Kazuhiro Nishii

Frequent expression of P‐glycoprotein/MDR1 by nasal T‐cell lymphoma cells

Background. Lethal midline granuloma is now considered to be a malignant lymphoma derived from peripheral T cells or from natural killer cells. The therapeutic outcome of nasal T‐cell lymphoma (NL) treated by conventional chemotherapy for non‐Hodgkins lymphoma is poor, although some patients have a good response to radiotherapy. To clarify the mechanisms of drug resistance, the expression of P‐glycoprotein (P‐gp)/MDR1, which is the product of the multidrug resistance (MDR) 1 gene, and MDR3 mRNA in NL cells, were examined.

Prospective monitoring of BCR-ABL1 transcript levels in patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia undergoing imatinib-combined chemotherapy.

The clinical significance of minimal residual disease (MRD) is uncertain in patients with Philadelphia chromosome‐positive acute lymphoblastic leukaemia (Ph+ ALL) treated with imatinib‐combined chemotherapy. Here we report the results of prospective MRD monitoring in 100 adult patients. Three hundred and sixty‐seven follow‐up bone marrow samples, collected at predefined time points during a uniform treatment protocol, were analysed for BCR‐ABL1 transcripts by quantitative reverse transcription polymerase chain reaction. Ninety‐seven patients (97%) achieved complete remission (CR), and the relapse‐free survival (RFS) rate was 46% at 3 years. Negative MRD at the end of induction therapy was not associated with longer RFS or a lower relapse rate (P = 0·800 and P = 0·964 respectively). Twenty‐nine patients showed MRD elevation during haematological CR. Of these, 10 of the 16 who had undergone allogeneic haematopoietic stem cell transplantation (HSCT) in first CR were alive without relapse at a median of 2·9 years after transplantation, whereas 12 of the 13 who had not undergone allogeneic HSCT experienced a relapse. These results demonstrate that, in Ph+ ALL patients treated with imatinib‐combined chemotherapy, rapid molecular response is not associated with a favourable prognosis, and that a single observation of elevated MRD is predictive of subsequent relapse, but allogeneic HSCT can override its adverse effect.

Activity of interleukin 6 in the differentiation of monocytes to macrophages and dendritic cells

Peripheral blood monocytes are common precursor cells of dendritic cells (DCs) and macrophages. We have searched for factors with the potential to regulate the differentiation of monocytes to DCs and macrophages. When CD14+ monocytes are cultured with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL) 4, the CD14+CD1a− population, which consists of macrophages, was found in the serum‐containing cultures but not in the serum‐free cultures. Addition of IL‐6 receptor‐neutralizing monoclonal antibody (mAb) or gp130‐neutralizing mAb to the serum‐containing cultures resulted in a decreased population of CD14+CD1a− cells. An increase in the CD14+CD1a− population with reduction in CD14−CD1a+ DCs was observed with the addition of IL‐6 to cultures, whereas IL‐11, leukaemia inhibitory factor, oncostatin M or macrophage colony‐stimulating factor did not affect the differentiation of monocytes in the presence of GM‐CSF plus IL‐4. This effect of IL‐6 was blocked by tumour necrosis factor α (TNF‐α), lipopolysaccharide (LPS), IL‐1β, CD40 ligand (CD40L) and transforming growth factor β1 (TGF‐β1). Among these factors, TNF‐α was most potent in interfering with the action of IL‐6. These results suggest that IL‐6 inhibits the differentiation of monocytes to DCs by promoting their differentiation toward macrophages, which is modulated by factors such as TNF‐α, LPS, IL‐1β, CD40L and TGF‐β1.

Survival of human leukaemic B-cell precursors is supported by stromal cells and cytokines: association with the expression of bcl-2 protein.

We searched for cytokines with the potential to support the survival of human B‐cell precursor acute lymphoblastic leukaemia (pre‐B ALL) cells. 47 patients with pre‐B ALL were classified into four stages: stage I, CD19+CD10−CD20−; stage II, CD19+CD10+CD20−; stage III, CD19+CD10+CD20+cytoplasmic μ‐heavy chain (cμ)−; stage IV, CD19+CD10+CD20+cμ+. Interleukin (IL)‐3 receptor α chain (IL‐3Rα) was expressed in all stages, whereas the expressions of IL‐7Rα and IL‐2Rα were pronounced in stages IV and II, respectively. Neither IL‐3, IL‐7 nor IL‐2 supported the survival of pre‐B ALL cells. When pre‐B ALL cells were layered on stromal, MS‐10, cells, viability of the pre‐B ALL cells increased. Addition of IL‐3 to culture containing MS‐10 cells enhanced the survival of pre‐B ALL cells in all cases, whereas addition of IL‐7 augmented the survival of pre‐B ALL cells of some cases of stage III and all cases of stage IV. The survival of pre‐B ALL cells was also supported by the conditioned media of MS‐10 cells. Stromal‐cell‐derived factor 1 (SDF‐1) supported the survival of pre‐B ALL cells. Effects of the conditioned media of MS‐10 cells were abrogated by an anti‐SDF‐1 neutralizing antibody. The extent of survival of pre‐B ALL cells supported by stromal cells and IL‐3 and IL‐7, correlated with the expression level of bcl‐2 protein. The effects of stromal cells may be in part related to SDF‐1.

A novel role for Notch ligand Delta-1 as a regulator of human Langerhans cell development from blood monocytes.

Human Langerhans cells (LCs) are of hematopoietic origin, but cytokine regulation of their development is not fully understood. Notch ligand Delta‐1 is expressed in a proportion of the skin. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and transforming growth factor‐β1 (TGF‐β1) are also secreted in the skin. We report here that Delta‐1, in concert with GM‐CSF and TGF‐β1, induces the differentiation of human CD14+ blood monocytes into cells that express LC markers: CD1a, Langerin, cutaneous lymphocyte‐associated antigen, CC chemokine receptor 6, E‐cadherin, and Birbeck granules. The resulting cells display phagocytic activity and chemotaxis to macrophage inflammatory protein‐1α (MIP‐1α). In response to CD40 ligand and tumor necrosis factor α, the cells acquire a mature phenotype of dendritic cells that is characterized by up‐regulation of human leukocyte antigen (HLA)‐ABC, HLA‐DR, CD80, CD86, CD40, and CD54 and appearance of CD83. These cells in turn show chemotaxis toward MIP‐1β and elicit activation of CD8+ T cells and T helper cell type 1 polarization of CD4+ T cells. Thus, blood monocytes can give rise to LCs upon exposure to the skin cytokine environment consisting of Delta‐1, GM‐CSF, and TGF‐β1, which may be, in part, relevant to the development of human epidermal LCs. Our results extend the functional scope of Notch ligand δ‐1 in human hematopoiesis.

Dual mutations in the AML1 and FLT3 genes are associated with leukemogenesis in acute myeloblastic leukemia of the M0 subtype.

Point mutations of the transcription factor AML1 are associated with leukemogenesis in acute myeloblastic leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and mutations in the second tyrosine kinase domain of the Fms-like tyrosine kinase 3 (FLT3) gene represent the most frequent genetic alterations in AML. However, such mutations per se appear to be insufficient for leukemic transformation. To evaluate whether both AML1 and FLT3 mutations contribute to leukemogenesis, we analyzed mutations of these genes in AML M0 subtype in whom AML1 mutations were predominantly observed. Of 51 patients, eight showed a mutation in the Runt domain of the AML1 gene: one heterozygous missense mutation with normal function, five heterozygous frameshift mutations and two biallelic nonsense or frameshift mutations, resulting in haploinsufficiency or complete loss of the AML1 activities. On the other hand, a total of 10 of 49 patients examined had the FLT3 mutation. We detected the FLT3 mutation in five of eight (63%) patients with AML1 mutation, whereas five of 41 (12%) without AML1 mutation showed the FLT3 mutation (P=0.0055). These observations suggest that reduced AML1 activities predispose cells to the acquisition of the activating FLT3 mutation as a secondary event leading to full transformation in AML M0.

Karyotype at diagnosis is the major prognostic factor predicting relapse-free survival for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia treated with imatinib-combined chemotherapy

In patients with Philadelphia chromosome-positive acute lymphoblastic leukemia treated with imatinib-combined chemotherapy, the presence of secondary chromosome aberrations in addition to (t9;22) at diagnosis represents an independent risk factor for relapse. To identify factors associated with relapse-free survival (RFS), 80 patients with newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia, enrolled in a phase II study of imatinib-combined chemotherapy, were analyzed. The median follow-up of surviving patients was 26.7 months (maximum, 52.5 months). Twenty-eight out of 77 patients who had achieved CR relapsed. The probability of RFS was 50.5% at 2 years. Multivariate analysis revealed that the presence of secondary chromosome aberrations in addition to t(9;22) at diagnosis constitute an independent predictive value for RFS (p=0.027), and increase the risk of treatment failure by 2.8-fold.

Gene expression profiling of peripheral T-cell lymphoma including γδ T-cell lymphoma

The gene expression profile of peripheral gammadelta T-cell lymphoma (gammadeltaTCL) has not been investigated. Using oligonucleotide microarrays, we analyzed total RNA from 7 patients with gammadeltaTCL (4 hepatosplenic, 1 cutaneous, 1 intestinal, and 1 thyroidal) and 27 patients with alphabetaTCL (11 peripheral TCL-unspecified, 15 angioimmunoblastic TCL, and 1 hepatosplenic). Unsupervised microarray analyses classified all hepatosplenic gammadeltaTCLs into a single cluster, whereas other gammadeltaTCLs were scattered within the alphabetaTCL distribution. We identified a T-cell receptor signature gene set, which accurately classified gammadeltaTCL and alphabetaTCL. A classifier based on gene expression under supervised analysis correctly identified gammadeltaTCL. One case of hepatosplenic alphabetaTCL was placed in the gammadeltaTCL grouping. gammadeltaTCL signature genes included genes encoding killer cell immunoglobulin-like receptors and killer cell lectin-like receptors. Our results indicate that hepatosplenic gammadeltaTCL is a distinct form of peripheral TCL and suggest that nonhepatosplenic gammadeltaTCLs are heterogeneous in gene expression.

Characteristics of t(8;21) acute myeloid leukemia (AML) with additional chromosomal abnormality: concomitant trisomy 4 may constitute a distinctive subtype of t(8;21) AML.

t(8;21)(q22;q22) is the most frequently observed karyotypic abnormality associated with acute myeloid leukemia (AML), especially in FAB M2. Clinically, this type of AML often shows eosinophilia and has a high complete remission rate with conventional chemotherapy. t(8;21) AML is also frequently associated with additional karyotypic aberrations, such as a loss of the sex chromosome; however, it is unclear whether these aberrations change the biological and clinical characteristics of t(8;21) AML. To investigate this issue, 94 patients with t(8;21) AML were categorized according to their additional karyotypic aberrations, which were detected in more than three cases, and then morphologic features, phenotypes, expression of cytokine receptors, and clinical features were compared to t(8;21) AML without other additional aberrant karyotypes. t(8;21) AML with loss of the sex chromosome and abnormality of chromosome 9 were found in 27 cases (29.3%) and 10 cases (10.6%), respectively; however, no differences were observed from the t(8;21) AML without other additional karyotypes in terms of morphological and phenotypic features. There was also no significant difference in the clinical outcome among these three groups. On the other hand, trisomy 4 was found in three cases (3.2%) and these cells showed low expressions of CD19 (P=0.06) and IL-7 receptor (P=0.05), and high expressions of CD33 (P=0.13), CD18 (P=0.03), and CD56 (P=0.03) when compared to t(8;21) AML without additional karyotypes. Moreover, all three t(8;21) AML cases with trisomy 4 did not show eosinophilia in their bone marrow and died within 2.4 years. These observations suggest that additional karyotypic aberration, t(8;21) with trisomy 4 is rare, but it may constitute a distinctive subtype of t(8;21) AML.

Hypermethylation of death-associated protein (DAP) kinase CpG island is frequent not only in B-cell but also in T- and natural killer (NK)/T-cell malignancies.

Death‐associated protein (DAP) kinase is a pro‐apoptotic serine/threonine kinase with a death domain, which is involved in apoptosis induced by interferon‐γ, tumor necrosis factor‐α, and Fas ligand. Down‐regulation of DAP kinase gene expression by hypermethylation of its promoter region might result in resistance to apoptotic cell death, and could provide a basis for tumor development. In the present study, we employed methylation‐specific polymerase chain reaction to examine the methylation status of CpG islands in the DAP kinase gene in 19 cases of T‐cell malignancies (including eight adult T‐cell leukemia/lymphoma), 24 of natural killer (NK)/T‐cell, and 34 of B‐cell. Frequency of methylation was significantly higher in B‐cell (27 of 34, 79.4%) than in T‐cell malignancies (nine of 19, 47.4%) (P<0.05). Fifteen of 24 (62.5%) NK/T‐cell lymphomas showed DNA methylation. One B‐cell lymphoma cell line with DNA methylation was resistant to apoptotic stimuli, and treatment of the cells with a demethylating agent restored apoptotic cell death. These findings suggested that suppression of DAP kinase expression by DNA methylation might play a substantial role in the development of not only B‐cell, but also T‐ and NK/T‐cell lymphomas. (Cancer Sci 2003; 94: 87–91)