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. IntroductionThe myelodysplastic syndromes (MDSs) are characterized by clonal hematopoiesis, a variety of chromosomal abnormalities, bone marrow (BM) failure, and a propensity for evolution to acute myeloid leukemia (AML). Because of their often protracted course, MDSs recapitulate the stages of acquisition of a malignant phenotype, thereby offering insights into leukemogenesis. Although, traditionally, histomorphology-based schemes have been applied to subclassify patients with MDS, 1,2 this approach is unlikely to be reflective of the underlying pathogenesis. Instead, a better molecular characterization of MDS on the genomic, epigenetic, and genetic levels probably more objectively diagnoses conditions, determines patients' prognosis and, based on the underlying molecular defects, directs the application of targeted therapies. The emerging realization of the molecular diversity of MDS parallels the clinical and phenotypic heterogeneity of this disease. Moreover, molecular defects have the potential to serve as biomarkers and probably are more suitable for the identification of therapy targets and responsiveness/refractoriness to treatment.The application of high-throughput molecular technologies, including high-density single nucleotide polymorphism arrays (SNP-As) 3 and new sequencing technologies 4,5 has led to the improved characterization of genomic lesions such as chromosomal aberrations and of somatic mutations affecting specific classes of genes, 6 including signal transducers (eg, CBL), 7-10 apoptotic genes (eg, TP53 and RAS), [11][12][13] genes involved in epigenetic regulation of DNA (eg, DNMT3A, IDH1/2, and TET2), [14][15][16][17][18] and histone modifiers (eg, EZH2, UTX, and ASXL1). [19][20][21][22][23][24] Although some mutations in these factors are activating, most are loss-of-function or hypomorphic mutations and affect bona fide t...

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IntroductionThe myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem-cell disorders characterized by cytopenias and frequent leukemic progression. MDS constitutes a prototype of age-related malignancy, with a prevalence in the United States that may be more than 100 000. 1 Its incidence in the United States, estimated to be more than 10 000 yearly, is likely to further increase due to the greater life expectancy of the general population (http://www.census.gov/).Chromosomal aberrations can be detected by metaphase cytogenetics (MC) in approximately 50% of MDS patients and are responsible for some of the observed clinical diversity. Based on the experience that certain chromosomal lesions have a major impact on survival in MDS, 2-5 cytogenetic results were included in The International Prognostic Scoring System (IPSS), the most commonly applied prognostic algorithm for MDS. Moreover, recent studies demonstrate that MDS patients with certain cytogenetic abnormalities may be candidates for targeted therapies. For example, lenalidomide results in a high remission rate in MDS patients with 5q-abnormalities. 6,7 High-resolution single nucleotide polymorphisms arrays (SNP-A) can be applied in karyotypic analysis. SNP-A-based karyotyping does not depend upon the availability of live, dividing cells, and consequently can yield results when routine MC is not informative. Moreover, due to the higher resolution of SNP-A as compared with MC, smaller, previously cryptic deletions and duplications can be detected. A major advantage of this technology over MC is its ability to identify loss of heterozygosity (LOH) that occurs without concurrent changes in the gene copy number (CN). Such defects are consistent with acquired uniparental disomy (UPD) and can be attributed to errors in mitotic recombination occurring in somatic cells. Acquired segmental UPD is being increasingly recognized in a variety of neoplasms. 8,9 UPD has been described in chronic lymphocytic leukemia 10 and polycythemia vera as a mechanism leading to homozygosity for the Jak2 mutation. 11 Recently, an extensive study of acute lymphoblastic leukemia using SNP-A revealed chromosomal deletions and amplifications, many of them involving genes encoding principal regulators of B-lymphocyte development. 12 SNP-A also has been used for detecting genomic lesions in smaller case series of myeloma, 13 leukemias, 14-16 and lymphoma. 17 Initially using 50K arrays, we have demonstrated the potential diagnostic value of this technology, in a smaller cohort of myelodysplastic syndrome (MDS) patients. 18 This preliminary study demonstrated frequent detection of UPD in MDS. Subsequent larger studies limited to low-risk MDS showed similar results. 19 MDS is a particularly suitable target for demonstrating the use of SNP-A, as acquired cytogenetic abnormalities are relatively frequent and mostly unbalanced. 20 Using this disease as a model, we tested the hypothesis that high-density SNP-A can complement routine MC and enhance its diagnostic re...

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.