Non-del(5q) myelodysplastic syndromes-associated loci detected by SNP-array genome-wide association meta-analysis.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell malignancies. Known predisposing factors to adult MDS include rare germline mutations, cytotoxic therapy, age-related clonal hematopoiesis, and autoimmune or chronic inflammatory disorders. To date, no published studies characterizing MDS-associated germline susceptibility polymorphisms exist. We performed a genome-wide association study of 2 sample sets (555 MDS cases vs 2964 control subjects; 352 MDS cases vs 2640 control subjects) in non-del(5q) MDS cases of European genomic ancestry. Meta-analysis identified 8 MDS-associated loci at 1q31.1 (PLA2G4A), 3p14.1 (FAM19A4), 5q21.3 (EFNA5), 6p21.33, 10q23.1 (GRID1), 12q24.32, 15q26.1, and 20q13.12 (EYA2) that approached genome-wide significance. Gene expression for 5 loci that mapped within or near genes was significantly upregulated in MDS bone marrow cells compared with those of control subjects (P < .01). Higher PLA2G4A expression and lower EYA2 expression were associated with poorer overall survival (P = .039 and P = .037, respectively). Higher PLA2G4A expression is associated with mutations in NRAS (P < .001), RUNX1 (P = .012), ASXL1 (P = .007), and EZH2 (P = .038), all of which are known to contribute to MDS development. EYA2 expression was an independently favorable risk factor irrespective of age, sex, and Revised International Scoring System score (relative risk, 0.67; P = .048). Notably, these genes have regulatory roles in innate immunity, a critical driver of MDS pathogenesis. EYA2 overexpression induced innate immune activation, whereas EYA2 inhibition restored colony-forming potential in primary MDS cells indicative of hematopoietic restoration and possible clinical relevance. In conclusion, among 8 suggestive MDS-associated loci, 5 map to genes upregulated in MDS with functional roles in innate immunity and potential biological relevance to MDS.

FLT3 and JAK2 Mutations in Acute Myeloid Leukemia Promote Interchromosomal Homologous Recombination and the Potential for Copy Neutral Loss of Heterozygosity.

Acquired copy neutral LOH (CN-LOH) is a frequent occurrence in myeloid malignancies and is often associated with resistance to standard therapeutic modalities and poor survival. Here, we show that constitutive signaling driven by mutated FLT3 and JAK2 confers interchromosomal homologous recombination (iHR), a precedent for CN-LOH. Using a targeted recombination assay, we determined significant iHR activity in internal tandem duplication FLT3 (FLT3-ITD) and JAK2V617F-mutated cells. Sister chromatid exchanges, a surrogate measure of iHR, was significantly elevated in primary FLT3-ITD normal karyotype acute myeloid leukemia (NK-AML) compared with wild-type FLT3 NK-AML. HR was harmonized to S phase of the cell cycle to repair broken chromatids and prevent iHR. Increased HR activity in G0 arrested primary FLT3-ITD NK-AML in contrast to wild-type FLT3 NK-AML. Cells expressing mutated FLT3-ITD demonstrated a relative increase in mutation frequency as detected by thymidine kinase (TK) gene mutation assay. Moreover, resistance was associated with CN-LOH at the TK locus. Treatment of FLT3-ITD- and JAK2V617F-mutant cells with the antioxidant N-acetylcysteine diminished reactive oxygen species (ROS), restoring iHR and HR levels. Our findings show that mutated FLT3-ITD and JAK2 augment ROS production and HR, shifting the cellular milieu toward illegitimate recombination events such as iHR and CN-LOH. Therapeutic reduction of ROS may thus prevent leukemic progression and relapse in myeloid malignancies. Cancer Res; 77(7); 1697-708. ©2017 AACR.

CSNK1A1 mutations and isolated del(5q) abnormality in myelodysplastic syndrome: a retrospective mutational analysis.

BACKGROUND: A mechanism for clonal growth advantage in isolated del(5q) disease remains elusive. CSNK1A1 resides on the critically deleted region, and deletion of this gene has been shown in mouse knockout and transplantation studies to produce some characteristics of bone marrow failure, including a proliferative advantage. We aimed to establish the frequency, nature, and clinical association of CSNK1A1 mutations in patients with myelodysplastic syndrome and associated myeloid neoplasms. METHODS: Between June 1, 2004, and May 31, 2014, in King's College (London, UK), we did whole-exome sequencing of five patients with isolated del(5q) followed by targeted screening for CSNK1A1 mutations and 20 myelodysplastic syndrome-associated mutations in 245 additional patients with myeloid neoplasms. All patients met present WHO diagnostic criteria for myelodysplastic syndrome and other related myeloid neoplasms. FINDINGS: 39 (16%) of 250 patients with myeloid neoplasms had isolated del(5q), of whom seven (18%) had CSNK1A1 mutations. All these mutations were missense and presented in a highly conserved region that is implicated in ATP catalysis. Serial sampling and response to lenalidomide treatment showed that CSNK1A1 mutations were highly associated with the del(5q) clone. Only one patient with a CSNK1A1 mutation showed complete cytogenetic response to lenalidomide. Four (57%) of the seven patients carrying a CSNK1A1 mutation showed disease progression coupled with an increase in mutant allele burden (all four were on lenalidomide). We detected coexisting myelodysplastic syndrome-related gene mutations in patients with CSNK1A1 mutations, including TP53. INTERPRETATION: Similar to the effect of TP53 mutations on progression of del(5q) abnormality, mutant CSNK1A1 also gives rise to a poor prognosis in del(5q) abnormality, for which a coupled increase in P53 activation is suggested. CSNK1A1 mutations in del(5q) disease are important in the context of therapeutic manipulation and need incorporation into future prospective studies. FUNDING: Leukaemia and Lymphoma Research.

Somatic mutations identify a subgroup of aplastic anemia patients who progress to myelodysplastic syndrome.

The distinction between acquired aplastic anemia (AA) and hypocellular myelodysplastic syndrome (hMDS) is often difficult, especially nonsevere AA. We postulated that somatic mutations are present in a subset of AA, and predict malignant transformation. From our database, we identified 150 AA patients with no morphological evidence of MDS, who had stored bone marrow (BM) and constitutional DNA. We excluded Fanconi anemia, mutations of telomere maintenance, and a family history of BM failure (BMF) or cancer. The initial cohort of 57 patients was screened for 835 known genes associated with BMF and myeloid cancer; a second cohort of 93 patients was screened for mutations in ASXL1, DNMT3A, BCOR, TET2, and MPL. Somatic mutations were detected in 19% of AA, and included ASXL1 (n = 12), DNMT3A (n = 8) and BCOR (n = 6). Patients with somatic mutations had a longer disease duration (37 vs 8 months, P < .04), and shorter telomere lengths (median length, 0.9 vs 1.1, P < .001), compared with patients without mutations. Somatic mutations in AA patients with a disease duration of >6 months were associated with a 40% risk of transformation to MDS (P < .0002). Nearly one-fifth of AA patients harbor mutations in genes typically seen in myeloid malignancies that predicted for later transformation to MDS.

Recent advances in understanding the molecular pathogenesis of myelodysplastic syndromes.

The advent of novel genomic sequencing technologies has aided the identification of somatically acquired genetic abnormalities up to 80% of myelodysplastic syndrome (MDS) patients. Novel recurrent genetic mutations in pathways such as RNA splicing, DNA methylation and histone modification and cohesion complexes, underscore the molecular heterogeneity seen in this clinically varied disease. Functional studies to establish a causative link between genomic aberrations and MDS biogenesis are still in their infancy. The deluge of this molecular information, once validated on a larger cohort, will be incorporated into prognostic systems and clinical practise, and also hopefully aid in MDS therapeutics, especially in guiding targeted therapy.

Utility of peripheral blood for cytogenetic and mutation analysis in myelodysplastic syndrome.

Recent studies have shown that more than 80% of bone marrow (BM) samples from patients with myelodysplastic syndrome (MDS) harbor somatic mutations and/or genomic aberrations, which are of diagnostic and prognostic importance. We investigated the potential use of peripheral blood (PB) and serum to identify and monitor BM-derived genetic markers using high-resolution single nucleotide polymorphism array (SNP-A) karyotyping and parallel sequencing of 22 genes frequently mutated in MDS. This pilot study showed a 100% SNP-A karyotype concordance and a 97% mutation concordance between the BM and PB. In contrast, mutation analysis using Sanger sequencing of PB and serum-derived DNA showed only 65% and 42% concordance to BM, respectively. Our results show the potential utility of PB as a surrogate for BM for MDS patients, thus avoiding the need for repeated BM aspirates particularly in elderly patients and those with fibrotic or hypocellular marrows.

Microsatellite instability induced mutations in DNA repair genes CtIP and MRE11 confer hypersensitivity to poly (ADP-ribose) polymerase inhibitors in myeloid malignancies.

Inactivation of the DNA mismatch repair pathway manifests as microsatellite instability, an accumulation of mutations that drives carcinogenesis. Here, we determined whether microsatellite instability in acute myeloid leukemia and myelodysplastic syndrome correlated with chromosomal instability and poly (ADP-ribose) polymerase (PARP) inhibitor sensitivity through disruption of DNA repair function. Acute myeloid leukemia cell lines (n=12) and primary cell samples (n=18), and bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients (n=63) were profiled for microsatellite instability using fluorescent fragment polymerase chain reaction. PARP inhibitor sensitivity was performed using cell survival, annexin V staining and cell cycle analysis. Homologous recombination was studied using immunocytochemical analysis. SNP karyotyping was used to study chromosomal instability. RNA silencing, Western blotting and gene expression analysis was used to study the functional consequences of mutations. Acute myeloid leukemia cell lines (4 of 12, 33%) and primary samples (2 of 18, 11%) exhibited microsatellite instability with mono-allelic mutations in CtIP and MRE11. These changes were associated with reduced expression of mismatch repair pathway components, MSH2, MSH6 and MLH1. Both microsatellite instability positive primary acute myeloid leukemia samples and cell lines demonstrated a downregulation of homologous recombination DNA repair conferring marked sensitivity to PARP inhibitors. Similarly, bone marrow mononuclear cells from 11 of 56 (20%) patients with de novo high-risk myelodysplastic syndrome exhibited microsatellite instability. Significantly, all 11 patients with microsatellite instability had cytogenetic abnormalities with 4 of them (36%) possessing a mono-allelic microsatellite mutation in CtIP. Furthermore, 50% reduction in CtIP expression by RNA silencing also down-regulated homologous recombination DNA repair responses conferring PARP inhibitor sensitivity, whilst CtIP differentially regulated the expression of homologous recombination modulating RecQ helicases, WRN and BLM. In conclusion, microsatellite instability dependent mutations in DNA repair genes, CtIP and MRE11 are detected in myeloid malignancies conferring hypersensitivity to PARP inhibitors. Microsatellite instability is significantly correlated with chromosomal instability in myeloid malignancies.

Spliceosome mutations exhibit specific associations with epigenetic modifiers and proto-oncogenes mutated in myelodysplastic syndrome.

The recent identification of acquired mutations in key components of the spliceosome machinery strongly implicates abnormalities of mRNA splicing in the pathogenesis of myelodysplastic syndromes. However, questions remain as to how these aberrations functionally combine with the growing list of mutations in genes involved in epigenetic modification and cell signaling/transcription regulation identified in these diseases. In this study, amplicon sequencing was used to perform a mutation screen in 154 myelodysplastic syndrome patients using a 22-gene panel, including commonly mutated spliceosome components (SF3B1, SRSF2, U2AF1, ZRSR2), and a further 18 genes known to be mutated in myeloid cancers. Sequencing of the 22-gene panel revealed that 76% (n=117) of the patients had mutations in at least one of the genes, with 38% (n=59) having splicing gene mutations and 49% (n=75) patients harboring more than one gene mutation. Interestingly, single and specific epigenetic modifier mutations tended to coexist with SF3B1 and SRSF2 mutations (P<0.03). Furthermore, mutations in SF3B1 and SRSF2 were mutually exclusive to TP53 mutations both at diagnosis and at the time of disease transformation. Moreover, mutations in FLT3, NRAS, RUNX1, CCBL and C-KIT were more likely to co-occur with splicing factor mutations generally (P<0.02), and SRSF2 mutants in particular (P<0.003) and were significantly associated with disease transformation (P<0.02). SF3B1 and TP53 mutations had varying impacts on overall survival with hazard ratios of 0.2 (P<0.03, 95% CI, 0.1-0.8) and 2.1 (P<0.04, 95% CI, 1.1-4.4), respectively. Moreover, patients with splicing factor mutations alone had a better overall survival than those with epigenetic modifier mutations, or cell signaling/transcription regulator mutations with and without coexisting mutations of splicing factor genes, with worsening prognosis (P<0.001). These findings suggest that splicing factor mutations are maintained throughout disease evolution with emerging oncogenic mutations adversely affecting patients' outcome, implicating spliceosome mutations as founder mutations in myelodysplastic syndromes.

TP53 mutations in myelodysplastic syndrome are strongly correlated with aberrations of chromosome 5, and correlate with adverse prognosis.

This study aimed to determine the incidence/prognostic impact of TP53 mutation in 318 myelodysplastic syndrome (MDS) patients, and to correlate the changes to cytogenetics, single nucleotide polymorphism array karyotyping and clinical outcome. The median age was 65 years (17-89 years) and median follow-up was 45 months [95% confidence interval (CI) 27-62 months]. TP53 mutations occurred in 30 (9.4%) patients, exclusively in isolated del5q (19%) and complex karyotype (CK) with -5/5q-(72%), correlated with International Prognostic Scoring System intermediate-2/high, TP53 protein expression, higher blast count and leukaemic progression. Patients with mutant TP53 had a paucity of mutations in other genes implicated in myeloid malignancies. Median overall survival of patients with TP53 mutation was shorter than wild-type (9 versus 66 months, P < 0.001) and it retained significance in multivariable model (Hazard Ratio 3.8, 95%CI 2.3-6.3,P < 0.001). None of the sequentially analysed samples showed a disappearance of the mutant clone or emergence of new clones, suggesting an early occurrence of TP53 mutations. A reduction in mutant clone correlated with response to 5-azacitidine, however clones increased in non-responders and persisted at relapse. The adverse impact of TP53 persists after adjustment for cytogenetic risk and is of practical importance in evaluating prognosis. The relatively common occurrence of these mutations in two different prognostic spectrums of MDS, i.e. isolated 5q- and CK with -5/5q-, possibly implies two different mechanistic roles for TP53 protein.

The effects of 5-azacytidine on the function and number of regulatory T cells and T-effectors in myelodysplastic syndrome.

Expansion of regulatory T cells occurs in high-risk myelodysplastic syndrome and correlates with a poor prognosis. DNA methyltransferase inhibitors, particularly 5-azacytidine, have been shown to increase the survival of patients with high-risk myelodysplastic syndrome. It is not entirely clear whether this improvement in patients' survival is related to the effects of DNA methyltransferase inhibitors on the immune system and/or the direct effect of these drugs on the dysplastic clone. In this study we investigated the effect of 5-azacytidine on the function and proliferation capability of regulatory T cells and T-helper cells. The number and function of CD4(+) T-cell subsets in 68 patients with intermediate-2/high-risk myelodysplastic syndrome were serially assessed at diagnosis and following treatment. The in-vitro effects of 5-azacytidine on CD4(+) T-cell subsets isolated from both healthy donors and patients with myelodysplastic syndrome were also investigated. The number of peripheral blood regulatory T cells was significantly higher in myelodysplastic syndrome patients than in healthy donors and responders to treatment (P=0.01). The absolute numbers of T-helper 1 and T-helper 2, but not T-helper 17, cells were significantly reduced following 12 months of treatment (P=0.03, P=0.03). The in vitro addition of 5-azacytidine to CD4(+) T cells reduced the proliferative capacity of regulatory T cells (P=0.03). In addition, the 5-azacytidine-treated regulatory T cells had reduced suppressive function and produced larger amounts of interleukin-17. The FOXP3 expression in 5-azacyti-dine-treated T-effectors was also increased. Interestingly, these FOXP3(+)/interleukin-17(+) cells originated mainly from effector T cells rather than regulatory T cells. Our data suggest that 5-azacytidine has profound effects on CD4(+) T cells, which correlate with disease status after treatment. Furthermore, despite the demethylation of the FOXP3 promoter and increased FOXP3 expression following 5-azacytidine treatment, these phenotypic regulatory T cell-like cells lack the regulatory function and cytokine profile of regulatory T cells. These findings are important in correlating the clinically relevant immunomodulatory effects of 5-azacytidine.

Loss of heterozygosity in 7q myeloid disorders: clinical associations and genomic pathogenesis.

Loss of heterozygosity affecting chromosome 7q is common in acute myeloid leukemia and myelodysplastic syndromes, pointing toward the essential role of this region in disease phenotype and clonal evolution. The higher resolution offered by recently developed genomic platforms may be used to establish more precise clinical correlations and identify specific target genes. We analyzed a series of patients with myeloid disorders using recent genomic technologies (1458 by single-nucleotide polymorphism arrays [SNP-A], 226 by next-generation sequencing, and 183 by expression microarrays). Using SNP-A, we identified chromosome 7q loss of heterozygosity segments in 161 of 1458 patients (11%); 26% of chronic myelomonocytic leukemia patients harbored 7q uniparental disomy, of which 41% had a homozygous EZH2 mutation. In addition, we describe an SNP-A-isolated deletion 7 hypocellular myelodysplastic syndrome subset, with a high rate of progression. Using direct and parallel sequencing, we found no recurrent mutations in typically large deletion 7q and monosomy 7 patients. In contrast, we detected a markedly decreased expression of genes included in our SNP-A defined minimally deleted regions. Although a 2-hit model is present in most patients with 7q uniparental disomy and a myeloproliferative phenotype, haplodeficient expression of defined regions of 7q may underlie pathogenesis in patients with deletions and predominant dysplastic features.

Prevalence and clinical outcomes of the 46/1 haplotype, Janus kinase 2 mutations, and ten-eleven translocation 2 mutations in Budd-Chiari syndrome and their impact on thrombotic complications post liver transplantation.

Latent myeloproliferative disorders (MPDs) can be identified by Janus kinase 2 (JAK2) mutations in patients with idiopathic Budd-Chiari syndrome (BCS). The incidence and clinical outcomes of JAK2 mutations, novel ten-eleven translocation 2 (TET2) mutations, and the 46/1 haplotype in BCS are unknown for liver transplantation (LT). We undertook molecular studies of 66 patients presenting with BCS and correlated the results with the clinical outcomes. An overt MPD was present in 20% of the cases, and a latent MPD confirmed by the presence of a JAK2 mutation was detected in 45%. Testing for a TET2 mutation identified MPDs at the molecular level in another 7% of the subset of patients with BCS who were evaluated. The 46/1 haplotype frequency was significantly greater in BCS patients versus the general population (P < 0.001). The presence of JAK2 and TET2 mutations had no impact on 1-year survival. Thirty-six patients underwent LT, and 12 developed liver-related thrombotic complications (33%). Ten of these 12 patients required retransplantation. Retransplantation was more likely in those patients who developed liver-related thrombotic complications (P < 0.001). A JAK2 mutation was highly associated with the development of thrombotic complications after LT (P = 0.005). In conclusion, the presence of JAK2V617F predicts hepatic and extrahepatic thrombotic complications after LT. Testing for TET2 mutations can identify another 7% of idiopathic BCS patients with molecular MPDs.

Topography, clinical, and genomic correlates of 5q myeloid malignancies revisited.

PURPOSE: Interstitial deletions of chromosome 5q are common in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), pointing toward the pathogenic role of this region in disease phenotype and clonal evolution. The higher level of resolution of single-nucleotide polymorphism array (SNP-A) karyotyping may be used to find cryptic abnormalities and to precisely define the topographic features of the genomic lesions, allowing for more accurate clinical correlations. PATIENTS AND METHODS: We analyzed high-density SNP-A karyotyping at diagnosis for a cohort of 1,155 clinically well-annotated patients with malignant myeloid disorders. results: We identified chromosome 5q deletions in 142 (12%) of 1,155 patients and uniparental disomy segments (UPD) in four (0.35%) of 1,155 patients. Patients with deletions involving the centromeric and telomeric extremes of 5q have a more aggressive disease phenotype and additional chromosomal lesions. Lesions not involving the centromeric or telomeric extremes of 5q are not exclusive to 5q- syndrome but can be associated with other less aggressive forms of MDS. In addition, larger 5q deletions are associated with either del(17p) or UPD17p. In 31 of 33 patients with del(5q) AML, either a deletion involving the centromeric and/or telomeric regions or heterozygous mutations in NPM1 or MAML1 located in 5q35 were present. CONCLUSION: Our results suggest that the extent of the affected region on 5q determines clinical characteristics that can be further modified by heterozygous mutations present in the telomeric extreme.

A functional assay for microRNA target identification and validation.

MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.

Functional characterization of CD4+ T cells in aplastic anemia.

The role of CD4(+) T cells in the pathogenesis of aplastic anemia (AA) is not well characterized. We investigate CD4(+) T-cell subsets in AA. Sixty-three patients with acquired AA were studied. Th1 and Th2 cells were significantly higher in AA patients than in healthy donors (HDs; P = .03 and P = .006). Tregs were significantly lower in patients with severe AA than in HDs (P < .001) and patients with non-severe AA (P = .01). Th17 cells were increased in severe AA (P = .02) but normal in non-severe AA. Activated and resting Tregs were reduced in AA (P = .004; P = .01), whereas cytokine-secreting non-Tregs were increased (P = .003). Tregs from AA patients were unable to suppress normal effector T cells. In contrast, AA effector T cells were suppressible by Tregs from HDs. Th1 clonality in AA, investigated by high-throughput sequencing, was greater than in HDs (P = .03). Our results confirm that Th1 and Th2 cells are expanded and Tregs are functionally abnormal in AA. The clonally restricted expansion of Th1 cells is most likely to be antigen-driven, and induces an inflammatory environment, that exacerbate the functional impairment of Tregs, which are reduced in number.

Prognostic impact of SNP array karyotyping in myelodysplastic syndromes and related myeloid malignancies.

Single nucleotide polymorphism arrays (SNP-As) have emerged as an important tool in the identification of chromosomal defects undetected by metaphase cytogenetics (MC) in hematologic cancers, offering superior resolution of unbalanced chromosomal defects and acquired copy-neutral loss of heterozygosity. Myelodysplastic syndromes (MDSs) and related cancers share recurrent chromosomal defects and molecular lesions that predict outcomes. We hypothesized that combining SNP-A and MC could improve diagnosis/prognosis and further the molecular characterization of myeloid malignancies. We analyzed MC/SNP-A results from 430 patients (MDS = 250, MDS/myeloproliferative overlap neoplasm = 95, acute myeloid leukemia from MDS = 85). The frequency and clinical significance of genomic aberrations was compared between MC and MC plus SNP-A. Combined MC/SNP-A karyotyping lead to higher diagnostic yield of chromosomal defects (74% vs 44%, P < .0001), compared with MC alone, often through detection of novel lesions in patients with normal/noninformative (54%) and abnormal (62%) MC results. Newly detected SNP-A defects contributed to poorer prognosis for patients stratified by current morphologic and clinical risk schemes. The presence and number of new SNP-A detected lesions are independent predictors of overall and event-free survival. The significant diagnostic and prognostic contributions of SNP-A-detected defects in MDS and related diseases underscore the utility of SNP-A when combined with MC in hematologic malignancies.

Next-generation sequencing of the TET2 gene in 355 MDS and CMML patients reveals low-abundance mutant clones with early origins, but indicates no definite prognostic value.

Mutations in the TET2 gene are frequent in myeloid disease, although their biologic and prognostic significance remains unclear. We analyzed 355 patients with myelodysplastic syndromes using "next-generation" sequencing for TET2 aberrations, 91 of whom were also subjected to single-nucleotide polymorphism 6.0 array karyotyping. Seventy-one TET2 mutations, with a relative mutation abundance (RMA) ≥ 10%, were identified in 39 of 320 (12%) myelodysplastic syndrome and 16 of 35 (46%) chronic myelomonocytic leukemia patients (P < .001). Interestingly, 4 patients had multiple mutations likely to exist as independent clones or on alternate alleles, suggestive of clonal evolution. "Deeper" sequencing of 96 patient samples identified 4 additional mutations (RMA, 3%-6.3%). Importantly, TET2 mutant clones were also found in T cells, in addition to CD34(+) and total bone marrow cells (23.5%, 38.5%, and 43% RMA, respectively). Only 20% of the TET2-mutated patients showed loss of heterozygosity at the TET2 locus. There was no difference in the frequency of genome-wide aberrations, TET2 expression, or the JAK2V617F 46/1 haplotype between TET2-mutated and nonmutated patients. There was no significant prognostic association between TET2 mutations and World Health Organization subtypes, International Prognostic Scoring System score, cytogenetic status, or transformation to acute myeloid leukemia. On multivariate analysis, age (> 50 years) was associated with a higher incidence of TET2 mutation (P = .02).

Novel TET2 mutations associated with UPD4q24 in myelodysplastic syndrome.

PURPOSE: Cryptic chromosomal aberrations, such as regions of uniparental disomy (UPD), have been shown to harbor homozygous mutations and are a common feature in myelodysplastic syndrome (MDS). We investigated the sequence integrity of 4q24 candidate tumor suppressor gene TET2 in MDS patients with UPD on chromosome 4. PATIENTS AND METHODS: The coding exons of TET2 were analyzed by 454 deep sequencing and Sanger sequencing in nine patients with UPD on 4q. Four patients had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS) and UPD4q24, and five patients (refractory anemia with excess blasts-II, n = 1; 5q- syndrome, n = 1; RCMD-RS, n = 1; refractory anemia, n = 1; refractory cytopenia with multilineage dysplasia, n = 1) had no UPD4q24. RESULTS: Mutations on TET2 were identified in all four patients with UPD4q24. These were localized to exons 3, 6, and 9 and resulted in two premature stop codons, one frameshift mutation, and one cysteine to glycine amino acid change. Mutant clone size varied between 30% and 85%. One patient with UPD outside of q24 (UPD4q28.3) displayed additional TET2 mutations, but these were at low clonal levels (13%, 4%, and 4% for a silent mutation, a 180-base pair deletion in exon 3, and a lysine to phenylalanine substitution in exon 11, respectively). The other patients who did not have UPD4q24 did not have verifiable TET2 mutations. CONCLUSION: Our data identify novel TET2 mutations in a dominant clone in patients with UPD4q24. The presence of UPD4q24 and mutations in RCMD-RS patients may suggest specificity to this subtype. Our preliminary results need to be confirmed in a large cohort of all MDS subtypes.