1Department of Translational Hematology and Oncology Research, Cleveland Clinic, Cleveland, OH, USA.
2Department of Hematologic Oncology and Blood Disorders, Cleveland Clinic, Cleveland, OH, USA.
3Department of Laboratory Medicine, Cleveland Clinic, Cleveland, OH, USA.
Myelodysplastic syndromes (MDS) are a group of clonal disorders arising from hematopoietic stem cells generally characterized by inefficient hematopoiesis, dysplasia in one or more myeloid cell lineages, and variable degrees of cytopenias. Most MDS patients are diagnosed in their late 60s to early 70s. The estimated incidence of MDS in the United States and in Europe are 4.3 and 1.8 per 100,000 individuals per year, respectively with lower rates reported in some Asian countries and less well estimated in other parts of the world. Evolution to acute myeloid leukemia can occur in 10-15% of MDS patients. Three drugs are currently approved for the treatment of patients with MDS: immunomodulatory agents (lenalidomide), and hypomethylating therapy [HMT (decitabine and 5-azacytidine)]. All patients will eventually lose their response to therapy, and the survival outcome of MDS patients is poor (median survival of 4.5 months) especially for patients who fail (refractory/relapsed) HMT. The only potential curative treatment for MDS is hematopoietic cell transplantation. Genomic/chromosomal instability and various mechanisms contribute to the pathogenesis and prognosis of the disease. High throughput genetic technologies like single nucleotide polymorphism array analysis and next generation sequencing technologies have uncovered novel genetic alterations and increased our knowledge of MDS pathogenesis. We will review various genetic and non-genetic causes that are involved in the pathogenesis of MDS.
Myelodysplastic syndromes (MDS) are clonal stem cell malignancies characterized by cytopenias, inefficient hematopoiesis, dysplasia in one or more myeloid cell lineages and increased risk of development of acute myeloid leukemia (AML). It is sub-classified based on percent of bone marrow (BM) and peripheral blood blasts, type/degree and number of dysplastic cell lineages, presence/absence of ring sideroblasts (RS) and presence of specific chromosomal abnormalities. The median age at the diagnosis of MDS is 71 years [1, 2]. Treatment options for MDS patients vary based on the disease severity. Transfusions, growth factors, and antibiotic therapy are part of the supportive care that is usually suggested for low-risk MDS patients. Chemotherapy regimens like hypomethylating therapy (HMT; decitabine and 5-azacytidine), immunomodulatory agents (lenalidomide), cytarabine, idarubicin, and daunorubicin are commonly used in high-risk MDS patients to delay the AML transformation. Ultimately, long term remission is achieved by using high dose chemotherapy and hematopoietic stem cell (HSC) transplantation.
Genetic defects (chromosomal aberrations, gene mutations, copy-number alterations, abnormal gene expression) are common in MDS. Clonal and recurrent cytogenetic abnormalities and their frequency are summarized in Table 1. They are often present at disease presentation; however abnormal clones can appear during the disease course and are associated with worsening conditions. Genomic instability (deletions, translocations) is important since these genetic events can encompass regions containing tumor suppressor genes (TSGs) relevant to MDS biology. Unbalanced translocations are found in ~50% of primary and 80% of therapy-related MDS (t-MDS) cases [3, 4]. Complex karyotype (3 or more defects) is usually correlated with dismal outcomes and can be seen following chemo/radiotherapy and toxic chemicals exposure. The recently published Revised International Prognostic Scoring System (R-IPSS) includes more specific cytogenetic prognostic subgroups to improve prognostic stratification in MDS .
Somatic mutations in multipotent stem cells are believed to contribute to MDS pathogenesis, even though no specific defect has been clearly identified. Genomic instability (genetic defects, mutations) increases the propensity to develop AML. It is believed that ~78% of MDS patients carry at least one somatic mutation . The importance of recurrent mutations resides in their potential clinical applications specifically in prognosis, diagnosis, risk stratification, and treatment response. For example,
Sanger sequencing, high resolution whole genome scanning technologies [single nucleotide polymorphism arrays (SNP-A) genotyping], and high-throughput next generation sequencing [HT-NGS, whole exome/genome sequencing (WE/WGS), deep sequencing] have brought to light the presence of mutations in genes of methylation, transcription, signaling, histone modification, RNA-splicing and other pathways. Ultimately, understanding the molecular alterations of genes relevant to MDS will hold the key of targeted therapy and improvement in therapeutic outcomes. Fig. 1 shows a schema of the histopathologic, cytogenetic, and molecular genetic tools for diagnosis, classification, and prognosis assessment in MDS.
Chromosomal abnormalities (-5/5q-, -7/7q-, +8, 20q-, +21, 12p-, 13q-, and 17p-) are detected in 40-60% of primary MDS and considered an important determinant in the prognostic scoring systems [12, 13]. However, conventional metaphase cytogenetics (MC) reaches 10% sensitivity and is informative in 46-59% of MDS patients due to limited results in non-viable cells or non-informative karyotypes . Some subtle chromosomal abnormalities can be undetectable or masked . Fluorescence
The low sensitivity of MC has been overcoming using a powerful method called SNP-A karyotyping. SNP-A, which uses DNA hybridization and fluorescence technique, is currently used in clinical centers as a diagnostic tool to improve the detection rate. SNP-A can detect cryptic lesions [copy neutral-loss of heterozygosity (CN-LOH) or acquired somatic uniparental disomy] in 50% of MDS patients with normal karyotype [19, 20, 21]. Part of these lesions can be pathogenic. Studies have demonstrated the value of combining MC with SNP-A and the prognostic importance of the number of SNP lesions in MDS [20, 22, 23].
In the latest 10 years, second-generation DNA sequencing such as HT-NGS has been implemented in the discovery of genetic alterations in cancer. These technologies refer to non-Sanger DNA sequencing methods where millions of DNA strands can be massively sequenced . More recently WE/WGS and deep sequencing have been helpful in discovering germ-line and somatic variants in MDS. These technologies are almost being considered as diagnostic tests compared to direct-sequencing. At the transcript level, RNA-sequencing has been used for a variety of studies (gene expression, transcript isoforms, small RNAs, TCRβ/BCR repertoires, exon usage/splicing patterns, and methylation/chromatin changes). These technologies rely on a high resolution, depth of coverage (number of times a nucleotide is sequenced) and variation/phred score (index of the quality of the variant calls) .
Genetic technologies including HT-NGS have discovered several mutated genes clustered in specific pathways. We will discuss the frequency, function, and prognostic significance of the main pathways and genetic alterations in MDS (Table 2).
Epigenetic regulation is one of the main mechanisms of controlling gene function. DNA methylation and histone modification are the 2 epigenetic processes that have been found to be altered in MDS. Aberrant methylation of TSG promoters is present in MDS . Indeed DNA methylation (addition of a methyl-group to DNA) occurs at CpG sites (regions in which a cytosine and a guanine are linked by a phosphate). Since the CpG dinucleotides are localized in upstream regulatory regions, the methylation of the CpG leads to a silencing mechanism. DNA methyltransferases (DNMTs) such as DNMTs 1, 3a, and 3b are enzymes responsible for DNA methylation and highly expressed in AML and other myeloid neoplasms.
A somatic frameshift mutation in
Mutations in the isocitrate dehydrogenase 1 (
Deletions of chromosome 7/7q are common in MDS and correlate with poor prognosis.
The Jumonji C (JmjC)-domain family of histone demethylases with the function of removing methyl-groups from the histone methylation site has been studied in MDS. Deletions in H3K27me2/3 (UTX/JMJD3), a demethylase on the X chromosome and in other JmjC members have been found at <1% in MDS with highest frequencies in chronic myelomonocytic leukemia (CMML) (8%) .
Somatic mutations in components of the RNA-splicing machinery were discovered using WE/WGS in myeloid and lymphoid disorders . Splicing factor genes are mutated in almost half of the MDS patients and are generally mutually exclusive and disease-type specific. Sometimes, splicing-gene mutations can also occur with other genetic mutations primarily involved in epigenetics as in the case of
Spliceosome inhibitors have been proposed to target the mutant allele in splicing factors in MDS. Pladienolide B, Sudemycins, and Spliceostatin A derived from bacterial fermentation products and small molecules (steroids, biflavonoid natural plant, indole derivatives, protein phosphatases and benzothiazole inhibitors) showed antitumoral effect against the spliceosome .
PRPF8 is a component of the catalytic core of the spliceosome and forms interactions with the 3' and 5' splice, the branch point, and the excised introns. Two mutant cases were reported by Makishima et al. and Gomez-Segui et al. [75, 81, 82]. In a larger cohort (N=447) the authors found mutations in 15/447 (3.3%) patients . Mutations were found along the gene, and D1598 was the most common amino acid change. A higher frequency was found in primary and sAML. In total, 60% of the patients appeared to have RS. Due to the genomic mapping of
Activating oncogenic mutations are rare in MDS.
Genome-wide mapping clarified the cohesin complex structure and identified its role in chromosome condensation in Saccharomyces cerevisiae . The structure of this complex resembles a ring constituted by 4 subunits (Scc1, Scc3, Smc1, Smc3) with the Smc1 and Smc3 being elements of the structural maintenance of chromosome family. Cohesins control that sister chromatids are connected during metaphase and segregate into right directions during cell division. Cohesins are important also in DNA-replication, DNA double-strand breaks repair, and chromosome condensation. Mutations in the cohesin complex (
Genetic alterations in
Although molecular mutations are frequent in MDS, the consequences of these mutations have not been clarified. Other non-genetic mechanisms including BM-microenvironment factors, apoptosis, cytokines, immunoregulation, T-cell repertoire and telomere length (TL) have been largely studied. In supporting to the involvement of apoptotic pathways, death receptors (Fas, TRAIL), mitochondrial pathways and caspase activation have been found modulated in MDS cells. Indeed, MDS precursors overexpress Fas and TRAIL receptors, which seem to induce death signaling. Tumor necrosis factor alpha (TNF-α) also seems to be released by cytotoxic T-cells inducing apoptosis. Cytochrome c release is observed in low-risk MDS patients while caspase-9 activity is increased. Recently, it was reported that
Abnormalities in suppressive cytokines [TNF-α, tumor growth factor-β (TGF-β), interferon-γ (IFN-γ), interleukins-3, 6, and 8, thrombopoietin (TPO)] have been described in MDS. Overactivation of TGF-β depends on a family of proteins called Sma- and Mad-related (SMAD). SMAD2, a downstream regulator of TGF-β receptor I kinase activation, is constitutively activated in MDS CD34+ cells, while SMAD7, a negative regulator, seems to be decreased in MDS cells . Multiplex-analysis of cytokines/chemokines showed similarity between MDS and AML cells with a higher expression of the VEGF in MDS. High levels of TNF-α have been correlated with worse OS in MDS . Higher levels of TPO and G-CSF, and lower levels of CD40L, CCL5, CCL11, VEGF, CXCL5, EGF, and CXCL11 were found by comparing plasma cytokines between aplastic anemia and MDS patients. Differences between low- and high-risk MDS were described with decreased CXCL5, CCL5, CD40L, EGF, and VEGF, and increased CCL4 in high-risk MDS . HSCs in high-risk MDS patients are believed to proliferate bypassing the immune system. Differences in immune-complexes were found between low- and high-risk MDS and attributable to clonal progression. Cytotoxicity of BM cells in low-risk MDS has been associated with high numbers of NK cells, Th17, and macrophages releasing IFN-γ. CD4+FOXP3+-regulatory T cells have been correlated with immune response suppression and found elevated in high-risk MDS patients. The BM failure of MDS patients has been related to autoimmunity. In trisomy 8 MDS, spectratyping of T-cell receptor β-chain variable (Vβ) families revealed that CD8+ T-lymphocytes are oligoclonal and selectively cytotoxic against trisomy 8 clones. Trisomy 8 clones seem to be resistant to the T-cell immune attack by up-regulating survivin. Microarray showed increased expression of WT1 in trisomy 8 CD34+ cells .
Cellular senescence in MDS has been related to telomere shortening. BM MDS cells appear to have erosive telomeric repeats without changes in telomerase activity. Southern blot showed a variability in TL in MDS with a lower TL correlating with leukemic progression and complex cytogenetics . Using multiplex-quantitative RT-PCR in 307 MDS patients, TL in BM cells was lower in MDS compared to healthy subjects with no correlation with age or gender. TL seems to be negatively correlated with IPSS, transfusion dependence, BM blasts and complex karyotype [115, 116]. Telomerase mutations/polymorphisms are sporadic in MDS. One patient with MDS and del(5q) harbored a
Aberrant methylation is another important mechanism in MDS. Hypermethylation of promoter-CpG island of TSGs is a silencing mechanism and contributes to clonal evolution in MDS. Level of methylation also correlated with survival outcomes. One study showed that MDS patients with higher levels of aberrant CpG methylation in relatively common genes had a shorter median OS and PFS (OS: 12.3 vs. 17.5 months;
MDS is a heterogeneous clonal disease with multifactorial causes. Molecular mutations in several pathways have been identified. Almost 78% of MDS patients carry at least one mutation in one gene. The presence of mutations has been associated with disease phenotypes and response to therapies. The use of NGS technologies has almost being implemented in clinical practice, although large clinical studies are needed to validate the possibility to use mutations as predictors of diagnosis, prognosis, and treatment response. Functional studies aim to decipher how genetic alterations may lead to the clinical phenotypes and whether targeting specific pathways or genes will be beneficial for MDS patients.
Diagram of the histopathologic, cytogenetic, and molecular genetic tools for comprehensive evaluation of diagnosis, classification and prognosis in myelodysplastic syndrome. (