Toru Kiguchi

Comprehensive analysis of genetic alterations and their prognostic impacts in adult acute myeloid leukemia patients.

To clarify the cooperative roles of recurrently identified mutations and to establish a more precise risk classification system in acute myeloid leukemia (AML), we comprehensively analyzed mutations in 51 genes, as well as cytogenetics and 11 chimeric transcripts, in 197 adult patients with de novo AML who were registered in the Japan Adult Leukemia Study Group AML201 study. We identified a total of 505 mutations in 44 genes, while only five genes, FLT3, NPM1, CEBPA, DNMT3A and KIT, were mutated in more than 10% of the patients. Although several cooperative and exclusive mutation patterns were observed, the accumulated mutation number was higher in cytogenetically normal AML and lower in AML with RUNX1-RUNX1T1 and CBFB-MYH11, indicating a strong potential of these translocations for the initiation of AML. Furthermore, we evaluated the prognostic impacts of each sole mutation and the combinations of mutations and/or cytogenetics, and demonstrated that AML patients could be clearly stratified into five risk groups for overall survival by including the mutation status of DNMT3A, MLL-PTD and TP53 genes in the risk classification system of the European LeukemiaNet. These results indicate that the prognosis of AML could be stratified by the major mutation status in combination with cytogenetics.

Life-threatening bleeding and acquired factor V deficiency associated with primary systemic amyloidosis

Acquired factor X deficiency has been described in patients with amyloidosis but acquired factor V deficiency is quite rare. We report here a case of life-threatening bleeding and acquired factor V deficiency associated with primary amyloidosis. A 50-year-old man who had no previous hemorrhagic diathesis was referred to our hospital because of recurrent epistaxis, gingival bleeding and hemospermia. The laboratory examination revealed that both the prothrombin time (PT) and the activated partial thromboplastin time (aPTT) were significantly prolonged, and factor V activities were markedly decreased to 14-39% of the normal value. Other coagulation factors such as fibrinogen, prothrombin, factor VII, factor VIII, factor IX and factor X were subnormal and normal. Transaminases were slightly elevated but serological tests of hepatitis B and hepatitis C were negative. Mild hepatosplenomegaly was noted without sign of liver cirrhosis. The PT and aPTT obtained 8 years ago when he received a cholecystectomy due to cholecystitis were both normal. Specific assays for the detection of factor V inhibitor were repeatedly performed but no factor V inhibitor was found. Furthermore, a significant recovery of the infused factor V was noted shortly after an intravenous administration of 5-10 U fresh frozen plasma, but it did not last more than 6 h. Melena, bleedings into the left shoulder and buttock, and finally mortal retroperitoneal hemorrhage developed despite repeated infusions of large amounts of fresh frozen plasma. Acquired factor V deficiency associated with primary amyloidosis was suspected but histological diagnosis was not obtained because of the severe bleeding tendency. Autopsy revealed hepatosplenomegaly and massive deposits of AL amyloid in the liver, spleen, heart and other parenchymal organs. Perivascular amyloid deposition and factor V deficiency are both thought to be the cause of the severe hemorrhagic tendency seen in this patient.

Simultaneous induction of matrix metalloproteinase-9 and interleukin 8 by all-trans retinoic acid in human PL-21 and NB4 myeloid leukaemia cells

Summary. All‐trans retinoic acid (ATRA) has been shown to induce differentiation of human acute promyelocytic leukaemia (APL) cells and eventual elimination of the malignant clone. Matrix metalloproteinase‐9 (MMP‐9) is produced by neutrophils and its expression appears to be linked with myeloid cell differentiation. We investigated effects of ATRA on MMP expression in two human myeloid leukaemia cell lines, PL‐21 and NB4. Both cells could differentiate into neutrophils after exposure to ATRA. Both the activity and antigen levels of MMP‐9 were much higher in NB4 cells than in PL‐21 cells. Stimulation with ATRA significantly increased MMP‐9 levels approximately three‐ to fivefold in both PL‐21 and NB4‐conditioned media. MMP‐9 mRNA levels increased in ATRA‐treated cells and was almost in parallel with the increase in MMP‐9 activity, suggesting that ATRA induced MMP‐9 by activating its gene expression. ATRA can induce interleukin 8 (IL‐8) in APL cells. IL‐8, chemokine for neutrophils and a potent inducer of MMP‐9, was also induced by ATRA in PL‐21 cells. However, recombinant IL‐8 did not induce MMP‐9 expression. In addition, a neutralizing antibody against IL‐8 did not inhibit ATRA‐induced MMP‐9 expression in either cell type. These observations suggest that ATRA can induce both MMP‐9 and IL‐8, but IL‐8 is not involved in ATRA‐induced MMP‐9 expression. As MMP‐9 can truncate and activate IL‐8, simultaneous induction of MMP‐9 and IL‐8 by ATRA could activate leucocytes excessively, causing the hyper‐inflammatory events in retinoic acid syndrome.

Induction of IL-8 and monoclyte chemoattractant protein-1 by doxorubicin in human small cell lung carcinoma cells

We previously demonstrated doxorubicin‐induced urokinase expression in human H69 SCLC cells by the microarray technique using Human Cancer CHIP version 2 (Takara Shuzo, Kyoto, Japan), in which 425 human cancer‐related genes were spotted on glass plates (Kiguchi et al., Int J Cancer 2001;93:792–7). Microarray analysis also revealed significant induction of IL‐8, a member of the CXC chemokines. We have, therefore, extended the observation by testing the effects of doxorubicin on expression of the chemokine family and provide here definitive evidence that doxorubicin induces IL‐8 and MCP‐1, one of the CC chemokines, at least in 2 human SCLC cells, H69 and SBC‐1. IL‐8 antigen levels, measured by ELISA, were markedly increased in both H69 and SBC‐1 conditioned media after doxorubicin treatment, in parallel with mRNA levels; and this was dependent on the dose of doxorubicin. The ribonuclease protection assay, using a multiprobe template set for human chemokines, revealed induction of not only IL‐8 but also MCP‐1 in doxorubicin‐treated H69 cells. MCP‐1 antigen levels increased approximately 100‐fold in doxorubicin‐treated H69 cells. RT‐PCR using specific primers for MCP‐1 suggested that doxorubicin also induced MCP‐1 expression in SBC‐1 and SBC‐3 SCLC cells. Futhermore, CAT analysis using IL‐8 promoter implicated the PEA3 transcriptional factor, whose binding site was located immediately upstream of the AP‐1 and NF‐κB binding sites. Thus, it is suggested that doxorubicin induces IL‐8 and MCP‐1 chemokines in human SCLC cells by activating gene expression, in which at least PEA3 is involved. IL‐8 and MCP‐1 are major chemoattractants for neutrophils and monocytes/macrophages, respectively; therefore, extensive induction of IL‐8 and MCP‐1 may provoke the interaction between inflammatory/immune cells and tumor cells under doxorubicin stimulation and influence many aspects of tumor cell biology.

Induction of urokinase‐type plasminogen activator by the anthracycline antibiotic in human RC‐K8 lymphoma and H69 lung‐carcinoma cells

Current evidence has suggested the possible involvement of ROS as signaling messengers in IL‐1β‐ or LPS‐induced gene expression. We previously reported that both IL‐1β and LPS induce uPA in RC‐K8 human lymphoma cells. Here, we provide evidence that ROS‐generating anthracycline antibiotics, including doxorubicin and aclarubicin, upregulate uPA expression in 2 human malignant cell lines, RC‐K8 and H69 small‐cell lung‐carcinoma cells. Both doxorubicin and aclarubicin markedly increased uPA accumulation in RC‐K8‐ and H69‐conditioned medium in a dose‐dependent manner. In each case, maximal induction was observed at a sublethal concentration, i.e., at a concentration where cell growth was slightly inhibited. Both doxorubicin and aclarubicin increased uPA mRNA levels, and induction in each case reached the maximal level 9 hr after stimulation. Doxorubicin barely changed the half‐life of uPA mRNA and activated uPA gene transcription. Antioxidants such as NAC and PDTC inhibited doxorubicin‐induced uPA mRNA accumulation. Microarray analysis, using Human Cancer CHIP version 2 (Takara Shuzo, Kyoto, Japan), in which 425 human cancer‐related genes were spotted on glass plates, revealed that uPA is 1 of 3 genes that were clearly upregulated in H69 cells by doxorubicin stimulation. These findings suggest that the anthracycline induces uPA in human malignant cells by activating gene transcription in which ROS may be involved. Therefore, by upregulating uPA expression, the anthracycline may influence many biologic cell functions mediated by the uPA/plasmin system.

The prophylactic effect of itraconazole capsules and fluconazole capsules for systemic fungal infections in patients with acute myeloid leukemia and myelodysplastic syndromes: A Japanese multicenter randomized, controlled study

We performed a randomized, controlled study comparing the prophylactic effects of capsule forms of fluconazole (n = 110) and itraconazole (n = 108) in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) during and after chemotherapy.There were 4 cases with possible systemic fungal infection in the itraconazole group, and there were 8 possible and 3 probable cases in the fluconazole group. Adverse events did not significantly differ in the 2 groups. In patients with MDS or in the remission-induction phase of chemotherapy, the numbers of cases with probable or possible infections were lower in the itraconazole group than in the fluconazole group, whereas no difference was seen in patients with AML or in the consolidation phase of therapy. In patients with neutrophil counts of <0.1 * 109/L lasting for more than 4 weeks, the frequency of infection in the fluconazole group (5 of 9 patients) was significantly higher than in the itraconazole group (0 of 7 patients; P = .03). Our results suggest that both drugs were well tolerated in patients with AML or MDS who received chemotherapy and that the efficacy of itraconazole for prophylaxis against systemic fungal disease is not inferior to that of fluconazole.

The effect of adding rituximab to CHOP-based therapy on clinical outcomes for Japanese patients with diffuse large B-cell lymphoma: a propensity score matching analysis.

We conducted a retrospective analysis to evaluate the impact on clinical outcomes of adding rituximab to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) treatment for diffuse large B-cell lymphoma (DLBCL) patients in Japan. A propensity score method was used to compensate for the non-randomized study design. From January 2000 to December 2004, 378 patients who were newly diagnosed with DLBCL at 13 institutes were enrolled: 123 in the rituximab plus CHOP-based chemotherapy (R+) group, and 255 in the CHOP-based chemotherapy only (R−) group. The complete response rate was significantly higher in the R+ group than in the R− group (77.7 vs. 69.4%, P < 0.001). The progression-free survival (PFS) at 2 years was 62.4% in the R+ group and 57.0% in the R− group. The 2-year overall survival (OS) was 76.9% for the R+ group and 70.5% for the R− group. A multivariate analysis revealed that the addition of rituximab was a strong independent prognostic factor for PFS (hazard ratio 0.64, 95% CI 0.43–0.96, P = 0.031). A subgroup analysis revealed that R+ particularly benefited younger patients (hazard ratio 0.25, 95% CI 0.08–0.75, P = 0.013). IPI also showed significant impact for PFS (hazard ratio 1.82, 95% CI 1.55–2.14 for one score increase, P < 0.001) as well as OS (hazard ratio 2.10, 95% CI 1.71–2.57, P < 0.001). In summary, the addition of rituximab to CHOP-based chemotherapy results in better outcomes for Japanese DLBCL patients, particularly younger patients.

Transfusion-transmitted hepatitis E in a patient with myelodysplastic syndromes

Patients with haematological diseases occasionally exhibit liver dysfunction during treatment. This liver dysfunction can have various causes such as therapy-related drugs and hepatitis B and C infections, although the cause is unclear in some cases. It was recently reported that some patients initially diagnosed with drug-induced liver dysfunction actually had hepatitis E1. Several cases of transfusion-transmitted hepatitis E infections have also been reported1,2. In Japan, screening for hepatitis E does not appear to be performed at the initial examination of patients with acute hepatitis. This might be because hepatitis E is believed to be orally transmitted and to occur mainly in developing countries and rarely in developed countries. However, hepatitis E is a zoonotic infectious disease. Cases of regional endemic hepatitis E virus (HEV) infection have been increasing in Europe, the United States, and Japan1. Although HEV usually causes self-limited acute hepatitis, it sometimes progresses to a chronic infection. Most cases of chronic infection occur in patients undergoing solid organ or haematopoietic stem cell transplantation, in those receiving anti-cancer or immunosuppressant drugs, and in patients with human immunodeficiency virus infection, in whom the condition may progress to liver cirrhosis3. HEV RNA persisted for a long period during treatment in a patient with T-cell lymphoma4. Reactivation of HEV hepatitis was reported after an allogeneic haematopoietic stem cell transplant in a patient with Philadelphia chromosome-positive acute lymphoblastic leukaemia5. On the other hand, a low risk of HEV reactivation after haematopoietic stem cell transplantation was also reported6. More studies on the risk of HEV reactivation are, therefore, required. Here, we report the case of a patient with a myelodysplastic syndrome (MDS) who developed acute hepatitis due to transfusion-transmitted HEV infection. We also review the literature on the topic.

Two Novel Gene Mutations in Type I Antithrombin Deficiency

We studied the molecular basis of type I antithrombin (AT) deficiency in 2 Japanese families, in which affected persons had histories of recurrent venous thrombosis and low (about 50% of normal) levels of AT protein according to measurements by both functional and antigen assays. Southern blotting of DNA isolated from peripheral leukocytes revealed no abnormalities in all the cases examined. Direct sequencing of the polymerase chain reaction (PCR) products from case 1 suggested a novel heterozygous nonsense mutation in exon 4 (GAG→TAG at nucleotide position 7627, leading to Glu306 stop). The sequencing of the subclones of the patient’s exon 4 products confirmed the nonsense mutation. No other sequence abnormalities were detected in the rest of the PCR products.The same mutation was detected in this patient’s brother, who had a history of recurrent venous thrombosis and a reduced level of AT activity. In case 2, the direct sequencing of PCR products suggested a novel heterozygous 9-bp deletion in exon 3a (—CACTTC at nucleotide position 5354-5362, leading to the deletion of 3 amino acids, His120, Phe121, and Phe122). The 9-bp deletion mutation in the region of a unique quasi palindrome was confirmed by sequencing several of the subclones of the patient’s exon 3a from the PCR products. No other mutations were found by direct sequencing of the rest of the coding regions. The 2 mutations found in this study are novel. The use of PCR and the sequencing of the PCR product subclones has simplified and confirmed the detection and characterization of the various AT mutations.

Tamibarotene for the treatment of acute promyelocytic leukemia

Introduction: Acute promyelocytic leukemia (APL) is characterized by a reciprocal translocation between chromosomes 15 and 17 yielding the PML-RARA fusion gene, which deregulates cell proliferation and blocks granulocyte differentiation. All-trans retinoic acid (ATRA) dramatically improved the prognosis of APL, but relatively, small number of relapsing patients achieves second remission with ATRA alone. Areas covered: Tamibarotene, a new synthetic retinoid, is about 10 times more potent than ATRA, chemically more stable than ATRA, with low affinity for cellular RA-binding protein and no detectable binding to RA receptor-γ. Tamibarotene has a significant effect on ATRA-resistant APLs. Some arsenic trioxide (ATO) and gemtuzumab ozogamicin-resistant APLs are reportedly tamibarotene-sensitive. Expert opinion: Tamibarotene was tested in APL-relapsed patients after ATRA-induced complete remission (CR), and 14/24 patients (58%) achieved CR. In an independent trial, 24/39 (61.5%) APL patients achieved CR including 5 newly diagnosed patients and 13 patients who had relapsed twice or more. A prospective randomized study compared tamibarotene with ATRA as maintenance therapy. Four-year relapse-free survival rate was 84% (ATRA) and 91% (tamibarotene) (p = 0.095). In high-risk patients, this became significant (58% ATRA, 87% tamibarotene, p = 0.028). These results suggest tamibarotene should be tested in induction or consolidation therapy and in combination with ATO.