demonstrated all four species of NFkappaB complex - high levels of p50 and lower levels of p55 and p75, in acute type cells; chronic type cells showed only the p50. As a control, normal resting T cells similarly showed only p50; control cells showed little change in activity when cultured without mitogenic stimulation, analogous to chronic type ATL. Northern blotting revealed enhancement of c-rel (encoding p85) and KBFI (encoding p50 and p55) expression in acute type cells during culture, while there was no significant enhancement of mRNAs in chronic type ATL cells or unstimulated normal T cells. Northern blotting also revealed that Tax is upregulated at the mRNA level in acute- but not chronic-type cells during culture. Expression of c-rel and KBF1 mRNAs in acute type cells appeared to be related to Tax mRNA expression. These results suggest that Tax is capable of inducing nuclear expression of all four NF-kappaB species in primary ATL cells of acute type patients, with marked effects on p55, p75, and p85. Tax induction of NF-kappaB species is regulated, at least in part, at a pretranslational level involving increases in c-rel and KBF1 mRNA.

Long-term Maintenance Combination Chemotherapy with OPECMPEC (Vincristine or Methotrexate, Prednisolone, Etoposide and Cyclophosphamide) or with Daily Oral Etoposide and Prednisolone Can Improve Survival and Quality of Life in Adult T-cell LeukemiaLymphoma

Acute leukemia and lymphoma varieties of adult T-cell leukemia/lymphoma (ATL) usually carry a poor prognosis. While etoposide is generally useful for treating ATL, especially as a daily oral maintenance regimen, etoposide has not proven effective in severe types of ATL efficient in some patients. Of 87 ATL patients whom we have treated, 51 had acute leukemia, 22 lymphoma and 14 progressive chronic leukemia. Seventy-nine patients were treated with a long term maintenance combination protocol, OPEC/MPEC (weekly doses of vincristine, 0.7 mg/m2 or methotrexate, 14 mg/m2; prednisolone, 20 mg/m2; etoposide, 70 mg/m2 and cyclophosphamide, 200 mg/m2). The other 8 patients, 3 with acute leukemia, 2 with lymphoma and 3 with progressive chronic leukemia, were treated with daily oral administration of 25 mg of etoposide and 10 mg of prednisolone (DOEP). The dose administered was modified in individual cases to maintain the granulocyte count and reduce the number of ATL cells. Considering both protocols, a complete response and a partial response were achieved in 31.0% and 58.6% patients, respectively. Median survival times (MST) of all patients and, acute leukemia, lymphoma and progressive chronic leukemia types were 7.5, 6.7, 9.6 and 12.4 months, respectively. Respective MST of patients treated with OPEC/MPEC or DOEP protocols were 7.1 and 18.0 months. Relatively normal WBC counts, lower lactate dehydrogenase concentration and normal calcium concentration, limited numbers of anatomic sites involved, good performance status and good response to chemotherapy were significantly associated with long survival time. Drug toxicity was not apparent, and about half of patients were treated in an outpatient setting.

Granulocyte‐colony stimulating factor‐induced proliferation of primary adult T‐cell leukaemia cells

Granulocyte‐colony stimulating factor (G‐CSF) is known to induce proliferation and differentiation of granulocyte progenitors, and is widely used to treat neutropenia induced by intensive chemotherapy for malignant lymphoma or adult T‐cell leukaemia/lymphoma (ATL). G‐CSF is thought not to stimulate malignant lymphoid cells. In the present study we examined the ability of G‐CSF to induce in vitro growth of primary ATL cells from 14 patients (nine acute‐type, two chronic‐type and three lymphoma‐type), and we analysed the in vivo counts of ATL cells in patients who received G‐CSF for neutropenia. FACS analysis using phycoerythrin‐labelled recombinant G‐CSF demonstrated that ATL cells from 11/14 patients express some G‐CSF receptor (G‐CSFR), with a range between 5.4% and 87.3%. Cells expressing G‐CSFR also expressed CD4. Reverse polymerase chain reaction (PCR) analysis demonstrated expression of G‐CSFR messenger RNA in G‐CSFR expressing cells. Leukaemic cells derived from seven (four acute‐type, one chronic‐type and two lymphoma‐type) of the 14 patients proliferated in vitro in response to G‐CSF, as measured by [3H]thymidine incorporation; maximum responses were at G‐CSF concentrations of 10–100 ng/ml. Nine of 14 patients receiving rG‐CSF for neutropenia were analysed retrospectively for ATL cell numbers. Four patients whose primary tumour cells proliferated in response to rG‐CSF in vitro showed a significant increase in ATL cell count after administration of rG‐CSF (P =0.038), whereas five patients whose leukaemic cells did not proliferate in vitro showed no significant increase in ATL cell count. G‐CSF can stimulate proliferation of ATL cells which may complicate therapy for this disease.

IL-2-induced growth of CD8+ T cell prolymphocytic leukemia cells mediated by NF-κB induction and IL-2 receptor α expression

Abstract The binding of interleukin-2 (IL-2) to its receptor on normal T cells induces nuclear expression of nuclear factor κB (NF-κB), activation of the IL-2 receptor (IL-2R) α chain gene, and cell proliferation. In the present study, the role of IL-2R signaling in the growth of CD8 + T cell prolymphocytic leukemia (T-PLL) cells has been investigated. Flow cytometry revealed that primary leukemia cells from a patient with CD8 + T-PLL expressed IL-2Rα and β chains, and the cells showed a proliferative response and an increase in IL-2Rα expression on culture with exogeneous IL-2. Northern blot analysis failed to detect IL-2 mRNA, suggesting that IL-2 may act in a paracrine manner in vivo . Electrophoretic mobility-shift assays revealed that recombinant IL-2 increased NF-κB binding activity in nuclear extracts of the leukemia cells, and Northern blot analysis showed that IL-2 increased the abundance of mRNAs encoding the NF-κB components c-Rel and KBF1 in these cells. IL-2 binding analysis demonstrated that IL-2 markedly increased the number of low affinity IL-2Rs on the leukemia cells, without an effect on the number of high-affinity IL-2Rs. These results show that IL-2 is capable of inducing the nuclear expression of NF-κB in primary CD8 + T-PLL cells, and that this effect is mediated, at least in part, at a pretranslational level.

Cytomegalovirus infection is not necessarily a poor prognostic factor in adult T-cell leukemia/lymphoma.

The relationship between cytomegalovirus (CMV) antigenemia and the clinical course was examined in 57 patients with adult T‐cell leukemia/lymphoma (ATLL). All patients included had the acute/lymphoma type of ATL according to the criteria of the Japan Lymphoma Study Group (LSG). CMV antigenemia was assessed on admission and at the time when the patients had fever higher than 37.5°C, which did not respond to antibiotics for longer than 3 days. The incidence of CMV antigenemia was 44%. Approximately 90% of patients with CMV antigenemia died of infections with viruses, bacteria, and/or fungi, while approximately 40% of patients without CMV antigenemia died of deterioration of ATLL (P<0.0001). In this study, the patients with CMV antigenemia tended to survive longer than those negative for it (321.4 days vs. 266.2 days), although there was no statistical significance (P=0.09). Kaplan‐Meier analysis revealed that CMV antigenemia was not a poor prognostic factor. When the disease status of ATLL was evaluated by thymidine kinase (TK) and soluble interleukin 2 receptor (sIL‐2R), both had lower titers during CMV antigenemia (TK: P=0.01, sIL‐2R: P=0.03, respectively). Therefore, CMV infections in ATLL patients seemed to have bimodal meanings; CMV infection at the end of clinical course were life‐threatening, but infection during the first half of clinical course seemed to suppress ATLL activity and to contribute to the longer survival of the patients. J. Med. Virol. 62:140–143, 2000.

Interferon-α Therapy following Autologous Peripheral Blood Stem Cell Transplantation for Adult T Cell Leukemia/Lymphoma

In the present report, we describe a case of adult T cell leukemia/lymphoma (ATLL), a 58-year-old woman, successfully treated with interferon (IFN)-α following autologous peripheral blood stem cell transplantation (auto-PBSCT). The patient remains in remission with full performance status for more than 12 months. Auto-PBSCT reduced the abdominal lymphoma mass and IFN-α eliminated residual tumor cells, possibly through the induction of specific T-cell subsets expressing CD3, CD8 on their surfaces and either IFN-γ or tumor necrosis factor (TNF)-α in cytoplasm. We have treated a total of 4 ATLL patients with auto-PBSCT, including the case presented herein. All other patients treated with auto-PBSCT were not followed by adjuvant chemotherapy or cytokine therapy and relapsed within 3 months after auto-PBSCT. This evidence suggests that the therapeutic success of the present case was attributable to the administration of IFN-α immunotherapy following auto-PBSCT.

Alteration of p16 (CDKN2) gene is associated with interleukin-2-induced tumor cell growth in adult T-cell leukemia.

Because tumorigenesis frequently involves the dysfunction of cell cycle-related proteins, we examined the effect of mutations in CDK inhibitor p16 and its linked genomic loci p15, cl.B, and 1063.7 on the growth of primary adult T-cell leukemia (ATL) cells. Southern blot analysis of primary ATL cells showed a significantly higher incidence of p16 gene alteration in acute ATL than in chronic ATL [67.7% (23/34) vs. 26.1% (6/23), respectively; p<0.003]. Similarly, polymerase chain reaction (PCR) analysis of p16 exon 2 revealed a higher incidence of alteration in acute ATL than in chronic ATL [52.9% (18/34) vs. 26.1% (6/23), respectively; p<0.05]. PCR-single strand conformation polymorphism analysis of exons 1 and 2 of p16 showed no mutations in the patients, with normal pattern by Southern blotting or PCR analysis. Notably five of six chronic ATL patients with abnormal p16 genes progressed to acute crisis within 4 months. PCR analysis of the p16 linked loci 1063.7, p15 exon 2, and cl.B found homozygous deletion in 55.9%, 20.6%, and 2.9% of acute ATL cells and 39.1%, 13.0%, and 0% of chronic ATL cells, respectively, showing no relationship of homozygous deletion in either loci with disease subtypes. In most cases, deletions were seen in multiple genes, including p16. Acute ATL cells had a higher frequency of multigene deletions than chronic ATL cells [44.1% vs. 17.4%; p<0.05]. When leukemic cells were analyzed for interleukin 2 (IL-2) responsive growth, only p16 gene alteration was directly associated with leukemic cell growth activity. Among leukemic cells showing high IL-2 responsiveness, 73.1% (19/26) had p16 gene alteration vs. 27.8% (5/18) of leukemic cells that showed low IL-2 responsiveness (p<0.005). p16 gene alteration was found in 73.3% (14/19) of leukemic cells showing high autonomous growth rates but in only 40.0% (10/25) of those leukemic cells showing low autonomous growth (p<0.03). These results suggest the following: alteration of p16-related genomic regions in ATL is usually a wide rearrangement including the p16 gene; within this region, only p16 gene alteration is associated with disease aggressiveness; and p16 gene deletion may be a proximate event in leukemogenesis.

Spontaneous regression associated with apoptosis in a patient with acute‐type adult T‐cell leukemia

We describe a 76‐year‐old man with acute‐type adult T‐cell leukemia, who demonstrated a spontaneous decrease in leukemic cell number, apparently coincident with apoptotic cell death. On admission the patients white blood cell count was 38.9 × 109/l with 77% abnormal lymphocytes. He also had hypoproteinemia (4.3 g/dl) from protein losing enteropathy. After admission the leukemic cell count decreased without chemotherapy, reaching 5.9 × 109/l after 2 months. Studies of peripheral lymphocytes demonstrated appearance of the apoptotic cells and DNA ladder formation from the beginning of regression. Same truncated proviral DNA was recognized in primary ATL cells through the whole clinical course. The hypoproteinemia improved with intravenous nutrition, followed by increase of the leukemic cells. This case is the first report that demonstrates tumor‐cell apoptosis induced clinical regression in adult T‐cell leukemia. Further, we speculate that the hypoproteinemia may have been involved in the leukemic cell apoptosis. Am. J. Hematol. 61:144–148, 1999.

Granulocyte-colony stimulating factor induces differentiation and apoptosis of CD2, CD7 positive hybrid leukemia cells in vivo and ex vivo

We report a case of a 70-year-old man with a hybrid leukemia treated successfully with granulocyte-colony stimulating factor (G-CSF) combined with a cytocine arabinoside regimen through the induction of differentiation of leukemic cells into monocytoid cells resulting in apoptosis. The leukemic cells demonstrated a TCR-gamma rearrangement, and expressed CD2, CD7, CD33 and G-CSF receptors but not CD11b on the cell surface nor non-specific esterase in cytoplasm. Several days following the administration of G-CSF, the cells with monocytoid characteristics such as CD11b and cytoplasmic non-specific esterase appeared in the peripheral blood replacing the blastic cells. The cells were shown to be derived from the same clone of the leukemic cell because of the identical TCR-gamma gene rearrangement. The short-term culture of leukemic cells with G-CSF induced the differentiation into a monocyte lineage, resulting in apoptosis. Although there is no denying the possibility that cytosine arabinoside is partly responsible, our results strongly suggest that G-CSF plays the main role in differentiation of leukemic cells into a monocyte lineage inducing apoptosis in vivo in this patient.

Interleukin-2-mediated growth of leukemic cells in lymph nodes of patients with adult T-cell leukemia/ lymphoma

We investigated the effect of interleukin-2 (IL-2) on tumor growth of primary adult T-cell leukemia/lymphoma (ATL) cells in biopsied lymph node cells obtained from 14 patients (seven [corrected] with acute-type disease, one with chronic-type disease and six [corrected] with lymphoma-type disease). Biological activity of IL-2 in culture supernatants of the cells was detected in six out of 12 cases. The IL-2 mRNA in the lymph node cells was detected in four out of nine patients by northern blotting. However, it was detected in all nine patients examined by reverse polymerase chain reaction (PCR) method. Lymph node cells from 12 out of 14 patients showed a high or moderate proliferative response to IL-2; the remaining two patients showed a slight response. These results suggest that malignant growth of primary tumor cells in lymph nodes may be associated with the IL-2-IL-2 receptor system in patients with ATL more frequently than had been previously thought.