Publications by authors named "Hideki Makishima"

112 Publications

Clonal evolution and clinical implications of genetic abnormalities in blastic transformation of chronic myeloid leukaemia.

Nat Commun 2021 05 14;12(1):2833. Epub 2021 May 14.

Laboratory of DNA Information Analysis, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Blast crisis (BC) predicts dismal outcomes in patients with chronic myeloid leukaemia (CML). Although additional genetic alterations play a central role in BC, the landscape and prognostic impact of these alterations remain elusive. Here, we comprehensively investigate genetic abnormalities in 136 BC and 148 chronic phase (CP) samples obtained from 216 CML patients using exome and targeted sequencing. One or more genetic abnormalities are found in 126 (92.6%) out of the 136 BC patients, including the RUNX1-ETS2 fusion and NBEAL2 mutations. The number of genetic alterations increase during the transition from CP to BC, which is markedly suppressed by tyrosine kinase inhibitors (TKIs). The lineage of the BC and prior use of TKIs correlate with distinct molecular profiles. Notably, genetic alterations, rather than clinical variables, contribute to a better prediction of BC prognosis. In conclusion, genetic abnormalities can help predict clinical outcomes and can guide clinical decisions in CML.
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http://dx.doi.org/10.1038/s41467-021-23097-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121838PMC
May 2021

[Predisposition and progression of myelodysplastic syndromes].

Authors:
Hideki Makishima

Rinsho Ketsueki 2021 ;62(4):278-288

Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University.

Recent advances in sequencing technologies have increased the detection rate for identifying germline mutations that predispose an individual to various myeloid neoplasms and somatic mutations acquired during progression from myelodysplastic syndromes (MDS) to acute myeloid leukemia (AML). In addition to pediatric subjects, adult patients were analyzed in order to obtain a complete spectrum of driver mutations in germline cells and/or somatic tumor samples. As shown in several recent studies, such driver mutations are acquired in a gene-specific fashion. DDX41 mutations are observed in germline cells long before MDS presentation. SAMD9/SAMD9L germline mutations associated with defective hematopoiesis account for recurrent and familial -7/del (7q) lesions, which result in the removal of the disadvantageous allele. Additionally, MDS cases in younger population display compound heterozygous germline mutations in the Shwachman-Diamond syndrome-associated SBDS gene. In peripheral blood samples from healthy elderly individuals, DNMT3A, TET2, and ASXL1 somatic mutations are usually detected due to age-related clonal hematopoiesis and are considered to be a risk factor for hematological neoplasms. In MDS, mutations of genes, such as NRAS and FLT3, designated as type-1 genes, are significantly associated with leukemic evolution. On the other hand, mutations in type-2 genes, including RUNX1 and GATA2, are related to progression from low risk MDS to high risk MDS.
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http://dx.doi.org/10.11406/rinketsu.62.278DOI Listing
May 2021

Landscape of driver mutations and their clinical impacts in pediatric B-cell precursor acute lymphoblastic leukemia.

Blood Adv 2020 10;4(20):5165-5173

Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan.

Recent genetic studies using high-throughput sequencing have disclosed genetic alterations in B-cell precursor acute lymphoblastic leukemia (B-ALL). However, their effects on clinical outcomes have not been fully investigated. To address this, we comprehensively examined genetic alterations and their prognostic impact in a large series of pediatric B-ALL cases. We performed targeted capture sequencing in a total of 1003 pediatric patients with B-ALL from 2 Japanese cohorts. Transcriptome sequencing (n = 116) and/or array-based gene expression analysis (n = 120) were also performed in 203 (84%) of 243 patients who were not categorized into any disease subgroup by panel sequencing or routine reverse transcription polymerase chain reaction analysis for major fusions in B-ALL. Our panel sequencing identified novel recurrent mutations in 2 genes (CCND3 and CIC), and both had positive correlations with ETV6-RUNX1 and hypodiploid ALL, respectively. In addition, positive correlations were also newly reported between TCF3-PBX1 ALL with PHF6 mutations. In multivariate Cox proportional hazards regression models for overall survival, TP53 mutation/deletion, hypodiploid, and MEF2D fusions were selected in both cohorts. For TP53 mutations, the negative effect on overall survival was confirmed in an independent external cohort (n = 466). TP53 mutation was frequently found in IGH-DUX4 (5 of 57 [9%]) ALL, with 4 cases having 17p LOH and negatively affecting overall survival therein, whereas TP53 mutation was not associated with poor outcomes among NCI (National Cancer Institute) standard risk (SR) patients. A conventional treatment approach might be enough, and further treatment intensification might not be necessary, for patients with TP53 mutations if they are categorized into NCI SR.
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http://dx.doi.org/10.1182/bloodadvances.2019001307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594377PMC
October 2020

Founder and subclonal mutations in myelodysplastic syndromes and related myeloid neoplasms.

Authors:
Hideki Makishima

Best Pract Res Clin Haematol 2020 09 5;33(3):101189. Epub 2020 Jun 5.

Department of Pathology and Tumor Biology, Kyoto University, Kyoto, Japan; Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA. Electronic address:

Somatic mutations constitute key elements of the pathogenesis of myelodysplastic syndromes (MDS), a group of clonal hematologic neoplasms characterized by cytopenias, dysplasia and leukemic evolution. Whole exome sequencing followed by targeted deep sequencing in patients with MDS and related diseases has been performed cross-sectionally and serially. Bioinformatic analysis and confirmatory sequencing led to detection of in 1458 genes affected by somatic alterations, and identification of known and new driver events. For each patient, mutation spectrum as well as clonal hierarchy was determined and for each significantly mutated gene, its role in the clonal succession established. This approach allowed for a dynamic definition of MDS mutatome, including the spectrum of founding mutations and subsequent secondary mutational patterns. We demonstrate that certain founder events determine the mode and speed of disease progression, while secondary mutations may further modulate phenotypic features. Combinations of founder and secondary mutations further contribute to the phenotypic diversity but categorical grouping of cases based on the type of founder mutations may better define molecular subtypes of MDS and correlates with clinical parameters.
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http://dx.doi.org/10.1016/j.beha.2020.101189DOI Listing
September 2020

Frequent mutations in HLA and related genes in extranodal NK/T cell lymphomas.

Leuk Lymphoma 2021 01 23;62(1):95-103. Epub 2020 Sep 23.

Department of Medicine, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, Bangkok, Thailand.

Extranodal NK/T cell lymphomas (ENKTCLs) are aggressive Epstein-Barr virus-associated T/NK neoplasms that predominantly affect Asians. To explore the causative somatic events, we conducted a comprehensive genetic analysis of 19 ENKTCL patients by whole-genome ( = 2), whole-exome ( = 16), and targeted sequencing ( = 15). Commonly deregulated gene pathways in ENKTCLs included epigenetic modifiers (58%, 11/19) followed by human leukocyte antigens (HLAs) and related genes including , , and (32%, 6/19), and JAK-STAT pathway (26%, 5/19). Conspicuously, loss-of-function mutations in were recurrently identified in ENKTCLs (16%, 3/19). HLA protein expression was examined by immunohistochemistry in 16 patients and lower expression was associated with advanced stages at presentation ( = .007). In conclusion, the defective antigen presenting pathway is common and related to disease progression, suggesting immune escape as a pathogenic mechanism of ENKTCLs.
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http://dx.doi.org/10.1080/10428194.2020.1821011DOI Listing
January 2021

Machine learning demonstrates that somatic mutations imprint invariant morphologic features in myelodysplastic syndromes.

Blood 2020 11;136(20):2249-2262

Department of Hematology and Medical Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH.

Morphologic interpretation is the standard in diagnosing myelodysplastic syndrome (MDS), but it has limitations, such as varying reliability in pathologic evaluation and lack of integration with genetic data. Somatic events shape morphologic features, but the complexity of morphologic and genetic changes makes clear associations challenging. This article interrogates novel clinical subtypes of MDS using a machine-learning technique devised to identify patterns of cooccurrence among morphologic features and genomic events. We sequenced 1079 MDS patients and analyzed bone marrow morphologic alterations and other clinical features. A total of 1929 somatic mutations were identified. Five distinct morphologic profiles with unique clinical characteristics were defined. Seventy-seven percent of higher-risk patients clustered in profile 1. All lower-risk (LR) patients clustered into the remaining 4 profiles: profile 2 was characterized by pancytopenia, profile 3 by monocytosis, profile 4 by elevated megakaryocytes, and profile 5 by erythroid dysplasia. These profiles could also separate patients with different prognoses. LR MDS patients were classified into 8 genetic signatures (eg, signature A had TET2 mutations, signature B had both TET2 and SRSF2 mutations, and signature G had SF3B1 mutations), demonstrating association with specific morphologic profiles. Six morphologic profiles/genetic signature associations were confirmed in a separate analysis of an independent cohort. Our study demonstrates that nonrandom or even pathognomonic relationships between morphology and genotype to define clinical features can be identified. This is the first comprehensive implementation of machine-learning algorithms to elucidate potential intrinsic interdependencies among genetic lesions, morphologies, and clinical prognostic in attributes of MDS.
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http://dx.doi.org/10.1182/blood.2020005488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7702479PMC
November 2020

Context dependent effects of ascorbic acid treatment in TET2 mutant myeloid neoplasia.

Commun Biol 2020 09 7;3(1):493. Epub 2020 Sep 7.

Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA.

Loss-of-function TET2 mutations (TET2) are common in myeloid neoplasia. TET2, a DNA dioxygenase, requires 2-oxoglutarate and Fe(II) to oxidize 5-methylcytosine. TET2 thus result in hypermethylation and transcriptional repression. Ascorbic acid (AA) increases dioxygenase activity by facilitating Fe(III)/Fe(II) redox reaction and may alleviate some biological consequences of TET2 by restoring dioxygenase activity. Here, we report the utility of AA in the prevention of TET2 myeloid neoplasia (MN), clarify the mechanistic underpinning of the TET2-AA interactions, and demonstrate that the ability of AA to restore TET2 activity in cells depends on N- and C-terminal lysine acetylation and nature of TET2. Consequently, pharmacologic modulation of acetyltransferases and histone deacetylases may regulate TET dioxygenase-dependent AA effects. Thus, our study highlights the contribution of factors that may enhance or attenuate AA effects on TET2 and provides a rationale for novel therapeutic approaches including combinations of AA with class I/II HDAC inhibitor or sirtuin activators in TET2 leukemia.
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http://dx.doi.org/10.1038/s42003-020-01220-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477582PMC
September 2020

Combined Cohesin-RUNX1 Deficiency Synergistically Perturbs Chromatin Looping and Causes Myelodysplastic Syndromes.

Cancer Discov 2020 06 5;10(6):836-853. Epub 2020 Apr 5.

Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

encodes a cohesin component and is frequently mutated in myeloid neoplasms, showing highly significant comutation patterns with other drivers, including . However, the molecular basis of cohesin-mutated leukemogenesis remains poorly understood. Here we show a critical role of an interplay between STAG2 and RUNX1 in the regulation of enhancer-promoter looping and transcription in hematopoiesis. Combined loss of STAG2 and RUNX1, which colocalize at enhancer-rich, CTCF-deficient sites, synergistically attenuates enhancer-promoter loops, particularly at sites enriched for RNA polymerase II and Mediator, and deregulates gene expression, leading to myeloid-skewed expansion of hematopoietic stem/progenitor cells (HSPC) and myelodysplastic syndromes (MDS) in mice. Attenuated enhancer-promoter loops in STAG2/RUNX1-deficient cells are associated with downregulation of genes with high basal transcriptional pausing, which are important for regulation of HSPCs. Downregulation of high-pausing genes is also confirmed in -cohesin-mutated primary leukemia samples. Our results highlight a unique STAG2-RUNX1 interplay in gene regulation and provide insights into cohesin-mutated leukemogenesis. SIGNIFICANCE: We demonstrate a critical role of an interplay between STAG2 and a master transcription factor of hematopoiesis, RUNX1, in MDS development, and further reveal their contribution to regulation of high-order chromatin structures, particularly enhancer-promoter looping, and the link between transcriptional pausing and selective gene dysregulation caused by cohesin deficiency..
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http://dx.doi.org/10.1158/2159-8290.CD-19-0982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269820PMC
June 2020

Molecular pathogenesis of progression to myeloid leukemia from TET-insufficient status.

Blood Adv 2020 03;4(5):845-854

Department of Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.

Loss-of-function mutations in ten-eleven translocation-2 (TET2) are recurrent events in acute myeloid leukemia (AML) as well as in preleukemic hematopoietic stem cells (HSCs) of age-related clonal hematopoiesis. TET3 mutations are infrequent in AML, but the level of TET3 expression in HSCs has been found to decline with age. We examined the impact of gradual decrease of TET function in AML development by generating mice with Tet deficiency at various degrees. Tet2f/f and Tet3f/f mice were crossed with mice expressing Mx1-Cre to generate Tet2f/wtTet3f/fMx-Cre+ (T2ΔT3), Tet2f/fTet3f/wtMx-Cre+ (ΔT2T3), and Tet2f/fTet3f/fMx-Cre+ (ΔT2ΔT3) mice. All ΔT2ΔT3 mice died of aggressive AML at a median survival of 10.7 weeks. By comparison, T2ΔT3 and ΔT2T3 mice developed AML at longer latencies, with a median survival of ∼27 weeks. Remarkably, all 9 T2ΔT3 and 8 ΔT2T3 mice with AML showed inactivation of the remaining nontargeted Tet2 or Tet3 allele, respectively, owing to exonic loss in either gene or stop-gain mutations in Tet3. Recurrent mutations other than Tet3 were not noted in any mice by whole-exome sequencing. Spontaneous inactivation of residual Tet2 or Tet3 alleles is a recurrent genetic event during the development of AML with Tet insufficiency.
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http://dx.doi.org/10.1182/bloodadvances.2019001324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065477PMC
March 2020

Frequent mutations that converge on the NFKBIZ pathway in ulcerative colitis.

Nature 2020 01 18;577(7789):260-265. Epub 2019 Dec 18.

Laboratory of DNA Information Analysis, Human Genome Centre, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer. However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer.
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http://dx.doi.org/10.1038/s41586-019-1856-1DOI Listing
January 2020

Invariant patterns of clonal succession determine specific clinical features of myelodysplastic syndromes.

Nat Commun 2019 11 26;10(1):5386. Epub 2019 Nov 26.

Department of Translational Hematology & Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA.

Myelodysplastic syndromes (MDS) arise in older adults through stepwise acquisitions of multiple somatic mutations. Here, analyzing 1809 MDS patients, we infer clonal architecture by using a stringent, the single-cell sequencing validated PyClone bioanalytic pipeline, and assess the position of the mutations within the clonal architecture. All 3,971 mutations are grouped based on their rank in the deduced clonal hierarchy (dominant and secondary). We evaluated how they affect the resultant morphology, progression, survival and response to therapies. Mutations of SF3B1, U2AF1, and TP53 are more likely to be dominant, those of ASXL1, CBL, and KRAS are secondary. Among distinct combinations of dominant/secondary mutations we identified 37 significant relationships, of which 12 affect clinical phenotypes, 5 cooperatively associate with poor prognosis. They also predict response to hypomethylating therapies. The clonal hierarchy has distinct ranking and the resultant invariant combinations of dominant/secondary mutations yield novel insights into the specific clinical phenotype of MDS.
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http://dx.doi.org/10.1038/s41467-019-13001-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879617PMC
November 2019

Novel DDX41 variants in Thai patients with myeloid neoplasms.

Int J Hematol 2020 Feb 11;111(2):241-246. Epub 2019 Nov 11.

Department of Medicine, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, Bangkok, 10330, Thailand.

Germline DDX41 mutations were recently reported to cause MDS/AML and donor-derived leukemia after transplantation. While previously described in Western countries, DDX41 variants have not been reported in a Southeast Asian population. We performed targeted sequencing of blood or bone marrow samples from 109 Thai patients with myeloid malignancies. Among the 109 patients (75 MDS, 8 MPN, 11 MDS/MPN and 15 AML), the most frequent mutations were in ASXL1 (17.4%), TET2 (16.5%) and SRSF2 (12.8%), respectively. DDX41 variants were detectable in six (5.5%) cases. Four patients exhibited three presumable germline DDX41 mutations: p.S21fs (n = 2), p.F235fs (n = 1), and p.R339H (n = 1). While p.S21fs was previously reported in myeloid neoplasm, the latter two variants have not been described. Two of these cases harbored concomitant probable germline/somatic DDX41 mutations (p.S21fs/p.R525H and p.R339H/p.K494T), while the other two patients carried only somatic mutations (p.R525H and p.F438L). The p.K494T and p.F438L variants have not been previously reported. In patients with DDX41 alterations, the diagnoses were MDS with excess blasts (4), secondary AML (1) and low-risk MDS (1). In conclusion, we identified DDX41 variants in Thai patients with myeloid malignancies in which these variants could be used to assess predisposition to MDS in Southeast Asia.
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http://dx.doi.org/10.1007/s12185-019-02770-3DOI Listing
February 2020

Distinct clinical and biological implications of in myeloid neoplasms.

Blood Adv 2019 07;3(14):2164-2178

Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH.

Somatic mutations of the CUT-like homeobox 1 () gene () can be found in myeloid neoplasms (MNs), in particular, in myelodysplastic syndromes (MDSs). The locus is also deleted in 3 of 4 MN cases with -7/del(7q). A cohort of 1480 MN patients was used to characterize clinical features and clonal hierarchy associated with and deletions () and to analyze their functional consequences in vitro. were present in 4% of chronic MNs. were preferentially found in advanced cases (6%). Most MDS and acute myeloid leukemia (AML) patients with -7/del(7q) and up to 15% of MDS patients and 5% of AML patients diploid for the locus exhibited downmodulated CUX1 expression. In 75% of mutant cases, were heterozygous, whereas microdeletions and homozygous and compound-heterozygous mutations were less common. were associated with worse survival compared with Within the clonal hierarchy, 1 of 3 served as founder events often followed by secondary and subclonal hits, whereas was the most common ancestral lesion, followed by subclonal Comet assay of patients' bone marrow progenitor cells and leukemic cell lines performed in various experimental conditions revealed that frameshift mutations, hemizygous deletions, or experimental knockdown decrease the repair of oxidized bases. These functional findings may explain why samples with either or low CUX1 expression coincided with significantly higher numbers of somatic hits by whole-exome sequencing. Our findings implicate the DNA repair dysfunction resulting from lesions in the pathogenesis of MNs, in which they lead to a mutator phenotype.
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http://dx.doi.org/10.1182/bloodadvances.2018028423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650742PMC
July 2019

[Genomic aberrations in myelodysplastic syndromes and related disorders].

Authors:
Hideki Makishima

Rinsho Ketsueki 2019 ;60(6):600-609

Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University.

Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are heterogeneous myeloid neoplasms that frequently evolve into secondary acute myeloid leukemia (sAML). Recent progress in next-generation sequencing technologies has allowed us to discover frequent mutations throughout the coding regions of MDS, MDS/MPN, and sAML, subsequently providing information on more than 60 driver genes in these diseases. As shown by many study groups recently, such driver mutations are acquired in a gene-specific fashion. DDX41 and SAMD9/SAMD9L mutations are observed in germline cells long before MDS presentation. In blood samples from healthy elderly individuals, somatic DNMT3A, TET2, and ASXL1 mutations are detected as age-related clonal hematopoiesis and supposed to be a risk factor for hematological neoplasms. Recent reports on MDS have shown that mutations in genes such as NRAS and FLT3, designated as Type I genes, were significantly associated with leukemic evolution. Another type (Type II) of genes, including RUNX1 and GATA2, has been shown to be related to the progression from low-risk to high-risk MDS. These driver mutations are significantly concomitant during disease progression. Overall, various types of driver mutations are sequentially acquired in MDS, accounting for the heterogeneity of these disorders.
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http://dx.doi.org/10.11406/rinketsu.60.600DOI Listing
August 2019

Genome analysis of myelodysplastic syndromes among atomic bomb survivors in Nagasaki.

Haematologica 2020 31;105(2):358-365. Epub 2020 Jan 31.

Department of Hematology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki

Ionizing radiation is a risk factor for myeloid neoplasms including myelodysplastic syndromes (MDS), and atomic bomb survivors have been shown to have a significantly higher risk of MDS. Our previous analyses demonstrated that MDS among these survivors had a significantly higher frequency of complex karyotypes and structural alterations of chromosomes 3, 8, and 11. However, there was no difference in the median survival time between MDS among survivors compared with those of origin. This suggested that a different pathophysiology may underlie the causative genetic aberrations for those among survivors. In this study, we performed genome analyses of MDS among survivors and found that proximally exposed patients had significantly fewer mutations in genes such as along the DNA methylation pathways, and they had a significantly higher rate of 11q deletions. Among the genes located in the deleted portion of chromosome 11, alterations of were significantly more frequent in proximally exposed group with mutations identified on the remaining allele in 2 out of 5 cases. , which is frequently mutated in therapy-related myeloid neoplasms, was equally affected between proximally and distally exposed patients. These results suggested that the genetic aberration profiles in MDS among atomic bomb survivors differed from those in therapy-related and origin. Considering the role of in DNA damage response after radiation exposure, further studies are warranted to elucidate how 11q deletion and aberrations of contribute to the pathogenesis of MDS after radiation exposure.
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http://dx.doi.org/10.3324/haematol.2019.219386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012461PMC
April 2021

Molecular heterogeneity in peripheral T-cell lymphoma, not otherwise specified revealed by comprehensive genetic profiling.

Leukemia 2019 12 15;33(12):2867-2883. Epub 2019 May 15.

Department of Hematology, Kobe City Medical Center General Hospital, Kobe, Japan.

Peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) is a diagnosis of exclusion, being the most common entity in mature T-cell neoplasms, and its molecular pathogenesis remains significantly understudied. Here, combining whole-exome and targeted-capture sequencing, gene-expression profiling, and immunohistochemical analysis of tumor samples from 133 cases, we have delineated the entire landscape of somatic alterations, and discovered frequently affected driver pathways in PTCL, NOS, with and without a T-follicular helper (TFH) cell phenotype. In addition to previously reported mutational targets, we identified a number of novel recurrently altered genes, such as KMT2C, SETD1B, YTHDF2, and PDCD1. We integrated these genetic drivers using hierarchical clustering and identified a previously undescribed molecular subtype characterized by TP53 and/or CDKN2A mutations and deletions in non-TFH PTCL, NOS. This subtype exhibited different prognosis and unique genetic features associated with extensive chromosomal instability, which preferentially affected molecules involved in immune escape and transcriptional regulation, such as HLA-A/B and IKZF2. Taken together, our findings provide novel insights into the molecular pathogenesis of PTCL, NOS by highlighting their genetic heterogeneity. These results should help to devise a novel molecular classification of PTCLs and to exploit a new therapeutic strategy for this group of aggressive malignancies.
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http://dx.doi.org/10.1038/s41375-019-0473-1DOI Listing
December 2019

Frequent germline mutations of in sporadic subcutaneous panniculitis-like T-cell lymphoma.

Blood Adv 2019 02;3(4):588-595

Department of Pathology and Tumor Biology, Kyoto University, Kyoto, Japan.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare subtype of peripheral T-cell lymphoma affecting younger patients and associated with hemophagocytic lymphohistiocytosis. To clarify the molecular pathogenesis of SPTCL, we analyzed paired tumor and germline DNAs from 13 patients by whole-exome sequencing. All cases were Asians and were phenotypically sporadic with no family history of SPTCL. Consistent with a recent report, germline mutations in , encoding T-cell immunoglobulin mucin 3 (TIM3), were identified in 11 of 13 (85%) cases. All mutated cases were primary SPTCL, whereas the 2 cases without mutation were secondary SPTCL associated with underlying diseases, including viral infection and autoimmune disease. Ten patients harbored homozygous p.Y82C mutations, and 1 showed compound heterozygous mutations (p.Y82C and p.T101I). Both missense mutations altered highly conserved residues located in the extracellular immunoglobulin variable-like domain. According to the Genome Aggregation Database of >138 500 general individuals, both mutations were documented with minor allele frequencies < 0.007, indicating remarkable enrichment of these alleles in SPTCL. SPTCL cells also harbored somatic mutations (6.2 per patient) that are frequently identified in genes associated with epigenetic regulation and signal transduction. In conclusion, individuals harboring biallelic (TIM3) germline mutations were highly susceptible to sporadic SPTCL, which was also associated with clonal somatic mutations.
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http://dx.doi.org/10.1182/bloodadvances.2018028340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391671PMC
February 2019

Frequent structural variations involving programmed death ligands in Epstein-Barr virus-associated lymphomas.

Leukemia 2019 07 25;33(7):1687-1699. Epub 2019 Jan 25.

Division of Immunology, Aichi Cancer Center Research Institute, Nagoya, Japan.

Viral infection induces potent cellular immunity and activated intracellular signaling, which may dictate the driver events involved in immune escape and clonal selection of virus-associated cancers, including Epstein-Barr virus (EBV)-positive lymphomas. Here, we thoroughly interrogated PD-L1/PD-L2-involving somatic aberrations in 384 samples from various lymphoma subtypes using high-throughput sequencing, particularly focusing on virus-associated lymphomas. A high frequency of PD-L1/PD-L2-involving genetic aberrations was observed in EBV-positive lymphomas [33 (22%) of 148 cases], including extranodal NK/T-cell lymphoma (ENKTL, 23%), aggressive NK-cell leukemia (57%), systemic EBV-positive T-cell lymphoproliferative disorder (17%) as well as EBV-positive diffuse large B-cell lymphoma (DLBCL, 19%) and peripheral T-cell lymphoma-not otherwise specified (15%). Predominantly causing a truncation of the 3'-untranslated region, these alterations represented the most prevalent somatic lesions in ENKTL. By contrast, the frequency was much lower in EBV-negative lymphomas regardless of histology type [12 (5%) of 236 cases]. Besides PD-L1/PD-L2 alterations, EBV-positive DLBCL exhibited a genetic profile distinct from EBV-negative one, characterized by frequent TET2 and DNMT3A mutations and the paucity of CD79B, MYD88, CDKN2A, and FAS alterations. Our findings illustrate unique genetic features of EBV-associated lymphomas, also suggesting a potential role of detecting PD-L1/PD-L2-involving lesions for these lymphomas to be effectively targeted by immune checkpoint blockade.
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http://dx.doi.org/10.1038/s41375-019-0380-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755969PMC
July 2019

Age-related remodelling of oesophageal epithelia by mutated cancer drivers.

Nature 2019 01 2;565(7739):312-317. Epub 2019 Jan 2.

Laboratory of Molecular Biosciences, Life Science Informatics Research Unit, Kyoto University, Kyoto, Japan.

Clonal expansion in aged normal tissues has been implicated in the development of cancer. However, the chronology and risk dependence of the expansion are poorly understood. Here we intensively sequence 682 micro-scale oesophageal samples and show, in physiologically normal oesophageal epithelia, the progressive age-related expansion of clones that carry mutations in driver genes (predominantly NOTCH1), which is substantially accelerated by alcohol consumption and by smoking. Driver-mutated clones emerge multifocally from early childhood and increase their number and size with ageing, and ultimately replace almost the entire oesophageal epithelium in the extremely elderly. Compared with mutations in oesophageal cancer, there is a marked overrepresentation of NOTCH1 and PPM1D mutations in physiologically normal oesophageal epithelia; these mutations can be acquired before late adolescence (as early as early infancy) and significantly increase in number with heavy smoking and drinking. The remodelling of the oesophageal epithelium by driver-mutated clones is an inevitable consequence of normal ageing, which-depending on lifestyle risks-may affect cancer development.
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http://dx.doi.org/10.1038/s41586-018-0811-xDOI Listing
January 2019

[Clonal hematopoiesis in aplastic anemia].

Authors:
Hideki Makishima

Rinsho Ketsueki 2018;59(10):1962-1968

Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University.

Aplastic anemia (AA) is an autoimmune-mediated bone marrow failure syndrome. While AA is not a malignant disease, clonal hematopoiesis is commonly detected via next-generation sequencing and single nucleotide polymorphism (SNP) array. Clonal hematopoiesis in AA has been confirmed by the detection of classic X chromosome skewing, PNH clones, UPD6p, and various mutations. The most frequent genetic events in AA are UPD6p and somatic mutations in BCOR/BCORL1, PIGA, DNMT3A, and ASXL1. While some mutations are common between patients with AA and healthy elderly donors, UPD6p and PIGA mutations are specific to clonal cells in AA, which need to manage their highly autoimmune extrinsic environment. During the evolution of AA into myelodysplastic syndrome (MDS), additional genetic events are frequently acquired that provide MDS cells with intrinsic survival benefits. Hematopoietic cells in AA appear to achieve clonal expansion by their escape from recognition and cytotoxicity by CD8 T-cells, accounting for the distinct landscape of genetic events observed in AA.
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http://dx.doi.org/10.11406/rinketsu.59.1962DOI Listing
July 2019

Molecular pathogenesis of disease progression in MLL-rearranged AML.

Leukemia 2019 03 12;33(3):612-624. Epub 2018 Sep 12.

Department of Pathology and Tumor Biology, Kyoto University, Kyoto, Japan.

Leukemic relapse is frequently accompanied by progressively aggressive clinical course. To understand the molecular mechanism of leukemic relapse, MLL/AF9-transformed mouse leukemia cells were serially transplanted in C57BL/6 mice (N = 96) by mimicking repeated recurrences, where mutations were monitored by exome sequencing (N = 42). The onset of leukemia was progressively promoted with advanced transplants, during which increasing numbers of somatic mutations were acquired (P < 0.005). Among these, mutations in Ptpn11 (p.G60R) and Braf (p.V637E) corresponded to those identified in human MLL-AML, while recurrent mutations affecting Msn (p.R295C) were observed only in mouse but not in human MLL-AML. Another mutated gene of interest was Gnb2 which was reported to be recurrently mutated in various hematological neoplasms. Gnb2 mutations (p.G77R) were significantly increased in clone size (P = 0.007) and associated with earlier leukemia onset (P = 0.011). GNB2 transcripts were significantly upregulated in human MLL-AML compared to MLL-negative AML (P < 0.05), which was supported by significantly increased Gnb2 transcript induced by MLL/AF9 overexpression (P < 0.001). In in vivo model, both mutation and overexpression of GNB2 caused leukemogenesis, and downregulation of GNB2 expression reduced proliferative potential and survival benefit, suggesting a driver role of GNB2. In conclusion, alterations of driver genes over time may play an important role in the progression of MLL-AML.
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http://dx.doi.org/10.1038/s41375-018-0253-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462875PMC
March 2019

Aberrant splicing and defective mRNA production induced by somatic spliceosome mutations in myelodysplasia.

Nat Commun 2018 09 7;9(1):3649. Epub 2018 Sep 7.

Department of Molecular Medicine, University of Pavia, 27100, Pavia, Italy.

Spliceosome mutations are frequently found in myelodysplasia. Splicing alterations induced by these mutations, their precise targets, and the effect at the transcript level have not been fully elucidated. Here we report transcriptomic analyses of 265 bone marrow samples from myelodysplasia patients, followed by a validation using CRISPR/Cas9-mediated gene editing and an assessment of nonsense-mediated decay susceptibility. Small but widespread reduction of intron-retaining isoforms is the most frequent splicing alteration in SF3B1-mutated samples. SF3B1 mutation is also associated with 3' splice site alterations, leading to the most pronounced reduction of canonical transcripts. Target genes include tumor suppressors and genes of mitochondrial iron metabolism or heme biosynthesis. Alternative exon usage is predominant in SRSF2- and U2AF1-mutated samples. Usage of an EZH2 cryptic exon harboring a premature termination codon is increased in both SRSF2- and U2AF1-mutated samples. Our study reveals a landscape of splicing alterations and precise targets of various spliceosome mutations.
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http://dx.doi.org/10.1038/s41467-018-06063-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128865PMC
September 2018

Clonal PIGA mosaicism and dynamics in paroxysmal nocturnal hemoglobinuria.

Leukemia 2018 11 25;32(11):2507-2511. Epub 2018 Apr 25.

Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA.

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http://dx.doi.org/10.1038/s41375-018-0138-5DOI Listing
November 2018

Fanconi Anemia germline variants as susceptibility factors in aplastic anemia, MDS and AML.

Oncotarget 2018 Jan 16;9(2):2050-2057. Epub 2017 Dec 16.

Department of Translational Hematology and Oncology Research, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.

Using next generation sequencing we have systematically analyzed a large cohort of 489 patients with bone marrow failure (BMF), including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), aplastic anemia (AA), and related conditions for the presence of germline (GL) alterations in Fanconi Anemia (FA) and telomerase genes. We have detected an increased frequency of heterozygous FA gene mutations in MDS and to lesser degree in AML suggesting that the presence of one normal allele may not be completely protective and indeed heterozygous FA lesions may have a long latency period before hematologic manifestation. In contrast, GL telomerase gene mutations were not associated with increased disease risk. When compared to large control cohorts, we have not detected an increased frequency of damaging variants among telomerase complex genes, including those previously believed to be involved in the pathogenesis of AA. Our results may suggest that while low penetrance and delayed disease onset can confound identification of genetic predisposition factors, GL FA alterations can be also associated with MDS.
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http://dx.doi.org/10.18632/oncotarget.23328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788620PMC
January 2018

Mutations Sensitize Acute Myeloid Leukemia to PARP Inhibition and This Is Reversed by IDH1/2-Mutant Inhibitors.

Clin Cancer Res 2018 04 16;24(7):1705-1715. Epub 2018 Jan 16.

Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio.

Somatic mutations in occur in approximately 20% of patients with myeloid neoplasms, including acute myeloid leukemia (AML). IDH1/2 enzymes produce -2-hydroxyglutarate (2HG), which associates with increased DNA damage and improved responses to chemo/radiotherapy and PARP inhibitors in solid tumor cells. Whether this also holds true for AML is not known. Well-characterized primary , , and AML cells were analyzed for DNA damage and responses to daunorubicin, ionizing radiation, and PARP inhibitors. caused increased DNA damage and sensitization to daunorubicin, irradiation, and the PARP inhibitors olaparib and talazoparib in AML cells. IDH1/2 inhibitors protected against these treatments. Combined treatment with a PARP inhibitor and daunorubicin had an additive effect on the killing of AML cells. We provide evidence that the therapy sensitivity of cells was caused by 2HG-mediated downregulation of expression of the DNA damage response gene and not by altered redox responses due to metabolic alterations in cells. AML cells are sensitive to PARP inhibitors as monotherapy but especially when combined with a DNA-damaging agent, such as daunorubicin, whereas concomitant administration of IDH1/2 inhibitors during cytotoxic therapy decrease the efficacy of both agents in AML. These results advocate in favor of clinical trials of PARP inhibitors either or not in combination with daunorubicin in AML. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-2796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5884732PMC
April 2018

Gene expression and risk of leukemic transformation in myelodysplasia.

Blood 2017 12 2;130(24):2642-2653. Epub 2017 Nov 2.

Department of Pathology and Tumor Biology, Kyoto University, Kyoto, Japan.

Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic disorders with a highly variable prognosis. To identify a gene expression-based classification of myelodysplasia with biological and clinical relevance, we performed a comprehensive transcriptomic analysis of myeloid neoplasms with dysplasia using transcriptome sequencing. Unsupervised clustering of gene expression data of bone marrow CD34 cells from 100 patients identified 2 subgroups. The first subtype was characterized by increased expression of genes related to erythroid/megakaryocytic (EMK) lineages, whereas the second subtype showed upregulation of genes related to immature progenitor (IMP) cells. Compared with the first so-called EMK subtype, the IMP subtype showed upregulation of many signaling pathways and downregulation of several pathways related to metabolism and DNA repair. The IMP subgroup was associated with a significantly shorter survival in both univariate (hazard ratio [HR], 5.0; 95% confidence interval [CI], 1.8-14; = .002) and multivariate analysis (HR, 4.9; 95% CI, 1.3-19; = .02). Leukemic transformation was limited to the IMP subgroup. The prognostic significance of our classification was validated in an independent cohort of 183 patients. We also constructed a model to predict the subgroups using gene expression profiles of unfractionated bone marrow mononuclear cells (BMMNCs). The model successfully predicted clinical outcomes in a test set of 114 patients with BMMNC samples. The addition of our classification to the clinical model improved prediction of patient outcomes. These results indicated biological and clinical relevance of our gene expression-based classification, which will improve risk prediction and treatment stratification of MDS.
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http://dx.doi.org/10.1182/blood-2017-05-783050DOI Listing
December 2017

Sequential acquisition of mutations in myelodysplastic syndromes.

Authors:
Hideki Makishima

Rinsho Ketsueki 2017 ;58(10):1828-1837

Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University.

Recent progress in next-generation sequencing technologies allows us to discover frequent mutations throughout the coding regions of myelodysplastic syndromes (MDS), potentially providing us with virtually a complete spectrum of driver mutations in this disease. As shown by many study groups these days, such driver mutations are acquired in a gene-specific fashion. For instance, DDX41 mutations are observed in germline cells long before MDS presentation. In blood samples from healthy elderly individuals, somatic DNMT3A and TET2 mutations are detected as age-related clonal hematopoiesis and are believed to be a risk factor for hematological neoplasms. In MDS, mutations of genes such as NRAS and FLT3, designated as Type-1 genes, may be significantly associated with leukemic evolution. Another type (Type-2) of genes, including RUNX1 and GATA2, are related to progression from low-risk to high-risk MDS. Overall, various driver mutations are sequentially acquired in MDS, at a specific time, in either germline cells, normal hematopoietic cells, or clonal MDS cells.
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http://dx.doi.org/10.11406/rinketsu.58.1828DOI Listing
December 2017