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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.
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 Corpor
Several highly specific gene mutations have been discovered in malignant lymphoma. Mutations of the MYD88 gene have been detected in lymphoplasmacytic lymphoma and in some cases of diffuse large ...B-cell lymphoma. We report here a case in which detection of such mutation led to a definitive diagnosis. A 76-year-old woman developed a fever of unknown origin. Physical findings revealed no evident signs of infection or lymphadenopathy. Although serum sIL-2R levels were high, computed tomography revealed only mild hepatosplenomegaly with no lesions from which a biopsy could be taken. We performed a bone marrow examination, which initially did not lead to a diagnosis. However, due to progressive worsening of the patient’s systemic condition and noted increased splenomegaly on computed tomography, we performed genetic testing which included the above. This testing detected mutation of the MYD88 gene in bone marrow cells. We strongly suspected malignant lymphoma and conducted further detailed examination, which led to the diagnosis of intravascular large B-cell lymphoma. Genetic testing may be an extremely useful method that can serve as a decisive factor in diagnosing malignant lymphoma that is otherwise difficult to diagnose.
Backgrounds Intravascular large B cell lymphoma (IVLBCL) is a rare subtype of extranodal diffuse large B-cell lymphoma (DLBCL) that features lymphoma cells lodged in lumina of small vessels. Because ...of the lack of mass formation, diagnosis by tumor biopsy is often difficult, and thus, cell-free DNA (cfDNA) analysis might be useful for assisting diagnosis of IVLBCL. In this study, we first aimed at investigating the prevalence of mutations in genes involved in the B-cell receptor, Toll-like receptor/interleukin-1 receptor, and nuclear factor kappa B pathways. We then evaluated the liquid biopsies for detection of hot-spot mutations by droplet digital polymerase chain reaction (ddPCR).
Patients and Methods Twenty-seven IVLBCL patients whose archived samples were available were included in this study. The patients were diagnosed as IVLBCL from January 2005 to May 2017 in University of Tsukuba Hospital, JA Toride Medical Center, and Kameda Medical Center. The study was approved by the review board in each institution, and conducted according to the Declaration of Helsinki. Tissue-derived DNA (tdDNA) was extracted from formalin-fixed paraffin-embedded specimens and/or cryo-preserved bone marrow samples, in which the lymphoma cell infiltration had been pathologically confirmed. cfDNA was extracted from 1 ml of serum or plasma obtained prior to chemotherapy. We performed targeted sequence for 8 genes (B2M, BTG2, CARD11, CD79B, MYD88, PIM1, PRDM1, and TNFAIP3) in paired tdDNA and cfDNA from 9 IVLBCL subjects by Ion Torrent Personal Genome Machine (Thermo Fisher Scientific). MYD88 L265P (c.794T>C) mutational analysis was performed by ddPCR assays (BioRad) for 46 samples in total collected from 27 subjects. Statistical analysis was performed with EZR version 1.3.2. Paired t-test was used to compare variant allele frequencies (VAFs) in paired samples.
Results All cases were categorized as the non-GCB by Hans classification. Random skin biopsies (RSB) and bone marrow biopsies (BMB) were performed in 21 and 27 patients. The diagnostic yield of RSB was 81.0% (17 of 21). Initial BMB detected lymphoma cells in 48.1% (13/27) patients, but repeated BMB increased successful detection up to 66.7% (18/27). tdDNA was available in 26 out of 27 subjects, and in 9 of which paired cfDNA was also available (Figure 1). In one subject, only cfDNA but not tdDNA was available. Targeted sequencing for 8 genes demonstrated at least one mutation in CD79B, MYD88, PIM1, or PRDM1 in both tdDNA and cfDNA, or only in cfDNA, in 8 of 9 subjects (Figure 2). We identified 3 missense CD79B mutations in 6 (66.7%; Y196C in 2, Y196H in 3, and L199P in 1). All MYD88 mutations were L265P, which were found in 5 of 9 (55.6%). Together, either Y196 CD79B or L265P MYD88 mutation was found in 7 of 9 (77.8%). PIM1 mutations and PRDM1 mutations were seen each in 3 (33.3%). VAFs were higher in cfDNA than in tdDNA (mean, 45.6% vs. 3.7%; P=0.0006, Figure 3). Four mutations in 3 patients were detected only in cfDNA. On the other hand, all variants detected in tdDNA were confirmed in cfDNA. ddPCR assay for the L265P MYD88 mutation with all 46 samples including tdDNA and cfDNA revealed the L265P MYD88 mutation in 24/46 (52.2%) samples from 16/27 (59.3%) IVLBCL patients. Furthermore, sequential analysis of cfDNA was performed in one of the MYD88 -mutated patients. The L265P MYD88 mutation was detected (VAF=30%) in cfDNA collected 17 weeks before the diagnosis of IVLBCL, whereas the mutation was not detected in DNA derived from the bone marrow sample that was collected at the same timing and resulted in the negative pathological finding.
Conclusion Mutations in MYD88 and CD79B are frequent in IVLBCL . Targeted sequencing suggested that tumor cell-derived DNA is abundant in serum/plasma. Liquid biopsy to detect L265P MYD88 and Y196 CD79B may provide a powerful tool for approaching diagnosis of IVLBCL.
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Chiba:Nippon Shinyaku: Honoraria, Research Funding.
To evaluate the fracture strength of TiN thin films deposited on the hard metal substrate WC-Co, and to investigate the influence of the deposition conditions (bias voltage VB) on the fracture ...strength of TiN thin films, the sphere indentation test was carried out to determine the ring crack initiation strength σf,m in TiN thin films deposited on two kinds of WC-Co substrates differing in hardness using sphere indenters of varying diameter. TiN thin films 2.5 μm thick were deposited by dc magnetron sputtering under various VB. Based on the probabilistic theory assuming a two-parameter Weibull distribution, the averages of the fracture strength σ~f of TiN thin films without residual stress under conditions of uniform tensile stress and the residual stress σ~R of thin films were predicted from the distribution characteristics of σf,m. The main results were as follows: the average σ~f is almost independent of sphere indenter diameter and substrate hardness, and decreases with increasing VB; the variation in σ~f is mainly due to the grain size of thin films; the residual stress σ~R increases with increasing VB, and this tendency is qualitatively consistent with the measurements obtained by the X-ray diffraction method.
The purpose of this study is to absolutely evaluate the fracture strength of ceramic films deposited on the hard substrate by considering the residual stress on films, and to clarify the effect of ...the deposition condition on the fracture strength of ceramic films. TiN films were deposited onto two kinds of WC-Co substrates with different hardness using dc magnetron sputtering using various bias voltages VB. Sphere indentation tests were carried out to obtain the micro fracture strength σf, m for ring crack initiation on TiN films using sphere indenters of varying diameter 2R. The main conclusions are the following. (1) σf, m depend on 2R, but are independent of the hardness of a substrate. Based on the probabilistic theory assuming a Weibull distribution, the relationship between the mean value of σf, m and 2R can be predicted. (2) σf, m increase as VB is increased. (3) Based on the probabilistic theory, the residual stress σR on TiN films and the fracture strength σf of TiN films under uniform tensile stress condition for various VB were estimated from the distribution characteristics of σf, m. Variation tendency in σR is consistent with that by X-ray stress measurement. But, variation tendency in σf is opposite to that in σf, m.
Angioimmunoblastic T-cell lymphoma (AITL) is a distinct subtype of T-cell lymphoma, characterized by generalized lymphadenopathy and frequent autoimmune-like manifestations. The diagnosis of AITL is ...sometimes challenging for hematopathologists, because the tumor cell content is generally low and relatively large reactive lymphocytes are confused as tumor cells. Possibly because of the low tumor cell frequency, clonal rearrangement of T-cell receptor gene is undetectable in 30% of the cases. We identified recurrent mutations in RHOA at c.G50T, predicting to generate p.G17V in 70% of AITL and PTCL-NOS harboring AITL features, which implies diagnostic properties for AITL (M S-Y and SC, unpublished).
To establish a novel cost-effective method to diagnose AITL, we performed allele-specific realtime PCR to detect RHOA G17V mutation.
Genomic DNA was extracted from 119 AITL and PTCL-NOS samples, which include 47 periodate-lysine-paraformaldehyde (PLP)-fixed, 12 formalin-fixed-paraffin- embedded (FFPE) and 60 frozen tissues. Forty-one out of 60 genomic DNA samples, purified from frozen tissue, were amplified by RepliG kit (QIAGEN). Allele-specific primers for RHOA G17V mutant and wild-type sequences were designed by Wangkumhang's algorithm. The mut and WT values were individually measured by realtime PCR using each primer set, and the mut/(mut+WT) values were calculated.
Mutant allele frequencies were determined by amplicon-based deep sequencing using MiSeq.
The mut values were distributed from 1.5×10-7 to 7.6×10-2, and the WT values were from 7.9×10-5 to 1.3×10-2. The mut/mut+WT values were distributed from 1.9×10-4 to 8.5×10-1. We set a cut-off value to determine existence of mutation as 2% for MiSeq, and 1.3×10-2 for the mut/(mut+WT) value. Then we compared these two methods to detect RHOA G17V mutation.
Forty-three cases were positive for the RHOA mutation in this study cohort by MiSeq, including 32 AITL and 11 PTCL-NOS cases. The mut/(mut+WT) values of DNA from FFPE samples tend to be lower than those from other samples, and 4 out of 12 FFPE samples determined as mutation-positive by MiSeq were not detected by our allele-specific realtime PCR. We therefore excluded the FFPE samples and analyzed the data of 107 DNA samples, purified from PLP-fixed and frozen tissues. Rank correlation coefficient was 0.753. Sensitivity was 97.4%, and specificity was 97.1%. Positive concordance rate was 94.9%, and negative concordance rate was 98.6%.
We established a method to detect RHOA G17V hotspot mutation for AITL. It is expected to be highly accurate and cost effective.
No relevant conflicts of interest to declare.
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