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Loss-of-function TET2 mutations (TET2 MT ) are common in myeloid neoplasia. TET2, a DNA dioxygenase, requires 2-oxoglutarate and Fe(II) to oxidize 5-methylcytosine. TET2 MT 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 MT by restoring dioxygenase activity. Here, we report the utility of AA in the prevention of TET2 MT 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 MT . 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 MT leukemia.

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., (Copyright © 2020. Published by Elsevier Ltd.)

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.

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.

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.

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.

Purpose: Somatic mutations in IDH1/2 occur in approximately 20% of patients with myeloid neoplasms, including acute myeloid leukemia (AML). IDH1/2 MUT enzymes produce D -2-hydroxyglutarate ( D 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 IDH1/2 MUT AML is not known. Experimental Design: Well-characterized primary IDH1 MUT , IDH2 MUT , and IDH1/2 WT AML cells were analyzed for DNA damage and responses to daunorubicin, ionizing radiation, and PARP inhibitors. Results: IDH1/2 MUT caused increased DNA damage and sensitization to daunorubicin, irradiation, and the PARP inhibitors olaparib and talazoparib in AML cells. IDH1/2 MUT inhibitors protected against these treatments. Combined treatment with a PARP inhibitor and daunorubicin had an additive effect on the killing of IDH1/2 MUT AML cells. We provide evidence that the therapy sensitivity of IDH1/2 MUT cells was caused by D 2HG-mediated downregulation of expression of the DNA damage response gene ATM and not by altered redox responses due to metabolic alterations in IDH1/2 MUT cells. Conclusions: IDH1/2 MUT 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 MUT inhibitors during cytotoxic therapy decrease the efficacy of both agents in IDH1/2 MUT AML. These results advocate in favor of clinical trials of PARP inhibitors either or not in combination with daunorubicin in IDH1/2 MUT AML. Clin Cancer Res; 24(7); 1705-15. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Myelodysplastic syndromes are typically diseases of older adults. Patients in whom the onset is early may have distinct molecular and clinical features or reflect a demographic continuum. The identification of differences between "early onset" patients and those diagnosed at a traditional age has the potential to advance understanding of the pathogenesis of myelodysplasia and may lead to formation of distinct morphological subcategories. We studied a cohort of 634 patients with various subcategories of myelodysplastic syndrome and secondary acute myeloid leukemia, stratifying them based on age at presentation and clinical parameters. We then characterized molecular abnormalities detected by next-generation deep sequencing of 60 genes that are commonly mutated in myeloid malignancies. The number of mutations increased linearly with age and on average, patients >50 years of age had more mutations. TET2 , SRSF2 , and DNMT3A were more commonly mutated in patients >50 years old compared to patients ≤50 years old. In general, patients >50 years of age also had more mutations in spliceosomal, epigenetic modifier, and RAS gene families. Although there are age-related differences in molecular features among patients with myelodysplasia, most notably in the incidence of SRSF2 mutations, our results suggest that patients ≤50 years old belong to a disease continuum with a distinct pattern of early onset ancestral events., (Copyright© Ferrata Storti Foundation.)

Genetic alterations, including mutations and copy-number alterations, are central to the pathogenesis of myelodysplastic syndromes and related diseases (myelodysplasia), but their roles in allogeneic stem cell transplantation have not fully been studied in a large cohort of patients. We enrolled 797 patients who had been diagnosed with myelodysplasia at initial presentation and received transplantation via the Japan Marrow Donor Program. Targeted-capture sequencing was performed to identify mutations in 69 genes, together with copy-number alterations, whose effects on transplantation outcomes were investigated. We identified 1776 mutations and 927 abnormal copy segments among 617 patients (77.4%). In multivariate modeling using Cox proportional-hazards regression, genetic factors explained 30% of the total hazards for overall survival; clinical characteristics accounted for 70% of risk. TP53 and RAS-pathway mutations, together with complex karyotype (CK) as detected by conventional cytogenetics and/or sequencing-based analysis, negatively affected posttransplant survival independently of clinical factors. Regardless of disease subtype, TP53 -mutated patients with CK were characterized by unique genetic features and associated with an extremely poor survival with frequent early relapse, whereas outcomes were substantially better in TP53 -mutated patients without CK. By contrast, the effects of RAS-pathway mutations depended on disease subtype and were confined to myelodysplastic/myeloproliferative neoplasms (MDS/MPNs). Our results suggest that TP53 and RAS-pathway mutations predicted a dismal prognosis, when associated with CK and MDS/MPNs, respectively. However, for patients with mutated TP53 or CK alone, long-term survival could be obtained with transplantation. Clinical sequencing provides vital information for accurate prognostication in transplantation., (© 2017 by The American Society of Hematology.)

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.

Epigenetic changes can contribute to development of acute myeloid leukemia (AML), a malignant disease of the bone marrow. A single-nucleotide polymorphism of transcription factor growth factor independence 1 (GFI1) generates a protein with an asparagine at position 36 (GFI1(36N)) instead of a serine at position 36 (GFI1(36S)), which is associated with de novo AML in humans. However, how GFI1(36N) predisposes to AML is poorly understood. To explore the mechanism, we used knock-in mouse strains expressing GFI1(36N) or GFI1(36S). Presence of GFI1(36N) shortened the latency and increased the incidence of AML in different murine models of myelodysplastic syndrome/AML. On a molecular level, GFI1(36N) induced genomewide epigenetic changes, leading to expression of AML-associated genes. On a therapeutic level, use of histone acetyltransferase inhibitors specifically impeded growth of GFI1(36N)-expressing human and murine AML cells in vitro and in vivo. These results establish, as a proof of principle, how epigenetic changes in GFI1(36N)-induced AML can be targeted., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Molecular predictors of outcome are increasingly important in determining optimal therapy for myeloid neoplasms. Mutations in the spliceosomal genes (U2AF1 and SRSF2) predict for poor outcomes in myelodysplastic syndromes (MDS) and related diseases. We investigated the effect of hematopoietic cell transplant (HCT) on the negative prognostic impact of U2AF1 and SRSF2 mutations. In total, 122 patients with MDS (30%), acute myeloid leukemia (51%), myeloproliferative neoplasms (MPN) (11%), and MDS/MPN (8%) receiving a HCT from 2003 to 2012 were evaluated for mutations in U2AF1 and SRSF2 by direct sequencing. Median time of follow up was 24 months (range 0.46-110). SRSF2 mutations were detected in 11 (10%) patients and U2AF1 in 3 (3%) patients. There were no significant differences in baseline characteristics between mutated and wild-type (WT) patients. Patients carrying SRSF2 and U2AF1 mutations had similar overall survival (P = 0.84), relapse mortality (P = 0.50), and non-relapse mortality (P = 0.72) compared to WT patients. However, taking into account disease status and cytogenetics in a subset of AML patients, SRSF2 and U2AF1 mutations were associated with worse survival (HR 3.71, P = 0.035)., (© 2016 Wiley Periodicals, Inc.)

Angioimmunoblastic T-cell lymphoma (AITL) is a nodal-based mature T-cell lymphoma with distinctive clinical symptomatology and histology. Research into its pathogenesis supports a cellular derivation from follicular helper T cells and overexpression of genes related to B cells, follicular dendritic cells, and vascular growth. Recently, a novel recurring somatic mutation in RHOA, encoding p.Gly17Val, was discovered in nearly 70% of AITLs and in a smaller proportion of peripheral T-cell lymphomas, not otherwise specified (PTCL-NOS). We investigated a series of AITLs to compare RHOA mutated with wild-type case for clinicopathologic differences. Targeted exome and Sanger sequencing was performed on 27 AITLs and 10 PTCL-NOS. The RHOA G17V mutation was identified in 63% of the AITL cases and in none of the PTCL-NOS cases. The median variant allelic frequency was 14%, with a range of 0.4 to 50% in positive cases. RHOA G17V-mutated cases had a significantly higher incidence of splenomegaly and B symptoms at diagnosis, but there was no difference in overall survival between mutated and wild-type subgroups. Cases with the RHOA G17V mutation had a significantly higher mean microvessel density (P<0.01) and expressed a greater number of follicular helper T-cell markers (P<0.05) than wild-type cases. RHOA G17V is present in a significant proportion of angioimmunoblastic lymphomas and is associated with classic pathologic features of AITL. Additional studies are needed to provide a biological or functional link between altered RHOA function and these pathologic features.

Acquired bone marrow failure syndromes consist of aplastic anemia, paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplastic syndromes (MDS). Clonal hematopoiesis is frequently observed in non-neoplastic disorders, aplastic anemia and PNH as well as a neoplastic phenotype, MDS. However, the significance of such clonal hematopoiesis, particularly in aplastic anemia, remains to be elucidated. Recent advancements in next generation sequencing technology have revealed a diverse clonal structure in these bone marrow failure syndromes, as well as in age-related clonal hematopoiesis in healthy people. In this review article, we describe gene mutations in bone marrow failure syndromes, together with those detected in healthy people.

Recent progress in next generation sequencing technologies has enabled us to investigate somatic mutations throughout the coding regions of myelodysplastic syndromes (MDS), and have likely provided us with almost the entire spectrum of driver mutations responsible for this disease. As shown by numerous recent studies, such frequent mutations play an important role in the leukemogenesis of MDS. In the first part of this review, we comprehensively describe the characteristics and frequencies of the most prevalent mutations already confirmed as drivers in MDS. Second, we focus on the associations between karyotypic abnormalities and somatic mutations involving chromosomes 5 and 7. In particular, CSNK1A1 mutations are the most recently identified among the 5q- syndromes. Finally, we summarize recently reported findings on the functional effects of frequent splice factor mutations on splicing defects, which have been clarified by multiple institutions.

Mutations in isocitrate dehydrogenase 1/2 (IDH1/2(MT)) are drivers of a variety of myeloid neoplasms. As they yield the same oncometabolite, D-2-hydroxyglutarate, they are often treated as equivalent, and pooled. We studied the validity of this approach and found IDH1/2 mutations in 179 of 2119 myeloid neoplasms (8%). Cross-sectionally, the frequencies of these mutations increased from lower- to higher risk disease, thus suggesting a role in clinical progression. Variant allelic frequencies indicated that IDH1(MT) and IDH2(MT) are ancestral in up to 14/74 (19%) vs 34/99 (34%; P=0.027) of cases, respectively, illustrating the pathogenic role of these lesions in myeloid neoplasms. IDH1/2(MT) was associated with poor overall survival, particularly in lower risk myelodysplastic syndromes. Ancestral IDH1(MT) cases were associated with a worse prognosis than subclonal IDH1(MT) cases, whereas the position of IDH2(MT) within clonal hierarchy did not impact survival. This may relate to distinct mutational spectra with more DNMT3A and NPM1 mutations associated with IDH1(MT) cases, and more ASXL1, SRSF2, RUNX1, STAG2 mutations associated with IDH2(MT) cases. Our data demonstrate important clinical and biological differences between IDH1(MT) and IDH2(MT) myeloid neoplasms. These mutations should be considered separately as their differences could have implications for diagnosis, prognosis and treatment with IDH1/2(MT) inhibitors of IDH1/2(MT) patients.

Next generation sequencing technologies have provided insights into the molecular heterogeneity of various myeloid neoplasms, revealing previously unknown somatic genetic events. In our cohort of 1444 cases analyzed by next generation sequencing, somatic mutations in the gene BRCA1-BRCA2-containing complex 3 (BRCC3) were identified in 28 cases (1.9%). BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases capable of cleaving Lys-63 linked polyubiquitin chains, and is implicated in DNA repair. The mutations were located throughout its coding region. The average variant allelic frequency of BRCC3 mutations was 30.1%, and by a serial sample analysis at two different time points a BRCC3 mutation was already identified in the initial stage of a myelodysplastic syndrome. BRCC3 mutations commonly occurred in nonsense (n=12), frameshift (n=4), and splice site (n=5) configurations. Due to the marginal male dominance (odds ratio; 2.00, 0.84-4.73) of BRCC3 mutations, the majority of mutations (n=23; 82%) were hemizygous. Phenotypically, BRCC3 mutations were frequently observed in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms and associated with -Y abnormality (odds ratio; 3.70, 1.25-11.0). Clinically, BRCC3 mutations were also related to higher age (P=0.01), although prognosis was not affected. Knockdown of Brcc3 gene expression in murine bone marrow lineage negative, Sca1 positive, c-kit positive cells resulted in 2-fold more colony formation and modest differentiation defect. Thus, BRCC3 likely plays a role as tumor-associated gene in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms., (Copyright© Ferrata Storti Foundation.)

Background: In patients with acquired aplastic anemia, destruction of hematopoietic cells by the immune system leads to pancytopenia. Patients have a response to immunosuppressive therapy, but myelodysplastic syndromes and acute myeloid leukemia develop in about 15% of the patients, usually many months to years after the diagnosis of aplastic anemia., Methods: We performed next-generation sequencing and array-based karyotyping using 668 blood samples obtained from 439 patients with aplastic anemia. We analyzed serial samples obtained from 82 patients., Results: Somatic mutations in myeloid cancer candidate genes were present in one third of the patients, in a limited number of genes and at low initial variant allele frequency. Clonal hematopoiesis was detected in 47% of the patients, most frequently as acquired mutations. The prevalence of the mutations increased with age, and mutations had an age-related signature. DNMT3A-mutated and ASXL1-mutated clones tended to increase in size over time; the size of BCOR- and BCORL1-mutated and PIGA-mutated clones decreased or remained stable. Mutations in PIGA and BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and longer and a higher rate of overall and progression-free survival; mutations in a subgroup of genes that included DNMT3A and ASXL1 were associated with worse outcomes. However, clonal dynamics were highly variable and might not necessarily have predicted the response to therapy and long-term survival among individual patients., Conclusions: Clonal hematopoiesis was prevalent in aplastic anemia. Some mutations were related to clinical outcomes. A highly biased set of mutations is evidence of Darwinian selection in the failed bone marrow environment. The pattern of somatic clones in individual patients over time was variable and frequently unpredictable. (Funded by Grant-in-Aid for Scientific Research and others.).

Whole exome next generation sequencing systematically applied as a discovery tool in myelodysplastic syndromes (MDS) has led to the identification of a large number of novel mutations. Despite hundreds of patients studied, mutational saturation has not been reached and it is expected that new driver mutations will be discovered in this very heterogeneous condition. Serial samples and deep sequencing of the identified alterations has allowed for a dynamic/chronologic analysis of clonal architecture and identification of a subset of ancestral and secondary molecular lesions. Chromosomal gains and losses have been incorporated into the mutational analyses because they can either cooperate with mutations or produce a functional phenocopy. In addition to the search for somatic defects in MDS, similar discovery studies have been also performed to identify germ line mutations/alterations. Clinical analysis showed applicability of multiplexed somatic mutational panels that would complement current pathomorphologic diagnosis, allow for subclassification of nosologic entities, and enhance predictive power of current prognostic algorithms. Overall, comprehensive genomic analysis in MDS has revealed a tremendous heterogeneity of somatic lesions and their combinations further enhanced by the heterogeneity of clonal architecture and chromosomal lesions.

Somatic cohesin mutations have been reported in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). To account for the morphologic and cytogenetic diversity of these neoplasms, a well-annotated cohort of 1060 patients with myeloid malignancies including MDS (n = 386), myeloproliferative neoplasms (MPNs) (n = 55), MDS/MPNs (n = 169), and AML (n = 450) were analyzed for cohesin gene mutational status, gene expression, and therapeutic and survival outcomes. Somatic cohesin defects were detected in 12% of patients with myeloid malignancies, whereas low expression of these genes was present in an additional 15% of patients. Mutations of cohesin genes were mutually exclusive and mostly resulted in predicted loss of function. Patients with low cohesin gene expression showed similar expression signatures as those with somatic cohesin mutations. Cross-sectional deep-sequencing analysis for clonal hierarchy demonstrated STAG2, SMC3, and RAD21 mutations to be ancestral in 18%, 18%, and 47% of cases, respectively, and each expanded to clonal dominance concordant with disease transformation. Cohesin mutations were significantly associated with RUNX1, Ras-family oncogenes, and BCOR and ASXL1 mutations and were most prevalent in high-risk MDS and secondary AML. Cohesin defects were associated with poor overall survival (27.2 vs 40 months; P = .023), especially in STAG2 mutant MDS patients surviving >12 months (median survival 35 vs 50 months; P = .017)., (© 2014 by The American Society of Hematology.)

We hypothesized that specific molecular mutations are important biomarkers for response to DNA methyltransferase inhibitors (DNMT inhibitors) and may have prognostic value in patients with myelodysplastic syndromes (MDS). Mutational analysis was performed in 92 patients with MDS and related disorders who received 5-azacytidine (n=55), decitabine (n=26) or both (n=11). Mutational status was correlated with overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) by univariate and multivariate analysis. Risk stratification models were created. TET2, DNMT3A, IDH1/IDH2, ASXL1, CBL, RAS and SF3B1 mutations were found in 18, 9, 8, 26, 3, 2 and 13% of patients, respectively. In multivariate analysis, TET2(MUT) and/or DNMT3A(MUT) (P=0.03), platelets > or = 100 × 10(9)/l (P=0.007) and WBC<3.0 × 10(9)/l (P=0.03) were independent predictors of better response. TET2(MUT) and/or DNMT3A(MUT) (P=0.04) status was also independently prognostic for improved PFS, as were good or intermediate cytogenetic risk (P<0.0001), age<60 (P=0.0001), treatment with both 5-azacytidine and decitabine (P=0.02) and hemoglobin > or = 10 g/dl (P=0.01). Better OS was associated with ASXL1(WT) (P=0.008) and SF3B1(MUT) (P=0.01), and, similar to PFS, cytogenetic risk (P=0.0002), age (P=0.02) and hemoglobin (P=0.04). These data support the role of molecular mutations as predictive biomarkers for response and survival in MDS patients treated with DNMT inhibitors.

Large granular lymphocyte leukemia (LGL) is often associated with immune cytopenias and can cooccur in the context of aplastic anemia (AA) and myelodysplastic syndromes (MDS). We took advantage of the recent description of signal transducer and activator of transcription 3 (STAT3) mutations in LGL clonal expansions to test, using sensitive methods, for the presence of these mutations in a large cohort of 367 MDS and 140 AA cases. STAT3 clones can be found not only in known LGL concomitant cases, but in a small proportion of unsuspected ones (7% AA and 2.5% MDS). In STAT3-mutated AA patients, an interesting trend toward better responses of immunosuppressive therapy and an association with the presence of human leukocyte antigen-DR15 were found. MDSs harboring a STAT3 mutant clone showed a lower degree of bone marrow cellularity and a higher frequency of developing chromosome 7 abnormalities. STAT3-mutant LGL clones may facilitate a persistently dysregulated autoimmune activation, responsible for the primary induction of bone marrow failure in a subset of AA and MDS patients.

Juvenile myelomonocytic leukemia (JMML) is an intractable pediatric leukemia with poor prognosis whose molecular pathogenesis is poorly understood, except for somatic or germline mutations of RAS pathway genes, including PTPN11, NF1, NRAS, KRAS and CBL, in the majority of cases. To obtain a complete registry of gene mutations in JMML, whole-exome sequencing was performed for paired tumor-normal DNA from 13 individuals with JMML (cases), which was followed by deep sequencing of 8 target genes in 92 tumor samples. JMML was characterized by a paucity of gene mutations (0.85 non-silent mutations per sample) with somatic or germline RAS pathway involvement in 82 cases (89%). The SETBP1 and JAK3 genes were among common targets for secondary mutations. Mutations in the latter were often subclonal and may be involved in the progression rather than the initiation of leukemia, and these mutations associated with poor clinical outcome. Our findings provide new insights into the pathogenesis and progression of JMML.

Here we report whole-exome sequencing of individuals with various myeloid malignancies and identify recurrent somatic mutations in SETBP1, consistent with a recent report on atypical chronic myeloid leukemia (aCML). Closely positioned somatic SETBP1 mutations encoding changes in Asp868, Ser869, Gly870, Ile871 and Asp880, which match germline mutations in Schinzel-Giedion syndrome (SGS), were detected in 17% of secondary acute myeloid leukemias (sAML) and 15% of chronic myelomonocytic leukemia (CMML) cases. These results from deep sequencing demonstrate a higher mutational detection rate than reported with conventional sequencing methodology. Mutant cases were associated with advanced age and monosomy 7/deletion 7q (-7/del(7q)) constituting poor prognostic factors. Analysis of serially collected samples indicated that SETBP1 mutations were acquired during leukemic evolution. Transduction with mutant Setbp1 led to the immortalization of mouse myeloid progenitors that showed enhanced proliferative capacity compared to cells transduced with wild-type Setbp1. Somatic mutations of SETBP1 seem to cause gain of function, are associated with myeloid leukemic transformation and convey poor prognosis in myelodysplastic syndromes (MDS) and CMML.

Chronic myelomonocytic leukemia is a heterogeneous disease with multifactorial molecular pathogenesis. Various recurrent somatic mutations have been detected alone or in combination in chronic myelomonocytic leukemia. Recently, recurrent mutations in spliceosomal genes have been discovered. We investigated the contribution of U2AF1, SRSF2 and SF3B1 mutations in the pathogenesis of chronic myelomonocytic leukemia and closely related diseases. We genotyped a cohort of patients with chronic myelomonocytic leukemia, secondary acute myeloid leukemia derived from chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia for somatic mutations in U2AF1, SRSF2, SF3B1 and in the other 12 most frequently affected genes in these conditions. Chromosomal abnormalities were assessed by nucleotide polymorphism array-based karyotyping. The presence of molecular lesions was correlated with clinical endpoints. Mutations in SRSF2, U2AF1 and SF3B1 were found in 32%, 13% and 6% of cases of chronic myelomonocytic leukemia, secondary acute myeloid leukemia derived from chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia, respectively. Spliceosomal genes were affected in various combinations with other mutations, including TET2, ASXL1, CBL, EZH2, RAS, IDH1/2, DNMT3A, TP53, UTX and RUNX1. Worse overall survival was associated with mutations in U2AF1 (P=0.047) and DNMT3A (P=0.015). RAS mutations had an impact on overall survival in secondary acute myeloid leukemia (P=0.0456). By comparison, our screening of juvenile myelomonocytic leukemia cases showed mutations in ASXL1 (4%), CBL (10%), and RAS (6%) but not in IDH1/2, TET2, EZH2, DNMT3A or the three spliceosomal genes. SRSF2 and U2AF1 along with TET2 (48%) and ASXL1 (38%) are frequently affected by somatic mutations in chronic myelomonocytic leukemia, quite distinctly from the profile seen in juvenile myelomonocytic leukemia. Our data also suggest that spliceosomal mutations are of ancestral origin.

Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

Chronic lymphoproliferative disorders of natural killer cells (CLPD-NKs) and T-cell large granular lymphocytic leukemias (T-LGLs) are clonal lymphoproliferations arising from either natural killer cells or cytotoxic T lymphocytes (CTLs). We have investigated for distribution and functional significance of mutations in 50 CLPD-NKs and 120 T-LGL patients by direct sequencing, allele-specific PCR, and microarray analysis. STAT3 gene mutations are present in both T and NK diseases: approximately one-third of patients with each type of disorder convey these mutations. Mutations were found in exons 21 and 20, encoding the Src homology 2 domain. Patients with mutations are characterized by symptomatic disease (75%), history of multiple treatments, and a specific pattern of STAT3 activation and gene deregulation, including increased expression of genes activated by STAT3. Many of these features are also found in patients with wild-type STAT3, indicating that other mechanisms of STAT3 activation can be operative in these chronic lymphoproliferative disorders. Treatment with STAT3 inhibitors, both in wild-type and mutant cases, resulted in accelerated apoptosis. STAT3 mutations are frequent in large granular lymphocytes suggesting a similar molecular dysregulation in malignant chronic expansions of NK and CTL origin. STAT3 mutations may distinguish truly malignant lymphoproliferations involving T and NK cells from reactive expansions.

Somatic CBL mutations have been reported in a variety of myeloid neoplasms but are rare in acute lymphoblastic leukemia (ALL). We analyzed 77 samples from hematologic malignancies, identifying a somatic mutation in CBL (p.C381R) in one patient with T-ALL that was associated with a uniparental disomy at the CBL locus and a germline heterozygous mutation in one patient with JMML. Two NOTCH1 mutations and homozygous deletions in LEF1 and CDKN2A were identified in T-ALL cells. The activation of the RAS pathway was enhanced, and activation of the NOTCH1 pathway was inhibited in NIH 3T3 cells that expressed p.C381R. This study appears to be the first to identify a CBL mutation in T-ALL., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Recurrent homozygous CBL-inactivating mutations in myeloid malignancies decrease ubiquitin ligase activity that inactivates SRC family kinases (SFK) and receptor tyrosine kinases (RTK). However, the most important SFK and RTK affected by these mutations, and hence, the most important therapeutic targets, have not been clearly characterized. We compared SFK and RTK pathway activity and inhibitors in acute myeloid leukemia cell lines containing homozygous R420Q mutation (GDM-1), heterozygous deletion (MOLM13) and wild-type (WT) CBL (THP1, U937). As expected with CBL loss, GDM-1 displayed high KIT expression and granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity. Ectopic expression of WT CBL decreased GDM-1 proliferation but not cell lines with WT CBL. GDM-1, but not the other cell lines, was highly sensitive to growth inhibition by dasatinib (dual SFK and RTK inhibitor, LD50 50 nM); there was less or no selective inhibition of GDM-1 growth by sunitinib (RTK inhibitor), imatinib (ABL, KIT inhibitor), or PP2 (SFK inhibitor). Phosphoprotein analysis identified phosphorylation targets uniquely inhibited by dasatinib treatment of GDM-1, including a number of proteins in the KIT and GM-CSF receptor pathways (for example, KIT Tyr721, STAT3 Tyr705). In conclusion, the promiscuous effects of CBL loss on SFK and RTK signaling appear to be best targeted by dual SFK and RTK inhibition.

Myelodysplastic syndromes (MDSs) are chronic and often progressive myeloid neoplasms associated with remarkable heterogeneity in the histomorphology and clinical course. Various somatic mutations are involved in the pathogenesis of MDS. Recently, mutations in a gene encoding a spliceosomal protein, SF3B1, were discovered in a distinct form of MDS with ring sideroblasts. Whole exome sequencing of 15 patients with myeloid neoplasms was performed, and somatic mutations in spliceosomal genes were identified. Sanger sequencing of 310 patients was performed to assess phenotype/genotype associations. To determine the functional effect of spliceosomal mutations, we evaluated pre-mRNA splicing profiles by RNA deep sequencing. We identified additional somatic mutations in spliceosomal genes, including SF3B1, U2AF1, and SRSF2. These mutations alter pre-mRNA splicing patterns. SF3B1 mutations are prevalent in low-risk MDS with ring sideroblasts, whereas U2AF1 and SRSF2 mutations are frequent in chronic myelomonocytic leukemia and advanced forms of MDS. SF3B1 mutations are associated with a favorable prognosis, whereas U2AF1 and SRSF2 mutations are predictive for shorter survival. Mutations affecting spliceosomal genes that result in defective splicing are a new leukemogenic pathway. Spliceosomal genes are probably tumor suppressors, and their mutations may constitute diagnostic biomarkers that could potentially serve as therapeutic targets.

Chronic myelomonocytic leukemia (CMML), a myelodysplastic/myeloproliferative neoplasm, is characterized by monocytic proliferation, dysplasia, and progression to acute myeloid leukemia. CMML has been associated with somatic mutations in diverse recently identified genes. We analyzed 72 well-characterized patients with CMML (N = 52) and CMML-derived acute myeloid leukemia (N = 20) for recurrent chromosomal abnormalities with the use of routine cytogenetics and single nucleotide polymorphism arrays along with comprehensive mutational screening. Cytogenetic aberrations were present in 46% of cases, whereas single nucleotide polymorphism array increased the diagnostic yield to 60%. At least 1 mutation was found in 86% of all cases; novel UTX, DNMT3A, and EZH2 mutations were found in 8%, 10%, and 5.5% of patients, respectively. TET2 mutations were present in 49%, ASXL1 in 43%, CBL in 14%, IDH1/2 in 4%, KRAS in 7%, NRAS in 4%, and JAK2 V617F in 1% of patients. Various mutant genotype combinations were observed, indicating molecular heterogeneity in CMML. Our results suggest that molecular defects affecting distinct pathways can lead to similar clinical phenotypes.

Progression of chronic myelogenous leukemia (CML) to accelerated (AP) and blast phase (BP) is because of secondary molecular events, as well as additional cytogenetic abnormalities. On the basis of the detection of JAK2, CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations in myelodysplastic/myeloproliferative neoplasms, we hypothesized that they may also contribute to progression in CML. We screened these genes for mutations in 54 cases with CML (14 with chronic phase, 14 with AP, 20 with myeloid, and 6 with nonmyeloid BP). We identified 1 CBLB and 2 TET2 mutations in AP, and 1 CBL, 1 CBLB, 4 TET2, 2 ASXL1, and 2 IDH family mutations in myeloid BP. However, none of these mutations were found in chronic phase. No cases with JAK2V617F mutations were found. In 2 cases, TET2 mutations were found concomitant with CBLB mutations. By single nucleotide polymorphism arrays, uniparental disomy on chromosome 5q, 8q, 11p, and 17p was found in AP and BP but not involving 4q24 (TET2) or 11q23 (CBL). Microdeletions on chromosomes 17q11.2 and 21q22.12 involved tumor associated genes NF1 and RUNX1, respectively. Our results indicate that CBL family, TET2, ASXL1, and IDH family mutations and additional cryptic karyotypic abnormalities can occur in advanced phase CML.

While a majority of patients with refractory anemia with ring sideroblasts and thrombocytosis harbor JAK2V617F and rarely MPLW515L, JAK2/MPL-negative cases constitute a diagnostic problem. 23 RARS-T cases were investigated applying immunohistochemical phospho-STAT5, sequencing and SNP-A-based karyotyping. Based on the association of TET2/ASXL1 mutations with MDS/MPN we studied molecular pattern of these genes. Two patients harbored ASXL1 and another 2 TET2 mutations. Phospho-STAT5 activation was present in one mutated TET2 and ASXL1 case. JAK2V617F/MPLW515L mutations were absent in TET2/ASXL1 mutants, indicating that similar clinical phenotype can be produced by various MPN-associated mutations and that additional unifying lesions may be present in RARS-T., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Mutations in NF1, PTPN11, NRAS, KRAS and CBL have been reported to play a pathogenetic role in juvenile myelomonocytic leukaemia (JMML), a rare myelodyplastic/myeloproliferative neoplasm occurring in children. Recently, mutations in ASXL1 were identified in chronic myelomonocytic leukaemia and other myeloid malignancies. We sequenced exon 12 of ASLX1 in 49 JMML patients, and found 2 novel heterozygous (nonsense and frameshift) mutations, one occurring as a sole lesion, the other was in conjunction with a PTPN11 mutation. ASXL1 cooperates with KDM1A in transcriptional repression and thereby ASXL1 mutations may synergize with or mimic other JMML-related mutations.

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. When we investigated the presence of recurrent molecular lesions in a cohort of 49 children with JMML, neurofibromatosis phenotype (and thereby NF1 mutation) was present in 2 patients (4%), whereas previously described PTPN11, NRAS, and KRAS mutations were found in 53%, 4%, and 2% of cases, respectively. Consequently, a significant proportion of JMML patients without identifiable pathogenesis prompted our search for other molecular defects. When we applied single nucleotide polymorphism arrays to JMML patients, somatic uniparental disomy 11q was detected in 4 of 49 patients; all of these cases harbored RING finger domain c-Cbl mutations. In total, c-Cbl mutations were detected in 5 (10%) of 49 patients. No mutations were identified in Cbl-b and TET2. c-Cbl and RAS pathway mutations were mutually exclusive. Comparison of clinical phenotypes showed earlier presentation and lower hemoglobin F levels in patients with c-Cbl mutations. Our results indicate that mutations in c-Cbl may represent key molecular lesions in JMML patients without RAS/PTPN11 lesions, suggesting analogous pathogenesis to those observed in chronic myelomonocytic leukemia (CMML) patients.

Purpose: Acquired somatic uniparental disomy (UPD) is commonly observed in myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), or secondary acute myelogenous leukemia (sAML) and may point toward genes harboring mutations. Recurrent UPD11q led to identification of homozygous mutations in c-Cbl, an E3 ubiquitin ligase involved in attenuation of proliferative signals transduced by activated receptor tyrosine kinases. We examined the role and frequency of Cbl gene family mutations in MPN and related conditions., Methods: We applied high-density SNP-A karyotyping to identify loss of heterozygosity of 11q in 442 patients with MDS, MDS/MPN, MPN, sAML evolved from these conditions, and primary AML. We sequenced c-Cbl, Cbl-b, and Cbl-c in patients with or without corresponding UPD or deletions and correlated mutational status with clinical features and outcomes., Results: We identified c-Cbl mutations in 5% and 9% of patients with chronic myelomonocytic leukemia (CMML) and sAML, and also in CML blast crisis and juvenile myelomonocytic leukemia (JMML). Most mutations were homozygous and affected c-Cbl; mutations in Cbl-b were also found in patients with similar clinical features. Patients with Cbl family mutations showed poor prognosis, with a median survival of 5 months. Pathomorphologic features included monocytosis, monocytoid blasts, aberrant expression of phosphoSTAT5, and c-kit overexpression. Serial studies showed acquisition of c-Cbl mutations during malignant evolution., Conclusion: Mutations in the Cbl family RING finger domain or linker sequence constitute important pathogenic lesions associated with not only preleukemic CMML, JMML, and other MPN, but also progression to AML, suggesting that impairment of degradation of activated tyrosine kinases constitutes an important cancer mechanism.

Two types of acquired loss of heterozygosity are possible in cancer: deletions and copy-neutral uniparental disomy (UPD). Conventionally, copy number losses are identified using metaphase cytogenetics, whereas detection of UPD is accomplished by microsatellite and copy number analysis and as such, is not often used clinically. Recently, introduction of single nucleotide polymorphism (SNP) microarrays has allowed for the systematic and sensitive detection of UPD in hematologic malignancies and other cancers. In this study, we have applied 250K SNP array technology to detect previously cryptic chromosomal changes, particularly UPD, in a cohort of 301 patients with myelodysplastic syndromes (MDS), overlap MDS/myeloproliferative disorders (MPD), MPD, and acute myeloid leukemia. We show that UPD is a common chromosomal defect in myeloid malignancies, particularly in chronic myelomonocytic leukemia (CMML; 48%) and MDS/MPD-unclassifiable (38%). Furthermore, we show that mapping minimally overlapping segmental UPD regions can help target the search for both known and unknown pathogenic mutations, including newly identified missense mutations in the proto-oncogene c-Cbl in 7 of 12 patients with UPD11q. Acquired mutations of c-Cbl E3 ubiquitin ligase may explain the pathogenesis of a clonal process in a subset of MDS/MPD, including CMML.