Myelodysplastic syndromes Prognostic biomarkers in myelodysplastic syndromes

M. Cazzola ABSTRACT Prognostic biomarkers in myelodysplastic syndromes (MDS) include cytogenetic abnormalities and Department of somatic gene mutations. Recurrent chromosomal abnormalities are found in approximately 50% of , Fondazione Istituto cases, and are mainly secondary genetic events. By contrast, somatic oncogenic mutations, responsible di Ricovero e Cura a Carattere for disease pathogenesis and progression, are found in up to 90% of MDS patients. Oncogenic muta - Scientifico (IRCCS) Policlinico tions can be classified as: i) founding or initiating driver mutations, which cause a selective advantage San Matteo, and Department in a hematopoietic cell with capacity for self-renewal and lead to formation of a clone of mutated of Molecular , myelodysplastic cells; ii) subclonal or cooperating driver mutations, which occur in cells of an already University of Pavia, Pavia, Italy established clone and generate subclones carrying both the founding and the newly acquired muta - tion. Driver mutant genes include those of RNA splicing, DNA methylation, histone modification, tran - Correspondence: Mario Cazzola scription regulation, DNA repair, signal transduction, and cohesin complex. Only 6 genes ( TET2, SF3B1, E-mail: [email protected] ASXL1, SRSF2, DNMT3A , and RUNX1 ) are consistently mutated in 10% or more of MDS patients, while Acknowledgments: a long tail of additional genes are mutated less frequently. Reliable genotype/phenotype relationships The studies on the genetic basis of have already been established, primarily the close association between SF3B1 mutation and ring sider - myeloid neoplasms conducted at the oblasts. Clonal and subclonal mutations appear to affect prognosis equally, and outcome correlates Department of Hematology with the number of driver mutations. Ongoing studies aim to develop molecular models for clinical Oncology, Fondazione IRCCS decision-making. Policlinico San Matteo, and the Learning goals Department of Molecular Medicine, University of Pavia, Italy, were sup - At the conclusion of this activity, participants should be able to: ported by grants from the - describe driver somatic mutations in myelodysplastic syndromes, including the main biological Associazione Italiana per la Ricerca pathways involved; sul Cancro (AIRC), Fondazione - explain the difference between founding and subclonal mutations; Cariplo, MIUR (PRIN 2010-2011), - describe at least an established genotype:phenotype relationship in myelodysplastic syndromes; and FIRB (project n. RBAP11CZLK). -describe the diagnostic and prognostic relevance of somatic mutations in myelodysplastic syndromes.

Hematology Education: the education program for the annual congress of the European Introduction ized by cytopenia, myelodysplasia, ineffective Hematology Association hematopoiesis, and increased risk of progres - sion to acute myeloid leukemia (AML). 3 The 2014;8:237-242 Diagnostic and prognostic biomarkers in current diagnostic approach to MDS includes myelodysplastic syndromes (MDS) have been peripheral blood and bone marrow morpholo - recently analyzed in this Education Program 1 gy to evaluate abnormalities of peripheral by Hellström-Lindberg. In the present paper, I blood cells and hematopoietic precursors, will focus on genetic biomarkers. We have bone marrow biopsy to assess marrow cellu - recently reviewed the genetic basis of MDS 2 larity and fibrosis, and cytogenetics to identify and its clinical relevance in Blood. The reader non-random chromosomal abnormalities. 4 is, therefore, referred to this open access Based on these parameters, MDS are currently review article for more detailed information categorized according to the 2008 World (http://bloodjournal.hematologylibrary.org/co Health Organization (WHO) classification, ntent/122/25/4021.long), while the present which is reported in Table 1. As shown in paper summarizes the crucial points only. As Figure 1, the current WHO classification of such, the present paper must be considered an MDS has valuable prognostic relevance. acceptable secondary publication intended to disseminate important information to the widest possible audience, in agreement with Pathophysiology of myelodysplastic the criteria of the International Committee of syndromes Medical Journal Editors (ICMJE, http://www. icmje.org/publishing_d.html). Our working model of the pathophysiology of MDS is reported in Figure 2. As demon - strated by Walter et al. 5 through studies of Classification of myelodysplastic whole genome sequencing, the vast majority syndromes of myeloid precursors (i.e. immature red cells, granulocytic/monocytic precursors, and Myelodysplastic syndromes are clonal megakaryocytes) are clonally derived in MDS. hematopoietic stem cell disorders character - The myelodysplastic clone originates from the Hematology Education: the education program for the annual congress of the European Hematology Association | 2014; 8(1) | 237 | 19 th Congress of the European Hematology Association occurrence in an immature hematopoietic stem cell of a somatic mutation that provides survival and growth advantage: typically this founding mutation involves genes of RNA splicing or DNA methylation. 2 During the natural course of the disease, the acquisition of additional driver mutations leads to formation of subclones of hematopoietic cells with further impaired differentiation and/or maturation capacity: the proportion of blast cells progressively increases over time, and overt AML eventu - ally develops.

Recurrent chromosomal abnormalities in myelodys - plastic syndromes

Recurrent chromosomal abnormalities are detected in approximately half of patients with MDS, 6 and the most common aberrations include del(5q), trisomy 8, del(20q), and monosomy 7 or del(7q). 6,7 While most of these abnor - malities are secondary genetic events, the isolated del(5q) of the 5q- syndrome represents a founding event, involv - ing haploinsufficiency for RPS14 and miR-145. 8-10 The recent study of 7012 patients aimed to develop the revised International Prognostic Scoring System (IPSS-R) for MDS has clearly demonstrated the prognostic rele - vance of chromosomal abnormalities with respect to sur - 11 vival and risk of evolution into AML. This cytogenetic Figure 1. Kaplan-Meier analysis of overall survival and risk classification has also been found to predict the out - leukemia-free survival of 1110 patients diagnosed with come of allogeneic hematopoietic stem cell transplanta - MDS at the Department of Hematology Oncology, 12 Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, tion (HSCT) in MDS patients. In particular, patients with between 1990 and 2012. MDS patients are stratified monosomal karyotype, defined as the karyotype of according to the 2008 WHO classification categories. patients who had two (or more) autosomal monosomies or Multilineage dysplasia and excess of blasts have a consid - erable impact on outcomes. Reproduced from Cazzola et one monosomy in combination with other structural al. 2 abnormalities, have a very poor outcome. 12

Table 1. 2008 WHO classification of myelodysplastic syndromes (from Swerdlow et al. 3).

MDS subtype Blood findings Bone marrow findings

Refractory cytopenia Unicytopenia or bicytopenia, Unilineage dysplasia ( ≥10% of the cells in one myeloid lineage) <5% blasts, with unilineage dysplasia (RCUD) no or rare blasts (<1%) <15% ringed sideroblasts within erythroid precursors Refractory with ringed Anemia, no blasts only, < 5% blasts, ≥15% ringed sideroblasts within erythroid precursors sideroblasts (RARS) Refractory cytopenia with Cytopenia(s), no or rare blasts Dysplasia in ≥10% of cells in 2 or more myeloid multilineage dysplasia (RCMD) (<1%), no Auer rods, cell lineages (erythroid precursors and/or neutrophil precursors and/or megakaryocytes), <1x10 9/L monocytes <5% blasts, no Auer rods (the percentage of ringed sideroblasts is irrelevant) Refractory anemia with Cytopenia(s), <5% blasts, no Unilineage or multilineage dysplasia, 5-9% blasts, no Auer roads excess blasts-1 (RAEB-1) Auer roads, <1x10 9/L monocytes Refractory anemia Cytopenia(s), 5-19% blasts, Unilineage or multilineage dysplasia, 10-19% blasts, occasional Auer rods with excess blasts-2 (RAEB-2) occasional Auer rods, <1x10 9/L monocytes Myelodysplastic syndrome associated Anemia, normal to increased Normal to increased megakaryocytes with hypolobated nuclei, <5% blasts, no Auer with isolated del(5q) platelet count, no or rare blasts rods, isolated del(5q) (<1%) Myelodysplastic syndrome, Cytopenia (various combinations), Unequivocal dysplasia in less than 10% of cells in one or more myeloid cell lines when unclassifiable (MDS-U) no or rare blasts (<1%) accompanied by a cytogenetic abnormality considered as presumptive evidence for a diagnosis of MDS, <5% blasts

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Somatic gene mutations in myelodysplastic Diagnostic, prognostic and predictive significance syndromes of driver mutations, and development of molecular models for clinical decision-making Our understanding of the molecular basis of MDS has improved dramatically in the last four years as a result of 13-22 The identification of founding and subclonal driver a few fundamental studies. A list of the most common mutations has the potential to considerably improve diag - recurrently mutated genes in patients with MDS or nosis and prognostication of MDS and, more generally, myelodysplastic/myeloproliferative neoplasm (MDS/ clinical decision-making in these disorders. In order to MPN) is reported in Table 2. Only 6 genes ( TET2 , SF3B1 , fully achieve these objectives, data should be collected in ASXL1 , SRSF2 , DNMT3A , and RUNX1 ) are consistently prospective clinical studies that include the molecular mutated in 10% or more of MDS patients, while a long tail characterization of the patient’s genome as an essential of additional genes are mutated less frequently. The driver component. genes whose mutations are responsible for MDS are fre - The very fact that somatic mutations can be detected in quently mutated also in other myeloid neoplasms, includ - approximately 90% of MDS patients, and that two-thirds ing acute myeloid leukemia (AML) and myeloprolifera - of these subjects have normal karyotype, 22 indicates that tive neoplasms. the vast majority of MDS patients may have a clonal marker that makes diagnosis of clonal disease more reli - Genotype/phenotype relationships able. We have previously shown that SF3B1 mutations are independent predictors of favorable clinical outcome. 23 As Genotype/phenotype relationships have been defined shown in Figure 3, these mutations are associated with not only in MDS but also in myeloid neoplasms. 2 In par - better overall survival (hazard ratio (HR) 0.15; P=0.025) ticular, SF3B1 mutation appears to be strictly associated and lower risk of evolution into AML (HR 0.33; P=0.049). with refractory anemia with ring sideroblasts with or with - On the contrary, somatic mutations of other genes of the out marked thrombocytosis, 17,23 and activating CSF3R spliceosome, primarily SRSF2 and U2AF1 , are associated mutation with chronic neutrophilic leukemia. 20 with unfavorable clinical outcome. 18,24 The prognostic rel -

Figure 2. Schematic representation of our current understanding of the pathophysiology of myelodysplasia. In this exam - ple, the founding driver mutation is assumed to occur in a hematopoietic cell located in the bone marrow of the right ilium, and the sternum is shown to illustrate the concept of mutated stem cell migration through peripheral blood. Bone marrow microphotographs: magnification from 600X, courtesy of Erica Travaglino. Reproduced from Cazzola et al .2

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evance of the most commonly mutant genes is summa - improve prognostication of MDS. 15,26 Haferlach et al .22 rized in Table 2. Of note, ASXL1 mutations involve unfa - have developed a model that combines 14 prognostic vorable clinical outcome in all myeloid neoplasms, includ - genes with conventional risk factors, such as age, gender ing MDS, MDS/MPN and myeloproliferative neoplasms, and the parameters used in the IPSS-R: this model suc - in particular, primary myelofibrosis. 2,25 In the study by cessfully discriminated four significant risk groups and Papaemmanuil et al. ,21 clonal and subclonal mutations better predicted overall survival than the IPSS-R. Solary were found to affect prognosis equally, and outcome cor - and co-workers 27 have proposed a new prognostic score related with the number of driver mutations. for chronic myelomonocytic leukemia (CMML) that A few studies have already suggested that incorporation includes not only age and hematologic parameters, but of somatic mutations into prognostic scoring systems can also ASXL1 mutation status. Similar models are being

Table 2. Recurrently mutated driver genes in patients with MDS and MDS/MPN.2

Biological pathways and genes Clinical relevance of mutant gene including prognosis

RNA splicing SF3B1 Strictly associated with ring sideroblast phenotype, associated with good overall survival and low risk of leukemic evolution SRSF2 Mainly found in MDS subtypes characterized by multilineage dysplasia and/or excess blasts, co-mutated with TET2 in chronic myelomonocytic leukemia, associated with poor overall survival and high risk of leukemic evolution U2AF1 Mainly found in MDS subtypes characterized by multilineage dysplasia and/or excess blasts, associated with high risk of leukemic evolution

DNA methylation TET2 Found in all MDS subtypes, high mutation frequency (50-60%) in chronic myelomonocytic leukemia, may predict response to hypomethylating agents DNMT3A Found in all MDS subtypes, co-mutated with SF3B1 in refractory anemia with ring sideroblasts, associated with unfavorable clinical outcome

Histone modification ASXL1 Mainly found in MDS subtypes characterized by multilineage dysplasia and/or excess blasts, high mutation frequency (40%) in chronic myelomonocytic leukemia, associated with unfavorable clinical outcome in all myeloid neoplasms EZH2 Mainly found in MDS subtypes characterized by multilineage dysplasia and/or excess blasts, associated with unfavorable clinical outcome in all myeloid neoplasms

Transcription RUNX1 Mainly found in MDS subtypes characterized by multilineage dysplasia and/or excess blasts, associated with unfavorable clinical outcome BCOR Associated with poor prognosis

DNA repair control TP53 Associated with advanced disease and complex karyotype, mutated in 20% of patients with MDS with del(5q), involves poor overall survival and high risk of leukemic evolution, may predict poor response to lenalidomide in MDS with del(5q)

Cohesin

STAG2 Mainly found in MDS subtypes characterized by multilineage dysplasia and/or excess blasts, mutated in approx. 10% of patients with AML

RAS pathway

CBL Found in different MDS subtypes, associated with juvenile myelomonocytic leukemia in children NRAS/KRAS Found in different MDS subtypes, associated with juvenile myelomonocytic leukemia in children

DNA replication SETBP1 Found in 25% of patients with atypical chronic myeloid leukemia and in subsets of patients with advanced MDS or chronic myelomonocytic leukemia, associated with poor overall survival and high risk of leukemic evolution

Receptors CSF3R Strictly associated with chronic neutrophilic leukemia, found in a subset of patients with aCML

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developed for myeloproliferative neoplasms. 28 associated with a lower response to lenalidomide and an Under the aegis of the MDS Foundation, the increased risk of progression to AML. 29 TET2 mutations International Working Group for Prognosis in MDS appear to predict response to hypomethylating agents, 30 (IWG-PM) has started a research project aimed at devel - while U2AF1 mutations would independently predict for oping a clinical/molecular IPSS that includes clinical, poor outcome after allogeneic stem cell transplantation. 31 hematologic and molecular parameters. This project is pri - marily based on the analysis of retrospective data. For fur - ther improvements, functional studies of the mutant genes Conclusions and prospective clinical trials that include the molecular characterization of the patient’s genome are now needed. Somatic gene mutations have considerable diagnostic Characterization of the patient’s genome may guide and prognostic relevance in MDS. From this point of view, therapeutic decision-making. TP53 mutated subclones SF3B1 mutation is so far the best characterized as it is may occur at an early disease stage in MDS with del(5q); strictly associated with ring sideroblast phenotype and their identification is of crucial importance as they are indolent clinical course. More generally, due to the high number of potentially mutated genes, detection of somatic mutations cannot yet be considered a clinical diagnostic/prognostic tool. Nonetheless, the Food and Drug Administration (FDA) has recently granted market - ing authorization for the first next generation genomic sequencer (Illumina’s MiSeqDx). 32 This means that this platform can be brought into use for clinical care. Thus, massive parallel sequencing of a panel of myeloid genes may soon become feasible in a clinical laboratory, allow - ing molecular diagnosis and prognostication of MDS. For these expectations to be fully realized, prospective clinical trials that include the molecular characterization of the patient’s genome are now needed.

References

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