Tatsuya Kawaguchi

Publications of Tatsuya Kawaguchi

• Efficacy and safety of nilotinib in Japanese patients with imatinib-resistant or -intolerant Ph+ CML or relapsed/refractory Ph+ ALL: a 36-month analysis of a phase I and II study.

  Authors: Kensuke Usuki, Arinobu Tojo, Yasuhiro Maeda, Yukio Kobayashi, Akira Matsuda, Kazuma Ohyashiki, Chiaki Nakaseko, Tatsuya Kawaguchi, Hideo Tanaka, Koichi Miyamura, Yasushi Miyazaki, Shinichiro Okamoto, Kenji Oritani, Masaya Okada, Noriko Usui, Tadashi Nagai, Taro Amagasaki, Aira Wanajo, Tomoki Naoe

  International journal of hematology. 02/2012;

  Although the tyrosine kinase inhibitor (TKI) imatinib is often used as first-line therapy for newly diagnosed chronic myelogenous leukemia (CML), some patients fail to respond, or become intolerantAlthough the tyrosine kinase inhibitor (TKI) imatinib is often used as first-line therapy for newly diagnosed chronic myelogenous leukemia (CML), some patients fail to respond, or become intolerant to imatinib. Nilotinib is a potent and selective second-generation TKI, with confirmed efficacy and tolerability in patients with imatinib-resistant or -intolerant CML. A phase I/II study was conducted in Japanese patients with imatinib-resistant or -intolerant CML or relapsed/refractory Ph+ acute lymphoblastic leukemia. Thirty-four patients were treated with nilotinib for up to 36 months. Major cytogenetic response was achieved in 15/16 patients (93.8%) with chronic-phase CML within a median of approximately 3 months. Major molecular response was achieved in 13/16 patients (81.3%). These responses were sustained at the time of the most recent evaluation in 13 patients and 11 patients, respectively. Hematologic and cytogenetic responses were also observed in patients with advanced CML. The BCR-ABL mutation associated with the most resistance to available TKIs, T315I, was observed in three patients. Common adverse events included rash, nasopharyngitis, leukopenia, neutropenia, thrombocytopenia, nausea, headache and vomiting. Most adverse events resolved following nilotinib dose interruptions/reductions. These results support the favorable long-term efficacy and tolerability of nilotinib in Japanese patients with imatinib-resistant or -intolerant chronic-phase chronic myeloid leukemia.

• Discontinuation of imatinib in Japanese patients with chronic myeloid leukemia.

  Authors: Naoto Takahashi, Taiichi Kyo, Yasuhiro Maeda, Takashi Sugihara, Kensuke Usuki, Tatsuya Kawaguchi, Noriko Usui, Shinichiro Okamoto, Yokiko Ohe, Shigeki Ohtake, Kunio Kitamura, Masahide Yamamoto, Hirofumi Teshima, Toshiko Motoji, Toshiharu Tamaki, Kenichi Sawada, Kazuma Ohyashiki

  Haematologica. 12/2011;

  It was recently recognized that some chronic myeloid leukemia patients with a complete molecular response could sustain that response after discontinuation of imatinib. To characterize the clinicalIt was recently recognized that some chronic myeloid leukemia patients with a complete molecular response could sustain that response after discontinuation of imatinib. To characterize the clinical outcomes and profiles of chronic phase chronic myeloid leukemia patients who could discontinue imatinib, we conducted a nationwide survey in Japan. Among 3,242 imatinib-treated chronic myeloid leukemia patients, 50 were identified who discontinued imatinib for at least 6 months; of those, 43 were analyzed. Molecular recurrence was detected in 19 patients, and a complete molecular response rate was estimated to be 47% following imatinib discontinuation. Based on multivariate regression analysis, imatinib dose intensity and prior interferon-α administration were independently predictive of molecular recurrence within 12 months. The depth of the molecular response should be a factor influencing long-term sustained complete molecular response after discontinuation of imatinib. Additionally, an immunological mechanism modified by interferon-α might control chronic myeloid leukemia stem cells.

• Association of genetic polymorphisms in the influx transporter SLCO1B3 and the efflux transporter ABCB1 with imatinib pharmacokinetics in patients with chronic myeloid leukemia.

  Authors: Yuji Yamakawa, Akinobu Hamada, Reiko Nakashima, Misato Yuki, Chie Hirayama, Tatsuya Kawaguchi, Hideyuki Saito

  Therapeutic drug monitoring. 02/2011; 33(2):244-50.

  This study explored the association of 14 single nucleotide polymorphisms in three genes coding for influx transporters (SLC22A1, SLCO1B1, and SLCO1B3), two genes coding for efflux transportersThis study explored the association of 14 single nucleotide polymorphisms in three genes coding for influx transporters (SLC22A1, SLCO1B1, and SLCO1B3), two genes coding for efflux transporters (ABCB1 and ABCG2), and four genes coding for enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A5) with the pharmacokinetics of imatinib in Japanese patients with chronic myeloid leukemia. Pharmacokinetic parameters were estimated by a population pharmacokinetic analysis based on 622 plasma samples from 34 patients at steady state. Approximately 4.6-fold variability in individual clearance was observed (range, 3.4-15.5 L/hr). The individual estimated clearance was significantly increased in patients with the SLCO1B3 334GG genotype (median value ± standard deviation, 9.5 ± 3.1 L/hr; n = 19) compared with SLCO1B3 334TT and TG genotypes (7.0 ± 3.1 L/hr; n = 15) (P = 0.019). Patients with the ABCB1 3435CC genotype had significantly higher imatinib clearance (12.7 ± 3.0 L/hr; n = 7) compared with patients with ABCB1 3435CT and TT genotypes (7.9 ± 2.7 L/hr; n = 27) (P = 0.035). In conclusion, the present study suggests that single nucleotide polymorphisms of the influx transporter SLCO1B3 and the efflux transporter ABCB1 were functionally associated with individual variability of imatinib pharmacokinetics in Japanese patients with chronic myeloid leukemia.

• Association of SLCO1B3 polymorphism with intracellular accumulation of imatinib in leukocytes in patients with chronic myeloid leukemia.

  Authors: Takeru Nambu, Akinobu Hamada, Reiko Nakashima, Misato Yuki, Tatsuya Kawaguchi, Hiroaki Mitsuya, Hideyuki Saito

  Biological & pharmaceutical bulletin. 01/2011; 34(1):114-9.

  Intracellular concentration of imatinib in leukemic cells is thought to affect the clinical efficacy of this drug in patients with chronic myeloid leukemia (CML); however, there is no report thatIntracellular concentration of imatinib in leukemic cells is thought to affect the clinical efficacy of this drug in patients with chronic myeloid leukemia (CML); however, there is no report that directly indicates the relationship between intracellular concentration and clinical outcome and/or, plasma concentration. In addition, the impacts of genetic variations of drug transporters, which mediate leukocyte concentration of imatinib, are unknown. In the present study, we investigated the correlation between intracellular imatinib concentrations in leukocytes, plasma imatinib levels, and genotypes of drug transporters, including ATP binding cassette B1 (ABCB), ABCG2, solute carrier 22A1 (SLC22A1), solute carrier organic anion transporter family members 1B1 (SLCO1B1) and SLCO1B3. The imatinib levels in leukocytes were determined using HPLC in 15 patients with chronic phase CML. No significant correlation between intracellular and plasma concentrations of imatinib was observed. The intracellular concentration was comparable in both patients with or without complete cytogenetic response. The intracellular imatinib concentration was significantly higher in patients with SLCO1B3 334TT than in those with 334TG/GG (p=0.0188). Plasma concentrations were similar in both SLCO1B3 genotypes (p=0.860), thereby resulting in the intracellular to plasma concentration ratio being higher in patients with SLCO1B3 334TT than those with 334 TG/GG (p=0.0502). These results suggested that the SLCO1B3 334T>G polymorphism could have a significant impact on the intracellular concentration of imatinib in leukocytes as a promising biomarker for personalized treatment of CML patients.

• Safety and efficacy of the terminal complement inhibitor eculizumab in Japanese patients with paroxysmal nocturnal hemoglobinuria: the AEGIS clinical trial.

  Authors: Yuzuru Kanakura, Kazuma Ohyashiki, Tsutomu Shichishima, Shinichiro Okamoto, Kiyoshi Ando, Haruhiko Ninomiya, Tatsuya Kawaguchi, Shinji Nakao, Hideki Nakakuma, Jun-ichi Nishimura, Taroh Kinoshita, Camille L Bedrosian, Marye Ellen Valentine, Gus Khursigara, Keiya Ozawa, Mitsuhiro Omine

  International journal of hematology. 01/2011; 93(1):36-46.

  Paroxysmal nocturnal hemoglobinuria (PNH) is a progressive and life-threatening disease characterized by complement-mediated chronic hemolysis, resulting in serious life-threatening complications andParoxysmal nocturnal hemoglobinuria (PNH) is a progressive and life-threatening disease characterized by complement-mediated chronic hemolysis, resulting in serious life-threatening complications and early mortality. Eculizumab, a humanized anti-C5 monoclonal antibody that inhibits terminal complement activation, has been shown to reduce hemolysis in PNH patients. The pivotal open-label, 12-week phase II registration study (AEGIS) was designed to evaluate the efficacy and safety of eculizumab in Japanese patients with PNH. This trial achieved its primary endpoint of reducing intravascular hemolysis with high statistical significance. Twenty-seven of the 29 patients responded to eculizumab treatment, resulting in an 87% reduction in hemolysis (P < 0.0001) and subsequent improvement in anemia (P = 0.0003) despite reduction in transfusion requirements (P = 0.006). Fatigue and dyspnea significantly improved within 1-2 weeks of eculizumab treatment and the improvement was independent of changes in hemoglobin. Chronic kidney disease (CKD) was common (66%) and eculizumab treatment improved CKD in 41% of patients at 12 weeks (P < 0.001). Elevated thrombotic risk was evident in Japanese PNH patients and eculizumab treatment normalized D: -dimer levels in 45% of patients with elevated D: -dimers at baseline (P < 0.001). The AEGIS results demonstrate that eculizumab is effective, safe and well tolerated in Japanese patients with PNH.

• High-performance liquid chromatographic assay for the determination of nilotinib in human plasma.

  Authors: Misato Yuki, Yuji Yamakawa, Takashi Uchida, Takeru Nambu, Tatsuya Kawaguchi, Akinobu Hamada, Hideyuki Saito

  Biological & pharmaceutical bulletin. 01/2011; 34(7):1126-8.

  A precise and convenient high-performance liquid chromatography (HPLC) method has been established to assay nilotinib in human plasma. Chromatographic separation of nilotinib was performed on aA precise and convenient high-performance liquid chromatography (HPLC) method has been established to assay nilotinib in human plasma. Chromatographic separation of nilotinib was performed on a LiChrosphere(®)100 RP-18(e) column (250 mm×4.0 mm, 5 µm) using a mixture of acetonitrile and 0.01 M phosphate buffer (pH 3.0) (42 : 58, v/v) under isocratic conditions at a flow rate of 1.0 ml/min with ultraviolet (UV) detection at 266 nm. The calibration curve showed linearity at concentrations between 250 ng/ml and 5000 ng/ml (r(2)>0.999). The mean±S.D. absolute recovery of nilotinib from plasma was 99.2±3.3%. The coefficients of variation of both intra- and inter-day precision were below 9.1%. These results indicate that this new HPLC-based quantification may be useful for therapeutic drug monitoring of nilotinib to help manage treatment in patients with chronic myeloid leukemia in clinical practice.

• Successful treatment of Aspergillus liver abscesses in a patient with acute monoblastic leukemia using combination antifungal therapy including micafungin as a key drug.

  Authors: Rie Yamada, Kentaro Horikawa, Sonoko Ishihara, Koyu Hoshino, Tatsuya Kawaguchi, Ken-ichi Iyama, Hiroaki Mitsuya, Norio Asou

  International journal of hematology. 03/2010; 91(4):711-5.

  While anti-cancer chemotherapy has improved the survival of patients with hematologic malignancies, it has also exposed such patients to the risk of life-threatening infection due to neutropenia. InWhile anti-cancer chemotherapy has improved the survival of patients with hematologic malignancies, it has also exposed such patients to the risk of life-threatening infection due to neutropenia. In intensive chemotherapy for leukemia, invasive aspergillosis resulting in death is infrequently observed. In such cases, aggressive diagnostic and therapeutic intervention is required. Herein, we report a case of Aspergillus liver abscesses in a patient with acute monoblastic leukemia. The patient presented with febrile neutropenia and concomitantly with an elevated serum beta-D: -glucan level during chemotherapy. The abscesses were finally diagnosed by liver biopsy. Although antifungal monotherapy of voriconazole or liposomal amphotericin B, both of which are recommended for invasive aspergillosis, showed a poor response, when combined with micafungin, an echinocandin, both had a highly favorable effect against the infection. Therefore, our clinical experience suggests that the serum test is useful for the rapid diagnosis of invasive aspergillosis, especially in deep tissues, and that combination antifungal therapy with micafungin should be considered when initial monotherapy for fungal infection shows an insufficient effect.

• Contribution of BCR-ABL-independent activation of ERK1/2 to acquired imatinib resistance in K562 chronic myeloid leukemia cells.

  Authors: Takeru Nambu, Norie Araki, Aiko Nakagawa, Akihiko Kuniyasu, Tatsuya Kawaguchi, Akinobu Hamada, Hideyuki Saito

  Cancer science. 09/2009;

  BCR-ABL tyrosine kinase, generated from the reciprocal chromosomal translocation t(9;22), causes chronic myeloid leukemia (CML). BCR-ABL is inhibited by imatinib; however, several mechanisms ofBCR-ABL tyrosine kinase, generated from the reciprocal chromosomal translocation t(9;22), causes chronic myeloid leukemia (CML). BCR-ABL is inhibited by imatinib; however, several mechanisms of imatinib resistance have been proposed that account for loss of imatinib efficacy in patients with CML. Previously, we showed that overexpression of the efflux drug transporter P-glycoprotein partially contributed to imatinib resistance in imatinib-resistant K562 CML cells having no BCR-ABL mutations. To explain an additional mechanism of drug resistance, we established a subclone (K562/R) of the cells and examined the BCR-ABL signaling pathway in these and wild-type K562 (K562/W) cells. We found the K562/R cells were 15 times more resistant to imatinib than their wild-type counterparts. In both cell lines, BCR-ABL and its downstream signaling molecules, such as ERK1/2, ERK5, STAT5, and AKT, were phosphorylated in the absence of imatinib. In both cell lines, imatinib effectively reduced the phosphorylation of all the above, except ERK1/2, whose phosphorylation was, interestingly, only inhibited in the wild-type cells. We then observed that phospho-ERK1/2 levels decreased in the presence of siRNA targeting BCR-ABL, again, only in the K562/W cells. However, using an ERK1/2 inhibitor, U0126, we found that we could reduce phospho-ERK1/2 levels in K562/R cells and restore their sensitivity to imatinib. Taken together, we conclude that the BCR-ABL-independent activation of ERK1/2 contributes to imatinib resistance in K562/R cells, and that ERK1/2 could be a target for the treatment of CML patients whose imatinib resistance is due to this mechanism. (Cancer Sci 2009).

• NKG2D-mediated immunity underlying paroxysmal nocturnal haemoglobinuria and related bone marrow failure syndromes.

  Authors: Nobuyoshi Hanaoka, Hideki Nakakuma, Kentaro Horikawa, Shoichi Nagakura, Yasuchika Tsuzuki, Masaya Shimanuki, Kensuke Kojima, Yuji Yonemura, Tatsuya Kawaguchi

  British journal of haematology. 08/2009;

  Summary It is considered that a similar immune mechanism acts in the pathogenesis of bone marrow (BM) failure in paroxysmal nocturnal haemoglobinuria (PNH) and its related disorders, such as aplasticSummary It is considered that a similar immune mechanism acts in the pathogenesis of bone marrow (BM) failure in paroxysmal nocturnal haemoglobinuria (PNH) and its related disorders, such as aplastic anaemia (AA) and myelodysplastic syndromes (MDS). However, the molecular events in immune-mediated marrow injury have not been elucidated. We recently reported an abnormal expression of stress-inducible NKG2D (natural-killer group 2, member D) ligands, such as ULBP (UL16-binding protein) and MICA/B (major histocompatibility complex class I chain-related molecules A/B), on granulocytes in some PNH patients and the granulocyte killing by autologous lymphocytes in vitro. The present study found that the expression of NKG2D ligands was common to both granulocytes and BM cells of patients with PNH, AA, and MDS, indicating their exposure to some incitement to induce the ligands. The haematopoietic colony formation in vitro by the patients' marrow cells significantly improved when their BM cells were pretreated with antibodies against NKG2D receptor, suggesting that the antibodies rescued haematopoietic cells expressing NKG2D ligands from damage by autologous lymphocytes with NKG2D. Clinical courses of patients with PNH and AA showed a close association of the expression of NKG2D ligands with BM failure and a favourable response to immunosuppressive therapy. We therefore propose that NKG2D-mediated immunity may underlie the BM failure in PNH and its-related marrow diseases.

• A Phase I/II study of nilotinib in Japanese patients with imatinib-resistant or -intolerant Ph+ CML or relapsed/refractory Ph+ ALL.

  Authors: Arinobu Tojo, Kensuke Usuki, Akio Urabe, Yasuhiro Maeda, Yukio Kobayashi, Itsuro Jinnai, Kazuma Ohyashiki, Miki Nishimura, Tatsuya Kawaguchi, Hideo Tanaka [......] Timothy Hughes, Susan Branford, Shinichiro Okamoto, Jun Ishikawa, Masaya Okada, Noriko Usui, Hiromi Tanii, Taro Amagasaki, Hiroko Natori, Tomoki Naoe

  International journal of hematology. 06/2009;

  Nilotinib is a second-generation BCR-ABL kinase inhibitor with improved potency and selectivity compared to imatinib. A Phase I/II dose-escalation study was designed to evaluate the efficacy, safety,Nilotinib is a second-generation BCR-ABL kinase inhibitor with improved potency and selectivity compared to imatinib. A Phase I/II dose-escalation study was designed to evaluate the efficacy, safety, and pharmacokinetics of nilotinib in Japanese patients with imatinib-resistant or -intolerant Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) or relapsed/refractory Ph+ acute lymphoblastic leukemia (ALL). A total of 34 patients were evaluated in this analysis and had a median duration of drug exposure of 293 (range 13-615) days. All 6 CML-CP patients without complete hematologic response (CHR) at baseline rapidly achieved CHR. A major cytogenetic response was achieved in 94% of patients with CML-CP, including a complete cytogenetic response in 69%. A major molecular response was achieved by 56%. These responses were also observed in patients with CML in advanced stages and Ph+ ALL. Non-hematologic adverse events were mostly mild to moderate. Grade 3 or 4 neutropenia and thrombocytopenia occurred in 50 and 28% of patients, respectively. Overall, the results of this study suggest that nilotinib induced significant responses in imatinib-resistant or -intolerant patients with CML-CP and CML in advanced stages and Ph+ ALL. The results of this study confirmed the efficacy and safety of nilotinib in Japanese patients.

• Relationship between an effective dose of imatinib, body surface area, and trough drug levels in patients with chronic myeloid leukemia.

  Authors: Tatsuya Kawaguchi, Akinobu Hamada, Chie Hirayama, Reiko Nakashima, Takeru Nambu, Yuji Yamakawa, Hiroshi Watanabe, Kentaro Horikawa, Hiroaki Mitsuya, Hideyuki Saito

  International journal of hematology. 05/2009;

  The standard dose of imatinib for the treatment of chronic-phase chronic myeloid leukemia (CML) is 400 mg/day. Some patients receive reduced doses of imatinib because of serious adverse effects.The standard dose of imatinib for the treatment of chronic-phase chronic myeloid leukemia (CML) is 400 mg/day. Some patients receive reduced doses of imatinib because of serious adverse effects. Recently, the effective plasma threshold for trough imatinib levels was demonstrated to be 1,002 ng/mL. In this study, we evaluated the association of an imatinib dose with trough plasma concentrations and clinical outcomes in 31 patients with chronic-phase CML who were treated at Kumamoto University Hospital. Twenty-seven patients were optimally treated with various doses of imatinib. The mean (+/-SD) trough plasma concentrations of imatinib were 1.40 +/- 0.57 mug/mL in 13 patients receiving 400 mg/day and 1.15 +/- 0.44 mug/mL in 9 patients receiving 300 mg/day as an effective dose. Mean trough levels of the two groups were not significantly different and exceeded the effective plasma threshold. Body surface area (BSA) was significantly smaller in patients receiving the reduced dose compared with those receiving the standard dose (p = 0.001). The effective imatinib dose was associated with age and gender as well as BSA. A reduced dose of 300 mg/day of imatinib may be sufficient for the treatment of CML patients with smaller body size, particularly when intolerability arises.

• [Clinical Values of Biomarkers in Hematopoietic Malignancies.]

  Authors: Tatsuya Kawaguchi, Kisato Nosaka

  Gan to kagaku ryoho. Cancer & chemotherapy. 02/2009; 36(1):26-31.

  Hematopoietic malignancies include leukemia, lymphoma and multiple myeloma. These diseases are primarily diagnosed on the basis of morphological features of affected cells, which appear in peripheralHematopoietic malignancies include leukemia, lymphoma and multiple myeloma. These diseases are primarily diagnosed on the basis of morphological features of affected cells, which appear in peripheral blood, bone marrow and lymphoid organs. By taking advantage of the repetitive accessibility of the neoplastic cells within the peripheral blood / and/or bone marrow aspirates, morphological tests are conducted not only for diagnosis but also for evaluation of clinical outcomes and prognosis, suggesting that the morphological features are considered as a clinical biomarker in hematopoietic malignancies. However, remarkable progress in molecular targeted therapy and allogeneic hematopoietic stem cell transplantation has improved the long-term prognosis of patients with hematopoietic malignancies, and some patients are curable. Under such modern strategies for therapy, monitoring of minimal residual disease(MRD), which is morphologically undetectable, is required to guide proper management of the disease by evaluation of an optimal response to therapy and early detection of disease relapse. At present, both immunophenotypes(surface markers)and chimeric fusion genes(e. g. BCR-ABL in chronic myeloid leukemia)characteristic of hematopoietic malignant cells are analyzed as clinically useful biomarkers to monitor MRD by two highly sensitive methods, multiparameter flow cytometry and real-time quantitative PCR, respectively. On the other hand, serum markers reflecting the size of the tumor mass are clinically available to monitor the disease progression in mass-forming hematopoietic malignancies: e. g., soluble IL-2 receptor for lymphoma and M-protein or free light chain for multiple myeloma.

• [Aspergillosis of masticator space including mandibular bone responding to itraconazole treatment in a diabetic adult: a case report]

  Authors: Kentaro Horikawa, Hironori Kudo, Sonoko Ishihara, Toshikazu Miyakawa, Tatsuya Kawaguchi, Hiroaki Mitsuya

  Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases. 06/2008; 82(3):220-3.

  Aspergillosis of the bone is rare and resistant to treatment. We report a case of Aspergillus infection of the masticator space including mandibular bone in a diabetic adult. After extraction of aAspergillosis of the bone is rare and resistant to treatment. We report a case of Aspergillus infection of the masticator space including mandibular bone in a diabetic adult. After extraction of a posterior tooth, the patient began to suffer from facial pain. The pain worsened in spite of antibiotic treatment. The results of serum tests and biopsy showed an invasive aspergillosis of the left masticator space including the mandibular bone six months after the onset. Although invasive aspergillosis can be fatal, the infection in our case responded to itraconazole treatment. Even in diabetes mellitus, invasive aspergillosis may occur after surgical interventions such as tooth extraction.

• Constitutive overexpression of P-glycoprotein, rather than breast cancer resistance protein or organic cation transporter 1, contributes to acquisition of imatinib-resistance in K562 cells.

  Authors: Chie Hirayama, Hiroshi Watanabe, Reiko Nakashima, Takeru Nanbu, Akinobu Hamada, Akihiko Kuniyasu, Hitoshi Nakayama, Tatsuya Kawaguchi, Hideyuki Saito

  Pharmaceutical research. 05/2008; 25(4):827-35.

  PURPOSE: The purpose of this study was to investigate the contribution of drug transporters in acquired imatinib-resistance. Specifically, we focused on the efflux transporters, P-glycoprotein (P-gp)PURPOSE: The purpose of this study was to investigate the contribution of drug transporters in acquired imatinib-resistance. Specifically, we focused on the efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), and an influx transporter, organic cation transporter 1 (OCT1). MATERIALS AND METHODS: We established imatinib-resistant K562 cells (K562/IM). Real-time PCR or Western blot analyses were performed to examine the mRNA or protein levels. Alamar blue method was used in the cytotoxicity assay. The transport activities and intracellular imatinib levels were measured by flow cytometry and HPLC, respectively. RESULTS: K562/IM displayed a 47-fold increase in resistance to imatinib over the parent K562 cells. Both P-gp and BCRP were overexpressed in K562/IM relative to K562. Furthermore, the intracellular imatinib level was markedly reduced in K562/IM. Interestingly, cyclosporin A, a P-gp inhibitor, but not fumitremorgin C, a BCRP inhibitor, restored both imatinib-sensitivity and the intracellular imatinib level. By contrast, no significant difference in the expression and function of OCT1 was observed between K562/IM and K562. CONCLUSIONS: P-gp, rather than BCRP or OCT1, is partially responsible for the development of imatinib-resistance due to constitutive and functional overexpression, leading to reduced intracellular accumulation of imatinib in K562/IM.

• New insights into molecular pathogenesis of bone marrow failure in paroxysmal nocturnal hemoglobinuria.

  Authors: Tatsuya Kawaguchi, Hideki Nakakuma

  International journal of hematology. 08/2007; 86(1):27-32.

  Paroxysmal nocturnal hemoglobinuria (PNH) is caused by the clonal expansion of hematopoietic stem cells with mutations of the phosphatidylinositol glycan-class A gene (PIGA). PNH clones then fail toParoxysmal nocturnal hemoglobinuria (PNH) is caused by the clonal expansion of hematopoietic stem cells with mutations of the phosphatidylinositol glycan-class A gene (PIGA). PNH clones then fail to generate glycosylphosphatidylinositol (GPI) or to express a series of GPI-linked membrane proteins including complement-regulatory proteins, resulting in complement-mediated intravascular hemolysis and thrombosis. Bone marrow failure is another characteristic feature of PNH. It is currently considered that immune-mediated injury of hematopoietic cells is implicated in PNH marrow failure as well as in aplastic anemia, a well-known PNH-related disorder. There is increasing evidence that the autoimmune attack allows PNH clones to selectively survive in the injured marrow, leading to clinical manifestations characteristic of PNH. As candidate molecules that trigger the immune attack on marrow cells, stress-inducible membrane proteins and Wilms' tumor protein WT1 have been proposed. Among the stress-inducible proteins, GPI-linked proteins, such as cytomegalovirus glycoprotein UL16-binding protein, are distinct candidates that not only induce immune attack, but also allow PNH clones to survive the attack. Here, we overview the current understanding of the molecular pathogenesis of bone marrow failure in PNH.

• Immunoselection by natural killer cells of PIGA mutant cells missing stress-inducible ULBP.

  Authors: Nobuyoshi Hanaoka, Tatsuya Kawaguchi, Kentaro Horikawa, Shoichi Nagakura, Hiroaki Mitsuya, Hideki Nakakuma

  Blood. 03/2006; 107(3):1184-91.

  The mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand is unknown. PNH clones harbor PIGA mutations and do not synthesize glycosylphosphatidylinositol (GPI), resulting inThe mechanism by which paroxysmal nocturnal hemoglobinuria (PNH) clones expand is unknown. PNH clones harbor PIGA mutations and do not synthesize glycosylphosphatidylinositol (GPI), resulting in deficiency of GPI-linked membrane proteins. GPI-deficient blood cells often expand in patients with aplastic anemia who sustain immune-mediated marrow injury putatively induced by cytotoxic cells, hence suggesting that the injury allows PNH clones to expand selectively. We previously reported that leukemic K562 cells preferentially survived natural killer (NK) cell-mediated cytotoxicity in vitro when they acquired PIGA mutations. We herein show that the survival is ascribable to the deficiency of stress-inducible GPI-linked membrane proteins ULBP1 and ULBP2, which activate NK and T cells. The ULBPs were detected on GPI-expressing but not on GPI-deficient K562 cells. In the presence of antibodies to either the ULBPs or their receptor NKG2D on NK cells, GPI-expressing cells were as less NK sensitive as GPI-deficient cells. NK cells therefore spared ULBP-deficient cells in vitro. The ULBPs were identified only on GPI-expressing blood cells of a proportion of patients with PNH but none of healthy individuals. Granulocytes of the patients partly underwent killing by autologous cytotoxic cells, implying ULBP-associated blood cell injury. In this setting, the lack of ULBPs may allow immunoselection of PNH clones.

• [Preventive effects of oren-gedoku-to on mucositis caused by anticancer agents in patients with acute leukemia]

  Authors: Fumiko Yuki, Tatsuya Kawaguchi, Kazuyo Hazemoto, Norio Asou

  Gan to kagaku ryoho. Cancer & chemotherapy. 10/2003; 30(9):1303-7.

  Most anticancer agents frequently cause mucositis, such as stomatitis and gastrointestinal mucosal injury, which is closely associated with decrease in quality of life, infections and discontinuationMost anticancer agents frequently cause mucositis, such as stomatitis and gastrointestinal mucosal injury, which is closely associated with decrease in quality of life, infections and discontinuation of chemotherapy in patients with malignancy. We retrospectively evaluated the preventive effect of oral administration of oren-gedoku-to on stomatitis and diarrhea induced by cytotoxic drugs in 40 patients with acute leukemia. Incidence of stomatitis was 27.9% in the group given oren-gedoku-to, which was significantly lower compared with 71.6% in those who received a gargle consisting of allopurinol, sodium gualenate, and povidone-iodine. Drug-induced diarrhea was observed in 9.3% of the oren-gedoku-to group compared with 31.7% of the control group. These observations indicate a significant preventive effect of oren-gedoku-to on mucositis caused by anticancer agents.

• Pathogenesis of selective expansion of PNH clones.

  Authors: Hideki Nakakuma, Tatsuya Kawaguchi

  International journal of hematology. 03/2003; 77(2):121-4.

  Hemolysis, a characteristic of paroxysmal nocturnal hemoglobinuria (PNH), is caused by the expansion of an affected stem cell with a mutation of the PIG-A gene. Increasing evidence has shown that theHemolysis, a characteristic of paroxysmal nocturnal hemoglobinuria (PNH), is caused by the expansion of an affected stem cell with a mutation of the PIG-A gene. Increasing evidence has shown that the presence of the PIG-A mutation alone does not induce the expansion. Two theories have been proposed. One, the growth advantage hypothesis, is supported by current data indicating the presence of several intrinsic alterations that might confer a proliferative advantage to PNH clones over normal cells. Alternatively, the PIG-A mutation might confer a relative survival advantage to PNH clones. This theory is supported by clinical observation indicating that PIG-A mutant cells survive immune-mediated bone marrow injury in patients with aplastic anemia, PNH, and myelodysplastic syndromes. The latter theory is also supported by current experimental data indicating that PIG-A mutant cells are relatively resistant to cytotoxic attack by natural killer cells and cytotoxic T-lymphocytes. The 2 theories appear complementary rather than mutually exclusive. Rapid progress in this field can be expected in the near future.

• Decreased susceptibility of leukemic cells with PIG-A mutation to natural killer cells in vitro.

  Authors: Shoichi Nagakura, Sonoko Ishihara, Daniel E Dunn, Jun-Ichi Nishimura, Tatsuya Kawaguchi, Kentaro Horikawa, Michihiro Hidaka, Tadashi Kagimoto, Nozomu Eto, Hiroaki Mitsuya, Taroh Kinoshita, Neal S Young, Hideki Nakakuma

  Blood. 09/2002; 100(3):1031-7.

  The cloning of the PIG-A gene has facilitated the unraveling of the complex pathophysiology of paroxysmal nocturnal hemoglobinuria (PNH). Of current major concern is the mechanism by which a PNHThe cloning of the PIG-A gene has facilitated the unraveling of the complex pathophysiology of paroxysmal nocturnal hemoglobinuria (PNH). Of current major concern is the mechanism by which a PNH clone expands. Many reports have suggested that an immune mechanism operates to cause bone marrow failure in some patients with PNH, aplastic anemia, and myelodysplastic syndromes. Because blood cells of PNH phenotype are often found in patients with these marrow diseases, one hypothesis is that the PNH clone escapes immune attack, producing a survival advantage by immunoselection. To test this hypothesis, we examined the sensitivity of blood cells, with or without PIG-A mutations, to killing by natural killer (NK) cells, using 51Cr-release assay in vitro. To both peripheral blood and cultured NK cells, PIG-A mutant cells prepared from myeloid and lymphoid leukemic cell lines were less susceptible than their control counterparts (reverted from the mutant cells by transfection with a PIG-A cDNA). NK activity was completely abolished with concanamycin A and by calcium chelation, indicating that killing was perforin-dependent. There were no differences in major histocompatibility (MHC) class I expression or sensitivity to either purified perforin or to interleukin-2-activated NK cells between PIG-A mutant and control cells. From these results, we infer that PIG-A mutant cells lack molecules needed for NK activation or to trigger perforin-mediated killing. Our experiments suggest that PIG-A mutations confer a relative survival advantage to a PNH clone, contributing to selective expansion of these cells in the setting of marrow injury by cytotoxic lymphocytes.

• Frequent detection of T cells with mutations of the hypoxanthine-guanine phosphoribosyl transferase gene in patients with paroxysmal nocturnal hemoglobinuria.

  Authors: Kentaro Horikawa, Tatsuya Kawaguchi, Sonoko Ishihara, Shoichi Nagakura, Michihiro Hidaka, Tadashi Kagimoto, Hiroaki Mitsuya, Hideki Nakakuma

  Blood. 01/2002; 99(1):24-9.

  Acquired mutations of the PIG-A gene result in the hemolysis characteristic of paroxysmal nocturnal hemoglobinuria (PNH). Although the etiology of the mutation(s) is unclear, mutable conditions haveAcquired mutations of the PIG-A gene result in the hemolysis characteristic of paroxysmal nocturnal hemoglobinuria (PNH). Although the etiology of the mutation(s) is unclear, mutable conditions have been suggested by the coexistence of multiple clones with different mutations of PIG-A and by the appearance of leukemic clones in patients with PNH. This study sought to test this hypothesis by examining the frequency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutations, identified by both resistance to 6-thioguanine (6-TG) and gene analysis. T-cell colonies resistant to 6-TG formed in methylcellulose culture were found in 8 (67%) of 12 PNH patients and 3 (18%) of 17 age-matched healthy volunteers (P <.02, Fisher exact probability test). The incidence of resistant colonies ranged from 40 to 367 (mean 149, x 10(-7)) in the 8 patients and from 1 to 16 (mean 7, x 10(-7)) in the 3 healthy donors. Thus, the HRPT gene mutated more frequently in patients with PNH than in healthy controls (P <.02, Mann-Whitney test). Analysis of bone marrow cells supported these findings. Like the PIG-A mutations in PNH, the HPRT mutations were widely distributed in the coding regions and consisted primarily of base deletions. Unlike PNH cells, 6-TG-resistant cells expressed CD59, indicating that the HPRT mutations did not occur in PNH clones. No correlation was noted between HPRT mutation frequency and content of therapy received by the patients. It is concluded that in PNH patients, conditions exist that favor the occurrence of diverse somatic mutations in blood cells.