We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K(m)(AdoMet), for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 microM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K(m)(DNA) values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 microM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT >/= CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.
We assessed the efficacy and safety of weekly cyclophosphamide-bortezomib-dexamethasone (CBD) induction prior to autologous stem cell transplantation (ASCT) in newly diagnosed Japanese patients with multiple myeloma (MM). This regimen consisted of four 28-day cycles of once-weekly oral cyclophosphamide (300 mg/m2), subcutaneous bortezomib (1.3 mg/m2), and oral dexamethasone (40 mg). Responding patients underwent stem cell collection followed by ASCT. The primary endpoint was the postinduction rate of achieving a near complete response (nCR) or better. Among the 38 enrolled patients, a complete response (CR), an nCR, a very good partial response (VGPR), and a partial response (PR) were achieved in 10.5, 2.6, 23.7, and 36.8% of cases, respectively. A grade 4 hematological adverse event (AE) was observed in 1 patient. Grade 3–4 infection, including febrile neutropenia, was observed in 4 patients (10.5%). Although 2 patients dropped out due to AE, 94.7% of the patients completed the induction phase. However, because of a poor response to induction chemotherapy (
Background: Treatment-free remission (TFR) is a new treatment goal for patients with chronic myeloid leukemia in chronic phase (CML-CP) with a sustained deep molecular response (DMR) by treatment with tyrosine kinase inhibitors (TKI). Although several guidelines have proposed clinical factors for successful TFR, they are based primarily on evidence with imatinib. Since 2 nd-generation TKI (2G-TKI) achieves a molecular response faster than imatinib, it may lead to TFR in a shorter treatment period. The multicenter phase II study D-FREE (Japan Registry of Clinical Trials: jRCTs031180332) was conducted to clarify optimal conditions for TFR in newly diagnosed patients with CML-CP treated with the 2G-TKI dasatinib. Methods: Newly diagnosed CML-CP patients were enrolled and treated with dasatinib in the induction phase. When patients achieved MR4.5 (BCR-ABL1 IS ≤0.0032%) based on assessment every three months during the induction phase for up to two years, they immediately entered the consolidation phase, where dasatinib is administered for 12 months. Patients with sustained MR4.5 throughout the consolidation phase discontinued dasatinib in the stop phase. Dasatinib was re-administered at molecular relapse, defined as loss of a major molecular response (BCR-ABL1 IS > 0.1%) or confirmed loss of MR4 (BCR-ABL1 IS > 0.01% on two consecutive assessments). The primary endpoint was the proportion of patients in TFR who showed no molecular relapse and did not need to resume dasatinib 12 months after treatment discontinuation. Results: Between July 2016 and May 2019, 181 patients with newly diagnosed CP-CML were enrolled in 41 centers in Japan. Four patients were excluded after screening, and no further information was available for 4 patients. Overall, 173 patients received study treatment. The median patient age was 54 years (18-83 years). The rates of Sokal low-, intermediate-, and high-risk groups were 28.6, 52.4, and 19.0%, respectively. The rates of EUTOS low- and high-risk groups were 81.0 and 19.0%, respectively. Of the 123 patients who completed the induction phase, 60 (48.8%) achieved MR4.5 for up to two years (median: 7.7 months, range: 3.0-21.1 months) and entered the consolidation phase. Single and multivariate analyses showed that the achievement of MMR at 3 months, but not sex, Sokal risk score, Hasford risk score, EUTOS risk score, or age (<60 vs. ≥60), was predictive of the achievement of MR4.5 within 2 years. During the consolidation phase, 15 patients could not sustain MR4.5 and finished study treatment. Among the first 21 patients who could sustain MR4.5 for 12 months and discontinued dasatinib treatment in the stop phase, 17 experienced molecular relapse within 12 months (median: 3.5 months, range: 2.0-6.4). In those patients, the median duration of dasatinib treatment was 18.9 months (range: 14.9-25.5) before the cessation of dasatinib. The study was terminated prematurely on December 3, 2019, based on the pre-specified interim analysis criterion that it would be stopped if the TFR rate was less than 25% in the first 20 patients of the stop phase. At termination, 46 patients were in the induction phase and 17 were in the consolidation phase. Four patients were in TFR in the stop phase. Of note, one patient remained in TFR for 18 months after receiving dasatinib treatment for only 18.6 months in induction and consolidation phases. All relapsing patients regained MR4 after a median of 2.1 months (range: 0.9-5.1) of dasatinib retreatment, except one patient who stopped study treatment because of an adverse event. No patients progressed to the accelerated/blastic phase or died due to CML. Conclusions: D-FREE was the first trial to discontinue TKI treatment in patients with newly diagnosed CML-CP who maintained MR4.5 for 1 year by dasatinib treatment regardless of the treatment duration. Hachhaus et al. previously reported in the ENSTfreedom trial (Leukemia 2017) that CML-CP patients who received at least 2 years of front-line nilotinib treatment, achieved MR 4.5, and then underwent consolidation of nilotinib treatment for 1 year had a TFR of 51.6% at 48 weeks after nilotinib discontinuation. The median duration of TKI treatment in the study was 43.5 months, compared with 18.9 months for D-FREE, suggesting that not only the duration of DMR but also that of TKI treatment is important for successful TFR even with 2G-TKI. Disclosures Yoshida: Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer Japan Inc.: Honoraria; Nippon Shinyaku: Honoraria; Novartis KK,: Honoraria; Janssen Pharmaceutical KK: Honoraria; AbbVie GK: Honoraria; Otsuka Pharmaceutical.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Yamaguchi: Bristol-Myers Squibb: Research Funding. Murai: Novartis Pharma: Honoraria; Pfizer: Honoraria; CHUGAI Pharmaceutical Co., Ltd.: Honoraria; TAKEDA Pharmaceutical Co., Ltd.: Honoraria; DAIICHI SANKYO COMPANY, LIMITED.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Asahi Kasei Pharma Corporation.: Honoraria; Bristol Myers Squibb: Honoraria. Hatta: Otsuka Pharmaceutical.: Honoraria; Novartis KK: Honoraria; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb: Honoraria. Yokose: Bristol Myers Squibb Company: Honoraria; Chugai Pharmaceutical Co., Ltd.: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding. Fujimaki: CSL Behring K.K.: Honoraria; Novartis KK: Honoraria; Janssen Pharmaceutical KK: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer Japan Inc.: Honoraria; Nippon Shinyaku: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Meiji Seika Pharma Co., Ltd.: Honoraria; AbbVie GK: Honoraria; Otsuka Pharmaceutical.: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Mundipharma K.K.: Honoraria. Oshikawa: Pfizer Japan Inc.: Honoraria; Otsuka Pharmaceutical.: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Eisai Co., Ltd.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; MSD K.K.: Honoraria; Meiji Seika Pharma Co., Ltd.: Honoraria; Sanofi K.K.: Honoraria; AbbVie GK: Honoraria; Janssen Pharmaceutical KK: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Celgene Corporation: Honoraria; Astellas Pharma Inc.: Honoraria; Novartis K.K.: Honoraria; Bristol-Myers Squibb: Honoraria; SymBio Pharmaceuticals.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria. Kumagai: Bristol-Myers Squibb: Honoraria; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria; Otsuka Pharmaceuticals: Honoraria, Speakers Bureau. Kimura: Bristol-Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Research Funding, Speakers Bureau; Ohara Pharmaceutical: Research Funding; Gilead: Research Funding; Nippon-Boehringer-Ingelheim: Research Funding; Sanifi: Speakers Bureau; Astellas: Speakers Bureau; Eisai: Speakers Bureau; PharmaEssentia: Speakers Bureau; Yakult: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; AbbVie: Research Funding, Speakers Bureau; Apellis: Research Funding; SymBio: Research Funding, Speakers Bureau; Mundi: Research Funding; Chugai: Research Funding, Speakers Bureau; Daiichi-Sankyo: Research Funding, Speakers Bureau; Janssen: Research Funding; Astellas-Amgen-Biopharma: Research Funding; Takeda: Research Funding, Speakers Bureau; Nippon-Shinyaku: Research Funding, Speakers Bureau; Amgen: Research Funding; Alexion: Research Funding, Speakers Bureau; Incyte: Research Funding; Ono: Research Funding, Speakers Bureau; Kyowa-Kirin: Research Funding, Speakers Bureau; MSD: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Sumitomo-Dainippon: Research Funding. Usuki: Bristol-Myers-Squibb K.K.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Research Funding, Speakers Bureau; Ono Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Research Funding; Celgene K.K.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Nippon-Boehringer-Ingelheim Co., Ltd.: Research Funding; Mundipharma K.K.: Research Funding; Amgen-Astellas Biopharma K.K.: Research Funding; Nippon-Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Kyowa-Kirin Co., Ltd.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; PharmaEssentia Japan KK: Research Funding, Speakers Bureau; Yakult Honsha Co., Ltd.: Research Funding, Speakers Bureau; SymBio Pharmaceuticals Ltd.: Research Funding, Speakers Bureau; Sumitomo-Dainippon Pharma Co., Ltd.: Research Funding; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau; Incyte Biosciences Japan G.K.: Research Funding; Apellis Pharmaceuticals, Inc.: Research Funding; Gilead Sciences, Inc.: Research Funding; AbbVie GK: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Sanofi K.K.: Speakers Bureau; Amgen K.K.: Research Funding. Yokoyama: Kowa: Consultancy; SymBio Pharmaceuticals: Honoraria; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Otsuka Pharmaceutical: Honoraria; Bayer: Honoraria; Nippon Shinyaku: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; Kyowa Kirin: Research Funding; Ono Pharmaceutical: Honoraria; Sanofi: Honoraria; Pfizer: Research Funding. Yamamoto: Bristol-Myers Squibb Company: Honoraria; Novartis Japan Co.: Honoraria; Kyowa Kirin Co.: Honoraria; Fujimoto Pharmaceutical Corporation: Honoraria; Sanofi K.K.: Honoraria; Otsuka Pharmaceutical Co.: Honoraria. Ishizawa: Sanofi: Research Funding; IQVIA: Research Funding; Eisai: Honoraria; Chugai: Honoraria; Ono: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Bayer: Research Funding; AbbVie: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Otsuka: Research Funding; SymBio: Honoraria, Research Funding; Kyowa Kirin: Consultancy; Pfizer: Research Funding; Bristol Myers Squibb: Speakers Bureau. Ishida: Ono: Honoraria; Takeda: Honoraria; Janssen: Honoraria; Sanofi: Honoraria; Bristol Myers Squibb: Honoraria. Yamamoto: NIPPON SINYAKU CO., LTD: Honoraria; Novartis Pharma: Honoraria; ONO PHARMACEUTICAL CO.: Honoraria; Otsuka Pharmaceutical: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Eisai Co., Ltd.: Honoraria; Bristol-Myers Squibb Company: Honoraria; Pfizer Japan Inc.: Honoraria; Takeda: Honoraria. Kondo: Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Novartis Pharma KK: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Bristol-Myers Squibb Company: Honoraria; Astellas Pharma Inc.: Consultancy, Honoraria; SANWA KAGAKU KENKYUSHO CO.,LTD.: Consultancy. Iriyama: Otsuka: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau. Oba: Eisai: Honoraria; Chugai pharma: Honoraria; Ono pharma: Honoraria; Asahi-Kasei pharma: Honoraria; Takeda pharma: Honoraria; Daiichi-Sankyo pharma: Honoraria; Bristol Myers Squibb: Honoraria. Inokuchi: Bristol-Myers Squibb: Research Funding.
A 78-year-old man who had a 20-year history of polycythemia vera (PV) with a JAK2 V617F mutation presented with gradually progressive disturbance of consciousness. Hyper-intense lesions in the peri-lateral ventricular area and left cerebellar hemisphere were observed by T2-weighted and fluid-attenuated inversion recovery magnetic resonance imaging. Cytologic and genetic analyses of the lymphoma cells obtained from his cerebrospinal fluid established the diagnosis of B-cell lymphoma. No lesions outside of the brain were recognized. Because of his poor general condition, he was not treated actively. A postmortem analysis revealed a JAK2 V617F mutation in the lymphoma cells, suggesting their origin was a PV clone.
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