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1.  Clonal diversity of recurrently mutated genes in myelodysplastic syndromes 
Leukemia  2013;27(6):1275-1282.
Recent studies suggest that most cases of myelodysplastic syndrome (MDS) are clonally heterogeneous, with a founding clone and multiple subclones. It is not known whether specific gene mutations typically occur in founding clones or subclones. We screened a panel of 94 candidate genes in a cohort of 157 patients with MDS or secondary acute myeloid leukemia (sAML). This included 150 cases with samples obtained at MDS diagnosis and 15 cases with samples obtained at sAML transformation (8 were also analyzed at the MDS stage). We performed whole-genome sequencing (WGS) to define the clonal architecture in eight sAML genomes and identified the range of variant allele frequencies (VAFs) for founding clone mutations. At least one mutation or cytogenetic abnormality was detected in 83% of the 150 MDS patients and 17 genes were significantly mutated (false discovery rate ≤0.05). Individual genes and patient samples displayed a wide range of VAFs for recurrently mutated genes, indicating that no single gene is exclusively mutated in the founding clone. The VAFs of recurrently mutated genes did not fully recapitulate the clonal architecture defined by WGS, suggesting that comprehensive sequencing may be required to accurately assess the clonal status of recurrently mutated genes in MDS.
doi:10.1038/leu.2013.58
PMCID: PMC3736571  PMID: 23443460
myelodysplastic syndromes; MDS; acute myeloid leukemia; genomics; clonality; sequencing
2.  A High Occurrence of Acquisition and/or Expansion of C-CBL Mutant Clones in the Progression of High-Risk Myelodysplastic Syndrome to Acute Myeloid Leukemia12 
Neoplasia (New York, N.Y.)  2011;13(11):1035-1042.
The molecular pathogenesis of myelodysplastic syndrome (MDS) and its progression to secondary acute myeloid leukemia (sAML) remain to be explored. Somatic C-CBL mutations were recently described in MDS. Our study aimed to determine the role of C-CBL mutations in the progression of MDS to sAML and sought to correlate with clinicohematological features and outcome. Bone marrow samples from 51 patients with high-risk MDS (13 with refractory cytopenia with multilineage dysplasia, 19 with refractory anemia with excess blast 1, and 19 with refractory anemia with excess blast 2) were analyzed for C-CBL mutations at both diagnosis and sAML in the same individuals. Mutational analysis was performed for exons 7 to 9 of C-CBL gene. Of the 51 paired samples, C-CBL mutations were identified in 6 patients at the sAML phase. One patient retained the identical C-CBL mutation (G415S) at sAML evolution and exhibited clonal expansion. The other five patients acquired C-CBL mutations (Y371S, F418S, L370_Y371 ins L, L399V, and C416W) during sAML evolution. Three of the six patients harboring C-CBL mutations at sAML had additional gene mutations including JAK2V617F, PTPN11, or N-RAS. There was no significant difference in clinicohematological features and overall survival with respect to C-CBL mutation status. Our results show that C-CBL mutation is very rare (0.6%) in MDS, but acquisition and/or expansion of C-CBL mutant clones occur in 11.8% of patients during sAML transformation. The findings suggest that C-CBL mutations play a role at least in part in a subset of MDS patients during sAML transformation.
PMCID: PMC3226941  PMID: 22131879
3.  Isoform-Specific Potentiation of Stem and Progenitor Cell Engraftment by AML1/RUNX1  
PLoS Medicine  2007;4(5):e172.
Background
AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo.
Methods and Findings
The human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo.
Conclusions
These data demonstrate that the “a” isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of leukaemia and also in cord blood transplantation in adults, in whom stem and progenitor cell numbers are often limiting.
The truncated "a" isoform of AML1 is shown to have the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation.
Editors' Summary
Background.
Blood contains red blood cells (which carry oxygen round the body), platelets (which help the blood to clot), and white blood cells (which fight off infections). All these cells, which are regularly replaced, are derived from hematopoietic stem cells, blood-forming cells present in the bone marrow. Like all stem cells, hematopoietic stem cells self-renew (reproduce themselves) and produce committed progenitor cells, which develop into mature blood cells in a process called hematopoiesis. Many proteins control hematopoiesis, some of which are called transcription factors; these factors bind to DNA through their DNA-binding domain and then control the expression of genes (that is, how DNA is turned into proteins) through particular parts of the protein (their transcription regulatory domains). An important hematopoietic transcription factor is AML1—a protein first identified because of its involvement in acute myelogenous leukemia (AML, a form of blood cancer). Mutations (changes) in the AML1 gene are now known to be present in other types of leukemia, which are often characterized by overproliferation of immature blood cells.
Why Was This Study Done?
Because of AML1′s crucial role in hematopoiesis, knowing more about which genes it regulates and how its activity is regulated could provide clues to treating leukemia and to improving hematopoietic cell transplantation. Many cancer treatments destroy hematopoietic stem cells, leaving patients vulnerable to infection. Transplants of bone marrow or cord blood (the cord that links mother and baby during pregnancy contains peripheral blood stem cells) can replace the missing cells, but cord blood in particular often contains insufficient stem cells for successful transplantation. It would be useful, therefore, to expand the stem cell content of these tissues before transplantation. In this study, the researchers investigated the effect of AML1 on self-renewal and differentiation of hematopoietic stem and progenitor cells in the laboratory (in vitro) and in animals (in vivo). In particular, they have asked how two isoforms (closely related versions) of AML1 affect the ability of these cells to grow and differentiate (engraft) in mice after transplantation.
What Did the Researchers Do and Find?
The researchers artificially expressed AML1a and AML1b (both isoforms contain a DNA binding domain, but only AML1b has transcription regulatory domains) in mouse hematopoietic stem and progenitor cells and then tested the cells' ability to engraft in mice. AML1a-expressing cells engrafted better than unaltered cells and outgrew unaltered cells when transplanted as a mixture. AML1b-expressing cells, however, did not engraft. In vitro, AML1a-expressing cells grew more than AML1b-expressing cells, whereas differentiation was promoted in AML1b-expressing cells. To investigate whether the isoforms have the same effects in human cells, the researchers measured the amount of AML1a and AML1b mRNA (the template for protein production) made by progenitor cells in human cord blood. Although AML1b (together with AML1c, an isoform with similar characteristics) mRNA predominated in all the progenitor cell types, the relative abundance of AML1a was greatest in the stem and progenitor cells. Furthermore, forced expression of AML1a in these cells improved their ability to divide in vitro and to engraft in mice.
What Do These Findings Mean?
These findings indicate that AML1a expression increases the self-renewal capacity of hematopoietic stem and progenitor cells and consequently improves their ability to engraft in mice, whereas AML1b expression encourages the differentiation of these cell types. These activities are consistent with the expression patterns of the two isoforms in normal hematopoietic cells and in leukemic cells—the mutated AML made by many leukemic cells resembles AML1a. Because the AML1 isoforms were expressed at higher than normal levels in these experiments, the physiological relevance of these findings needs to be confirmed by showing that normal levels of AML1a and AML1b produce similar results. Nevertheless, these results suggest that manipulating the balance of AML1 isoforms made by hematopoietic cells might be useful clinically. In leukemia, a shift toward AML1b expression might slow the proliferation of leukemic cells and encourage their differentiation. Conversely, in cord blood transplantation, a shift toward AML1a expression might improve patient outcomes by expanding the stem and progenitor cell populations.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040172.
Wikipedia has pages on hematopoiesis and hematopoietic stem cells (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The US National Cancer Institute has a fact sheet on bone marrow and peripheral blood stem cell transplantation (in English and Spanish) and information for patients and professionals on leukemia (in English)
The American Society of Hematology provides patient information about blood diseases, including information on bone marrow and stem cell transplantation
doi:10.1371/journal.pmed.0040172
PMCID: PMC1868041  PMID: 17503961
4.  Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2 
Nature  2010;468(7325):839-843.
TET2 is a close relative of TET1, an enzyme that converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in DNA1,2. The gene encoding TET2 resides at chromosome 4q24, in a region showing recurrent microdeletions and copy-neutral loss of heterozygosity (CN-LOH) in patients with diverse myeloid malignancies3. Somatic TET2 mutations are frequently observed in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukemia (CMML), acute myeloid leukemias (AML) and secondary AML (sAML)4–12. We show here that TET2 mutations associated with myeloid malignancies compromise TET2 catalytic activity. Bone marrow samples from patients with TET2 mutations displayed uniformly low levels of 5-hmC in genomic DNA compared to bone marrow samples from healthy controls. Moreover, small hairpin RNA (shRNA)-mediated depletion of Tet2 in mouse haematopoietic precursors skewed their differentiation towards monocyte/macrophage lineages in culture. There was no significant difference in DNA methylation between bone marrow samples from patients with high 5-hmC versus healthy controls, but samples from patients with low 5-hmC showed hypomethylation relative to controls at the majority of differentially-methylated CpG sites. Our results demonstrate that TET2 is important for normal myelopoiesis, and suggest that disruption of TET2 enzymatic activity favours myeloid tumorigenesis. Measurement of 5-hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anti-cancer drugs.
doi:10.1038/nature09586
PMCID: PMC3003755  PMID: 21057493
5.  NPM1 Deletion Is Associated with Gross Chromosomal Rearrangements in Leukemia 
PLoS ONE  2010;5(9):e12855.
Background
NPM1 gene at chromosome 5q35 is involved in recurrent translocations in leukemia and lymphoma. It also undergoes mutations in 60% of adult acute myeloid leukemia (AML) cases with normal karyotype. The incidence and significance of NPM1 deletion in human leukemia have not been elucidated.
Methodology and Principal Findings
Bone marrow samples from 145 patients with myelodysplastic syndromes (MDS) and AML were included in this study. Cytogenetically 43 cases had isolated 5q-, 84 cases had 5q- plus other changes and 18 cases had complex karyotype without 5q deletion. FISH and direct sequencing investigated the NPM1 gene. NPM1 deletion was an uncommon event in the “5q- syndrome” but occurred in over 40% of cases with high risk MDS/AML with complex karyotypes and 5q loss. It originated from large 5q chromosome deletions. Simultaneous exon 12 mutations were never found. NPM1 gene status was related to the pattern of complex cytogenetic aberrations. NPM1 haploinsufficiency was significantly associated with monosomies (p<0.001) and gross chromosomal rearrangements, i.e., markers, rings, and double minutes (p<0.001), while NPM1 disomy was associated with structural changes (p = 0.013). Interestingly, in complex karyotypes with 5q- TP53 deletion and/or mutations are not specifically associated with NPM1 deletion.
Conclusions and Significance
NPM1/5q35 deletion is a consistent event in MDS/AML with a 5q-/-5 in complex karyotypes. NPM1 deletion and NPM1 exon 12 mutations appear to be mutually exclusive and are associated with two distinct cytogenetic subsets of MDS and AML.
doi:10.1371/journal.pone.0012855
PMCID: PMC2943467  PMID: 20877721
6.  Prognosis and Therapy When Acute Promyelocytic Leukemia and Other “Good Risk” Acute Myeloid Leukemias Occur as a Therapy-Related Myeloid Neoplasm 
Treatment for a pre-existing condition using chemotherapy, radiation therapy, immunosuppressive therapy, or a combination of these modalities may lead to the devastating complication of therapy-related myelodysplastic syndrome or acute myeloid leukemia (t-MDS/t-AML), collectively known as therapy-related myeloid neoplasm (t-MN). This disorder arises as a direct consequence of mutational events induced by the primary treatment. The outcomes for these patients have been historically poor compared to people who develop AML de novo. Currently comprising 10–20% of all cases of AML, t-MN is relatively resistant to conventional leukemia therapies, and is associated with s ort survival times. Median life expectancy from diagnosis is about 8–10 months in most series. Although the spectrum of cytogenetic abnormalities in t-AML is similar to AML de novo, the frequency of unfavorable cytogenetics, such as a complex karyotype or deletion or loss of chromosomes 5 and/or 7, is considerably higher in t-MN. Two distinct groups of patients with t-MN have been described. The more common subtype, seen in about 75% of patients, typically occurs 5–7 years after first exposure to alkylating agents or radiation, is often preceded by a myelodysplastic syndrome (MDS), and is frequently accompanied by clonal cytogenetic abnormalities such as the loss of all or part of chromosomes 5 or 7. Mutations of the P53 tumor suppressor gene are also common. The risk is related to total cumulative exposure over time to alkylating agents. In contrast, among individuals who develop t-AML after treatment with topoisomerase II inhibitors, the latency period to the development of t-AML is often only 1–3 years, antecedent MDS is rare, and gene rearrangements involving MLL at 11q23 or RUNX1/AML1 at 21q22 are common. It is now well recognized that APL and other subtypes of AML with balanced translocations sometimes occur as therapy-related myeloid neoplasms (t-MN) in patients who have previously received cytotoxic therapy or ionizing radiation therapy (RT). The most of this review will focus on these “good risk” leukemias, i.e. those with APL or inv(16)/t(16;16) or t(8;21).
doi:10.4084/MJHID.2011.032
PMCID: PMC3152454  PMID: 21869918
7.  Clonal evolution in relapsed acute myeloid leukemia revealed by whole genome sequencing 
Nature  2012;481(7382):506-510.
Summary
Most patients with acute myeloid leukemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level1,2. To determine the mutational spectrum associated with relapse, we sequenced the primary tumor and relapse genomes from 8 AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to precisely define clonality and clonal evolution patterns at relapse. Besides discovering novel, recurrently mutated genes (e.g. WAC, SMC3, DIS3, DDX41, and DAXX) in AML, we found two major clonal evolution patterns during AML relapse: 1) the founding clone in the primary tumor gained mutations and evolved into the relapse clone, or 2) a subclone of the founding clone survived initial therapy, gained additional mutations, and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific vs. primary tumor mutations in all 8 cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped in part by the chemotherapy that the patients receive to establish and maintain remissions.
doi:10.1038/nature10738
PMCID: PMC3267864  PMID: 22237025
8.  Mutations of E3 Ubiquitin Ligase Cbl Family Members Constitute a Novel Common Pathogenic Lesion in Myeloid Malignancies 
Journal of Clinical Oncology  2009;27(36):6109-6116.
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.
doi:10.1200/JCO.2009.23.7503
PMCID: PMC3040009  PMID: 19901108
9.  Cytoplasmic Localization of Nucleophosmin in Bone Marrow Blasts of Acute Myeloid Leukemia Patients is not Completely Concordant with NPM1 Mutation and is not Predictive of Prognosis 
Cancer  2009;115(20):4737-4744.
Background
NPM1 mutations are reported to predict a favorable prognosis in acute myeloid leukemia (AML) patients. Aberrant cytoplasmic localization of NPM protein is reported be a surrogate for NPM1 gene mutation.
Methods
Using immunohistochemical analysis (IHC), we assessed for NPM (clone 376) expression in formalin-fixed, formic acid-decalcified bone marrow biopsy specimens. DNA sequencing of the exon 12 of NPM1 gene was performed in 104 patients.
Results
The study included 252 AML patients: 192 de novo AML, 33 AML following a myelodysplastic syndrome or chronic myelomonocytic leukemia, and 27 therapy-related AML. The median age was 62 years and 115 patients were ≤ 60 years old. All patients received intensive chemotherapy. Cytoplasmic NPM was detected in 59 of 252 (23%) patients, including 48 of 192 (25%) de novo AML and 33 of 94 (35%) with a normal karyotype. DNA sequencing identified NPM1 mutations in 30 of 38 cases with cytoplasmic NPM and 10 of 66 cases with nuclear NPM. Cytoplasmic NPM was associated with young patient age (p=0.024), FLT3/ITD (p=0.005), CD34- negative blasts (p<0.001), high peripheral blood blast counts (p=0.041), and high serum albumin level (p=0.028). No statistical differences in overall or event-free survival were found on the basis of NPM localization. Similar results were obtained in patients ≤ 60 years old with normal karyotype and wild-type FLT3 (p=0.768).
Conclusions
Immunohistochemical assessment for NPM localization did not predict prognosis in this patient cohort. The discordance between immunohistochemistry and DNA sequencing results indicates that DNA sequencing cannot be replaced by IHC assessment.
doi:10.1002/cncr.24543
PMCID: PMC4199225  PMID: 19637342
AML; NPM1 mutation; immunohistochemical analysis; prognosis; FLT3/ITD
10.  RepSox Slows Decay of CD34+ Acute Myeloid Leukemia Cells and Decreases T Cell Immunoglobulin Mucin-3 Expression 
To facilitate development of therapies that target leukemic stem/progenitor cells (LPCs), in vitro ways to enhance the survival and immunogenicity of a patient's CD34+ acute myeloid leukemia (AML) cells were explored. RepSox was identified as a candidate cell-engineering tool because it slows in vitro decay of CD34+ AML cells (which often contain LPCs) and accelerates loss of the immune checkpoint receptor T cell immunoglobulin mucin-3 (Tim-3).
Despite initial response to therapy, most acute myeloid leukemia (AML) patients relapse. To eliminate relapse-causing leukemic stem/progenitor cells (LPCs), patient-specific immune therapies may be required. In vitro cellular engineering may require increasing the “stemness” or immunogenicity of tumor cells and activating or restoring cancer-impaired immune-effector and antigen-presenting cells. Leukapheresis samples provide the cells needed to engineer therapies: LPCs to be targeted, normal hematopoietic stem cells to be spared, and cancer-impaired immune cells to be repaired and activated. This study sought to advance development of LPC-targeted therapies by exploring nongenetic ways to slow the decay and to increase the immunogenicity of primary CD34+ AML cells. CD34+ AML cells generally displayed more colony-forming and aldehyde dehydrogenase activity than CD34− AML cells. Along with exposure to bone marrow stromal cells and low (1%–5%) oxygen, culture with RepSox (a reprogramming tool and inhibitor of transforming growth factor-β receptor 1) consistently slowed decline of CD34+ AML and myelodysplastic syndrome (MDS) cells. RepSox-treated AML cells displayed higher CD34, CXCL12, and MYC mRNA levels than dimethyl sulfoxide-treated controls. RepSox also accelerated loss of T cell immunoglobulin mucin-3 (Tim-3), an immune checkpoint receptor that impairs antitumor immunity, from the surface of AML and MDS cells. Our results suggest RepSox may reduce Tim-3 expression by inhibiting transforming growth factor-β signaling and slow decay of CD34+ AML cells by increasing CXCL12 and MYC, two factors that inhibit AML cell differentiation. By prolonging survival of CD34+ AML cells and reducing Tim-3, RepSox may promote in vitro immune cell activation and advance development of LPC-targeted therapies.
doi:10.5966/sctm.2013-0193
PMCID: PMC4073822  PMID: 24855276
Acute myeloid leukemia; Cancer stem cells; Immunotherapy; Immunogenicity; Tim-3; CD34+
11.  CD30 Expression in High-Risk Acute Myeloid Leukemia and Myelodysplastic Syndromes 
Background
We assessed for CD30 expression in patients with acute myeloid leukemia (AML) or high-grade myelodysplastic syndrome (MDS) to examine the possibility that anti-CD30 could be targeted therapy in these patients.
Methods
Multicolor flow cytometry immunophenotypic analysis was performed on bone marrow aspirates of 135 AML/MDS patients and peripheral blood (PB) samples in a subset of 33 patients. Immunohistochemistry was performed on BM aspirate clot specimens of 84 patients.
Results
The median patient age was 63 years (range, 13–92). One hundred and two (75%) patients had refractory or recurrent disease, and 68 (50%) had high-risk cytogenetics. Overall, the median percentage of blasts positive for CD30 was 14% (range, 0–91). Using an arbitrary 20% cutoff, 49 (36%) patients were considered to have CD30-expression. Monocytic cells, either mature or immature, were consistently negative for CD30. Therefore, CD30 expression was less in AML with monocytic differentiation (P=0.006). Patients with persistent disease who had been actively treated had a higher level of CD30 expression than untreated patients (p=0.031). In paired samples, CD30 expression was consistently higher in BM blasts than PB blasts (p=0.002). Immunohistochemistry demonstrated CD30 expression by myeloblasts in a subset of patients, but reactivity was generally weaker and focal compared.
Conclusions
CD30 is expressed by myeloblasts in a substantial subset of AML/MDS patients. As the study group was composed mostly of high-risk AML/MDS patients in whom very few treatment options are available, these data raise the possibility that anti-CD30- targeted therapy could be a potential option for this patient group.
doi:10.1016/j.clml.2012.12.006
PMCID: PMC4188388  PMID: 23313069
CD30; acute myeloid leukemia; myelodysplastic syndromes; flow cytometry; immunohistochemistry
12.  Clinical Effect of Point Mutations in Myelodysplastic Syndromes 
The New England journal of medicine  2011;364(26):2496-2506.
BACKGROUND
Myelodysplastic syndromes are clinically heterogeneous disorders characterized by clonal hematopoiesis, impaired differentiation, peripheral-blood cytopenias, and a risk of progression to acute myeloid leukemia. Somatic mutations may influence the clinical phenotype but are not included in current prognostic scoring systems.
METHODS
We used a combination of genomic approaches, including next-generation sequencing and mass spectrometry–based genotyping, to identify mutations in samples of bone marrow aspirate from 439 patients with myelodysplastic syndromes. We then examined whether the mutation status for each gene was associated with clinical variables, including specific cytopenias, the proportion of blasts, and overall survival.
RESULTS
We identified somatic mutations in 18 genes, including two, ETV6 and GNAS, that have not been reported to be mutated in patients with myelodysplastic syndromes. A total of 51% of all patients had at least one point mutation, including 52% of the patients with normal cytogenetics. Mutations in RUNX1, TP53, and NRAS were most strongly associated with severe thrombocytopenia (P<0.001 for all comparisons) and an increased proportion of bone marrow blasts (P<0.006 for all comparisons). In a multivariable Cox regression model, the presence of mutations in five genes retained independent prognostic significance: TP53 (hazard ratio for death from any cause, 2.48; 95% confidence interval [CI], 1.60 to 3.84), EZH2 (hazard ratio, 2.13; 95% CI, 1.36 to 3.33), ETV6 (hazard ratio, 2.04; 95% CI, 1.08 to 3.86), RUNX1 (hazard ratio, 1.47; 95% CI, 1.01 to 2.15), and ASXL1 (hazard ratio, 1.38; 95% CI, 1.00 to 1.89).
CONCLUSIONS
Somatic point mutations are common in myelodysplastic syndromes and are associated with specific clinical features. Mutations in TP53, EZH2, ETV6, RUNX1, and ASXL1 are predictors of poor overall survival in patients with myelodysplastic syndromes, independently of established risk factors. (Funded by the National Institutes of Health and others.)
doi:10.1056/NEJMoa1013343
PMCID: PMC3159042  PMID: 21714648
13.  Recurrent DNMT3A Mutations in Patients with Myelodysplastic Syndromes 
Alterations in DNA methylation have been implicated in the pathogenesis of myelodysplastic syndromes (MDS), although the underlying mechanism remains largely unknown. Methylation of CpG dinucleotides is mediated by DNA methyltransferases, including DNMT1, DNMT3A, and DNMT3B. DNMT3A mutations have recently been reported in patients with de novo acute myeloid leukemia (AML), providing a rationale for examining the status of DNMT3A in MDS samples. Here, we report the frequency of DNMT3A mutations in patients with de novo MDS, and their association with secondary AML. We sequenced all coding exons of DNMT3A using DNA from bone marrow and paired normal cells from 150 patients with MDS and identified 13 heterozygous mutations with predicted translational consequences in 12/150 patients (8.0%). Amino acid R882, located in the methyltransferase domain of DNMT3A, was the most common mutation site, accounting for 4/13 mutations. DNMT3A mutations were expressed in the majority of cells in all tested mutant samples regardless of blast counts, suggesting that DNMT3A mutations occur early in the course of MDS. Patients with DNMT3A mutations had worse overall survival compared to patients without DNMT3A mutations (p=0.005) and more rapid progression to AML (p=0.007), suggesting that DNMT3A mutation status may have prognostic value in de novo MDS.
doi:10.1038/leu.2011.44
PMCID: PMC3202965  PMID: 21415852
myelodysplastic syndrome; DNMT3A; mutation
14.  Recurrent DNMT3A R882 Mutations in Chinese Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome 
PLoS ONE  2011;6(10):e26906.
Somatic mutations of DNMT3A gene have recently been reported in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We examined the entire coding sequences of DNMT3A gene by high-resolution melting analysis and sequencing in Chinese patients with myeloid malignancies. R882 mutations were found in 12/182 AML and in 4/51 MDS, but not in either 79 chronic myeloid leukemia (CML), or 57 myeloproliferative neoplasms (MPNs), or 4 chronic monomyelocytic leukemia. No other DNMT3A mutations were detected in all patients. R882 mutations were associated with old age and more frequently present in monoblastic leukemia (M4 and M5, 7/52) compared to other subtypes (5/130). Furthermore, 14/16 (86.6%) R882 mutations were observed in patients with normal karyotypes. The overall survival of mutated MDS patients was shorter than those without mutation (median 9 and 25 months, respectively). We conclude that DNMT3A R882 mutations are recurrent molecular aberrations in AML and MDS, and may be an adverse prognostic event in MDS.
doi:10.1371/journal.pone.0026906
PMCID: PMC3204995  PMID: 22066015
15.  The Frequency and Clinical Significance of IDH1 Mutations in Chinese Acute Myeloid Leukemia Patients 
PLoS ONE  2013;8(12):e83334.
Objective
Mutations in the gene encoding isocitrate dehydrogenease 1 (IDH1) occur in various hematopoietic tumors including acute myeloid leukemia (AML), myeloproliferative neoplasms and myelodysplastic syndromes. IDH1 mutations are significant in both diagnosis and prognosis of these conditions. In the present study we determined the prevalence and clinical significance of IDH1 mutations in 349 samples from newly diagnosed AML patients.
Results
Of the 349 AML patient specimens analyzed, 35 (10.03%) were found to have IDH1 mutations including 4 IDH1 R132 mutations and 31 non-R132 mutations. IDH1 non-R132 mutations were largely concentrated within AML-M1 (35.72%, p<0.01). We identified five IDH1 mutations that were novel to AML: (1) c.299 G>A, p.R100Q; (2) c.311G>T, p.G104V; (3) c.322T>C, p.F108L; (4) c.356G>A, p.R119Q; and (5) c.388A>G, p.I130V. In addition, we identified three IDH1 mutations that were previously described in AML. The frequency of IDH1 mutations in AML patients with normal karyotype was 9.9%. IDH1 non-R132 mutations were concurrent with mutations in FLT3-ITD (p<0.01), CEBPA (p<0.01), and NRAS (p<0.01), as well as the overexpression of MN1 (p<0.01) and WT1(p<0.01). The overall survival (OS) in the patients with IDH1 non-R132 mutations compared to patients without IDH1 mutations don't reach statistically significance (median 521 days vs median: not reached; n.s.).
Conclusion
IDH1 non-R132 mutations occurred frequently in newly diagnosed adult Chinese AML patients, and these mutations were associated with genetic alterations. The OS was not influenced by IDH1 non-R132 mutations in the present study.
doi:10.1371/journal.pone.0083334
PMCID: PMC3869765  PMID: 24376688
16.  U2AF1 Mutations in Chinese Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome 
PLoS ONE  2012;7(9):e45760.
Somatic mutations of U2AF1 gene have recently been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we analyzed the frequency and clinical impact of U2AF1 mutations in a cohort of 452 Chinese patients with myeloid neoplasms. Mutations in U2AF1 were found in 2.5% (7/275) of AML and 6.3% (6/96) of MDS patients, but in none of 81 CML. All mutations were heterozygous missense mutations affecting codon S34 or Q157. There was no significant association of U2AF1 mutation with blood parameters, FAB subtypes, karyotypes and other gene mutations in AML. The overall survival (OS) of AML patients with U2AF1 mutation (median 3 months) was shorter than those without mutation (median 7 months) (P = 0.035). No difference in the OS was observed between MDS patients with and without U2AF1 mutations. Our data show that U2AF1 mutation is a recurrent event at a low frequency in AML and MDS.
doi:10.1371/journal.pone.0045760
PMCID: PMC3446943  PMID: 23029227
17.  DNMT3A Mutations in Patients with Acute Myeloid Leukemia in South Brazil 
Advances in Hematology  2012;2012:697691.
Acute myeloid leukemia (AML) is a complex and heterogeneous hematopoietic tissue neoplasm. Several molecular markers have been described that help to classify AML patients into risk groups. DNA methyltransferase 3A (DNMT3A) gene mutations have been recently identified in about 22% of AML patients and associated with poor prognosis as an independent risk factor. Our aims were to determine the frequency of somatic mutations in the gene DNMT3A and major chromosomal translocations in a sample of patients with AML. We investigated in 82 samples of bone marrow from patients with AML for somatic mutations in DNMT3A gene by sequencing and sought major fusion transcripts by RT-PCR. We found mutations in the DNMT3A gene in 6 patients (8%); 3 were type R882H. We found fusion transcripts in 19 patients, namely, AML1/ETO (n = 5; 6.1%), PML/RARα (n = 12; 14.6%), MLL/AF9 (0; 0%), and CBFβ/MYH11 (n = 2; 2.4%). The identification of recurrent mutations in the DNMT3A gene and their possible prognostic implications can be a valuable tool for making treatment decisions. This is the first study on the presence of somatic mutations of the DNMT3A gene in patients with AML in Brazil. The frequency of these mutations suggests a possible ethnogeographic variation.
doi:10.1155/2012/697691
PMCID: PMC3502761  PMID: 23193409
18.  Gene expression profiling of the DNMT3A R882 mutation in acute leukemia 
Oncology Letters  2013;6(1):268-274.
DNA methyltransferase 3A (DNMT3A) is one of two human de novo DNA methyltransferases essential for the regulation of gene expression. DNMT3A mutations and deletions have been previously observed in acute myeloid leukemia (AML), myelodysplastic sydromes and myeloproliferative neoplasms. However, the involvement of DNMT3A in acute lymphoblastic leukemia (ALL) has rarely been reported. In the present study, PCR and direct sequencing was performed to analyze mutations of DNMT3A amino acid residue 882 in 99 acute leukemia patients, including 57 AML patients, 41 ALL patients and a single biphenotypic acute leukemia (BAL) patient. DNMT3A expression was detected in mono-nuclear cells of the bone marrow in these patients and in normal individuals using real-time quantitative polymerase chain reaction, and 17.5% (10/57) of AML patients were found to exhibit DNMT3A mutations. Four missense mutations were observed in the DNMT3A-mutated AML patients, including R882 mutations and a novel single nucleotide polymorphism resulting in the M880V amino acid substitution. However, the ALL and BAL patients were not found to exhibit DNMT3A mutations. The DNMT3A expression levels in the AML patients were significantly higher compared with those of the ALL patients or normal controls. The reduced expression levels of DNMT3A were associated with a significantly lower complete remission rate in the AML patients. However, in the ALL patients, no statistical significance was identified. The results of the present study indicate that DNMT3A may play varying roles in the regulation of DNA methylation in AML and ALL.
doi:10.3892/ol.2013.1347
PMCID: PMC3742503  PMID: 23946816
DNMT3A; R882 mutations; acute myeloid leukemia; acute lymphoblastic leukemia; gene expression
19.  Clinical and Pathologic Features of Secondary Acute Promyelocytic Leukemia 
Acute promyelocytic leukemia (APL) is a relatively common form of acute myeloid leukemia (AML) that has an excellent prognosis. In contrast, secondary acute myeloid leukemias, including therapy-related AML and AML with myelodysplasia-related changes, have a relatively poor prognosis. We identified 9 cases of APL at our institution in which there was a history of chemotherapy, radiotherapy, chronic immunosuppression, or antecedent myelodysplastic syndrome. The clinical and pathologic findings in these cases of secondary APL were compared with the clinical and pathologic findings in cases of de novo APL. We found that secondary and de novo APL had abnormal promyelocytes with similar morphologic and immunophenotypic features, comparable cytogenetic findings, comparable rates of FMS-like tyrosine kinase mutations, and similar rates of recurrent disease and death. These data suggest that secondary APL is similar to de novo APL and, thus, should be considered distinct from other secondary acute myeloid neoplasms.
doi:10.1309/AJCPE0MV0YTWLUUE
PMCID: PMC3578661  PMID: 22338051
Acute promyelocytic leukemia; APL; Therapy-related acute myeloid leukemia; Therapy-related myeloid neoplasm; Acute myeloid leukemia with myelodysplasia-related changes; Flow cytometry
20.  A case of acute myeloid leukemia (AML) with an unreported combination of chromosomal abnormalities: gain of isochromosome 5p, tetrasomy 8 and unbalanced translocation der(19)t(17;19)(q23;p13) 
Background
Acute myeloid leukemia (AML) comprises a spectrum of myeloid malignancies which are often associated with distinct chromosomal abnormalities, and the analysis of such abnormalities provides us with important information for disease classification, treatment selection and prognosis. Some chromosomal abnormalities albeit recurrent are rare such as tetrasomy 8 or isochromosome 5p. In addition, erratic chromosomal rearrangements may occur in AML, sometimes unbalanced and also accompanied by other abnormalities. Knowledge on the contribution of rare abnormalities to AML disease, progression and prognosis is limited.
Here we report a unique case of acute monoblastic leukemia with gain of i(5)(p10), tetrasomy 8, an unbalanced translocation der(19)t(17;19)(q23;p13.3) and mutated NPM1.
Results
Bone marrow cells were examined by conventional karyotyping, fluorescence in situ hybridization (FISH) and mutation analysis at diagnosis and follow-up. At diagnosis we detected trisomy 8, an unbalanced translocation der(19)t(17;19)(q23;p13.3) and mutated NPM1. During the course of the disease we observed clonal evolution with gain of i(5)(p10), tetrasomy 8 and eventually duplication of der(19)t(17;19)(q23;p13.3). By using the der(19)t(17;19) as clonal marker, we found that i(5)(p10) and tetrasomy 8 were secondary genetic events and that tetrasomy 8 had clonally evolved from trisomy 8.
Conclusions
This case of acute monoblastic leukemia presents a combination of rare chromosomal abnormalities including the unbalanced translocation der(19)t(17;19)(q23;p13.3), hitherto un-reported in AML. In addition, our case supports the hypothesis of a step-wise clonal evolution from trisomy 8 to tetrasomy 8 in AML. Reporting and collecting data of rare chromosomal abnormalities will add information to AML disease, progression and prognosis, and may eventually translate to improved patient management.
doi:10.1186/1755-8166-6-40
PMCID: PMC3852770  PMID: 24079663
AML; Acute monoblastic leukemia; Chromosomal abnormalities; Fluorescence in situ hybridization (FISH); Multicolor FISH; Tetrasomy 8; Isochromosome 5p; Clonal evolution
21.  RECURRENT MUTATIONS IN THE U2AF1 SPLICING FACTOR IN MYELODYSPLASTIC SYNDROMES 
Nature Genetics  2011;44(1):53-57.
Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole genome sequencing to perform an unbiased comprehensive screen to discover all the somatic mutations in a sAML sample and genotyped these loci in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (S34) in U2AF1 was recurrently mutated in 13/150 (8.7%) de novo MDS patients, with suggestive evidence of an associated increased risk of progression to sAML. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3′ end of introns and mutations are located in highly conserved zinc fingers in U2AF11,2. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This novel, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.
doi:10.1038/ng.1031
PMCID: PMC3247063  PMID: 22158538
22.  Familial Aggregation of Acute Myeloid Leukemia and Myelodysplastic Syndromes 
Journal of Clinical Oncology  2011;30(2):179-183.
Purpose
Apart from rare pedigrees with multiple cases of acute myeloid leukemia (AML), there is limited data on familial aggregation of AML and myelodysplastic syndromes (MDSs) in the population.
Patients and Methods
Swedish population-based registry data were used to evaluate risk of AML, MDS, and other malignancies among 24,573 first-degree relatives of 6,962 patients with AML and 1,388 patients with MDS compared with 106,224 first-degree relatives of matched controls. We used a marginal survival model to calculate familial aggregation.
Results
AML and/or MDS did not aggregate significantly in relatives of patients with AML. There was a modest risk ratio (RR, 1.3; 95% CI, 0.9 to 1.8) in myeloproliferative/myeloid malignancies combined. The risks for any hematologic or any solid tumor were modestly but significantly increased. Relatives of patients with MDS did not show an increased risk for any hematologic tumors. In contrast, we found a significantly increased risk (RR, 6.5; 95% CI, 1.1 to 38.0) of AML/MDS and of all myeloid malignancies combined (RR, 3.1; 95% CI, 1.0 to 9.8) among relatives of patients diagnosed at younger than age 21 years.
Conclusion
We did not find evidence for familial aggregation of the severe end of the spectrum of myeloid malignancies (AML and MDS). The risks of myeloproliferative neoplasms were modestly increased with trends toward significance, suggesting a possible role of inheritance. In contrast, although limited in sample size, relatives of young patients with AML were at increased risk of AML/MDS, suggesting that germline genes may play a stronger role in these patients. The increased risk of all hematologic malignancies and of solid tumors among relatives of patients with AML suggests that genes for malignancy in general and/or other environmental factors may be shared.
doi:10.1200/JCO.2011.37.1203
PMCID: PMC3255562  PMID: 22162584
23.  Heritable GATA2 Mutations Associated with Familial Myelodysplastic Syndrome and Acute Myeloid Leukemia 
Nature genetics  2011;43(10):1012-1017.
We report the discovery of the GATA2 gene as a new myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) predisposition gene. We found the same, novel heterozygous c.1061C>T (p.Thr354Met) missense mutation in the GATA2 transcription factor gene segregating with the multigenerational transmission of MDS/AML in three families, and a GATA2 c.1063_1065delACA (p.Thr355del) mutation at an adjacent codon in a fourth MDS/AML family. The mutations reside within the second zinc finger of GATA2 which mediates DNA-binding and protein-protein interactions. We show differential effects of the mutants on transactivation of target genes, cellular differentiation, apoptosis and global gene expression. Identification of such predisposing genes to familial forms of MDS and AML is critical for more effective diagnosis and prognosis, counselling, selection of related bone marrow transplant donors, and development of therapies.
doi:10.1038/ng.913
PMCID: PMC3184204  PMID: 21892162
24.  Genetic analysis of leukemic transformation of chronic myeloproliferative neoplasms 
Cancer research  2010;70(2):447-452.
The genetic events which contribute to transformation of myeloproliferative neoplasms (MPN) to acute myeloid leukemia (AML) are not well characterized. We investigated the role of JAK2, TET2, ASXL1, and IDH1 mutations in leukemic transformation of MPNs through mutational analysis of 63 patients with AML secondary to a preexisting MPN (sAML). We identified frequent TET2 (26.3%), ASXL1 (19.3%), IDH1 (9.5%), and JAK2 (36.8%) mutations in sAML; all possible mutational combinations of these genes were observed. Analysis of 14 patients for which paired samples from MPN and sAML were available demonstrated TET2 mutations were frequently acquired at leukemic transformation (6/14=43%). In contrast, ASXL1 mutations were almost always detected in both the MPN and AML clones from individual patients. A case was also observed where TET2 and ASXL1 mutations were found before the patient acquired a JAK2 mutation or developed clinical evidence of MPN. We conclude that mutations in TET2, ASXL1, and IDH1 are common in sAML derived from a pre-existing MPN. Although TET2/ASXL1 mutations may precede acquisition of JAK2 mutations by the MPN clone, mutations in TET2, but not ASXL1, are commonly acquired at the time of leukemic transformation. These data suggest the mutational order of events in MPN and sAML varies in different patients, and that TET2 and ASXL1 mutations have distinct roles in MPN pathogenesis and leukemic transformation. The presence of sAML with no pre-existing JAK2/TET2/ASXL1/IDH1 mutations indicates the existence of other mutations necessary for leukemic transformation.
doi:10.1158/0008-5472.CAN-09-3783
PMCID: PMC2947340  PMID: 20068184
25.  Recurring Mutations Found by Sequencing an Acute Myeloid Leukemia Genome 
The New England journal of medicine  2009;361(11):1058-1066.
BACKGROUND
The full complement of DNA mutations that are responsible for the pathogenesis of acute myeloid leukemia (AML) is not yet known.
METHODS
We used massively parallel DNA sequencing to obtain a very high level of coverage (approximately 98%) of a primary, cytogenetically normal, de novo genome for AML with minimal maturation (AML-M1) and a matched normal skin genome.
RESULTS
We identified 12 acquired (somatic) mutations within the coding sequences of genes and 52 somatic point mutations in conserved or regulatory portions of the genome. All mutations appeared to be heterozygous and present in nearly all cells in the tumor sample. Four of the 64 mutations occurred in at least 1 additional AML sample in 188 samples that were tested. Mutations in NRAS and NPM1 had been identified previously in patients with AML, but two other mutations had not been identified. One of these mutations, in the IDH1 gene, was present in 15 of 187 additional AML genomes tested and was strongly associated with normal cytogenetic status; it was present in 13 of 80 cytogenetically normal samples (16%). The other was a nongenic mutation in a genomic region with regulatory potential and conservation in higher mammals; we detected it in one additional AML tumor. The AML genome that we sequenced contains approximately 750 point mutations, of which only a small fraction are likely to be relevant to pathogenesis.
CONCLUSIONS
By comparing the sequences of tumor and skin genomes of a patient with AML-M1, we have identified recurring mutations that may be relevant for pathogenesis.
doi:10.1056/NEJMoa0903840
PMCID: PMC3201812  PMID: 19657110

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