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Perspectives

MYELOID NEOPLASIA

The genetic basis of myelodysplasia and its clinical relevance


Mario Cazzola,1,2 Matteo G. Della Porta,1,3 and Luca Malcovati1,2
1
Department of Hematology Oncology, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Policlinico San Matteo, Pavia, Italy; and Departments
of 2Molecular Medicine and 3Internal Medicine, University of Pavia, Pavia, Italy

Myelodysplasia is a diagnostic feature of and EZH2), transcription regulation (RUNX1), mutation with refractory anemia with ring
myelodysplastic syndromes (MDSs) but DNA repair (TP53), signal transduction (CBL, sideroblasts, TET2/SRSF2 comutation with
is also found in other myeloid neoplasms. NRAS, and KRAS), and cohesin complex chronic myelomonocytic leukemia, and
Its molecular basis has been recently elu- (STAG2). Only 4 to 6 genes are consistently activating CSF3R mutation with chronic
cidated by means of massive parallel mutated in 10% MDS patients, whereas neutrophilic leukemia. Although both
sequencing studies. About 90% of MDS a long tail of 50 genes are mutated less founding and subclonal driver muta-
patients carry 1 oncogenic mutations, frequently. At presentation, most patients tions have been shown to have prog-
and two thirds of them are found in in- typically have 2 or 3 driver oncogenic mu- nostic significance, prospective clinical
dividuals with a normal karyotype. Driver tations and hundreds of background muta- trials that include the molecular charac-
mutant genes include those of RNA splic- tions. MDS driver genes are also frequently terization of the patients genome are now
ing (SF3B1, SRSF2, U2AF1, and ZRSR2), mutated in other myeloid neoplasms. Reli- needed. (Blood. 2013;122(25):4021-4034)
DNA methylation (TET2, DNMT3A, and able genotype/phenotype relationships
IDH1/2), chromatin modification (ASXL1 include the association of the SF3B1

Introduction
Myelodysplasia is a term used in pathology for describing mor- the reader is referred to recent landmark studies of genomic and
phologic abnormalities, or dysplasia, in $1 of the major myeloid cell epigenomic landscapes of AML,3,4 and a review article in Blood.5 A
lines of hematopoiesis and is a typical feature of myelodysplastic very detailed analysis of what has been learned about cancer genomes
syndromes (MDSs).1 In the World Health Organization (WHO) in the last few years has been published recently by Vogelstein et al.6
classication of tumors of hematopoietic and lymphoid tissues, MDSs Denitions for basic terms used in studies of the genetic basis of
are dened as clonal hematopoietic stem cell disorders characterized myeloid neoplasms are reported in the Appendix.
by cytopenia, myelodysplasia, ineffective hematopoiesis, and
increased risk of progression to acute myeloid leukemia (AML).1
Representative examples of morphologic abnormalities of myelo-
dysplasia are reported in Figure 1. Clonal expansion of myelodysplastic stem
Myelodysplasia is not restricted to MDS but may be found cells, ineffective hematopoiesis, and leukemic
also in other myeloid neoplasms of the WHO classication transformation: a working model of the
(Table 1). Although the different subtypes of myeloid neoplasms pathophysiology of myelodysplasia
have distinctive characteristics, they may share morphologic
abnormalities. The paradigmatic example is refractory anemia For a better understanding of the pathophysiology of myelodys-
with ring sideroblasts associated with marked thrombocytosis plasia, we summarized the current concepts in the model reported
(RARS-T), which has both the myelodysplastic features of RARS in Figure 2. Some steps of this model are still working hypotheses,
and the myeloproliferative characteristics of essential thrombo- which will hopefully be veried in the near future.
cythemia. This suggests that the myelodysplastic features of An essential component of the WHO denition of MDS is the
various myeloid neoplasms may reect common underlying genetic clonal nature of myelodysplastic hematopoiesis.1 Although various
lesions and that these latter contribute to determining clinical approaches can be used to prove the existence of a clonal population
phenotypes. of hematopoietic cells, the more straightforward one is the use of
In this article, we will review the most recent advances in our chromosomal abnormalities or discrete gene rearrangements to
understanding of the genetic basis of myelodysplasia and will discuss show the uniform presence of these markers in puried hematopoi-
its clinical relevance. The Chronic Myeloid Disorders Working etic cell populations.7 Using this approach, chromosomal aberra-
Group of the International Cancer Genome Consortium has just tions were found to be restricted to committed myeloid progenitor
completed a study of targeted gene sequencing in a large cohort of cells in MDS patients, suggesting that the genetic lesion occurred in
patients with MDS and closely related neoplasms.2 For additional a hematopoietic cell with the capacity to differentiate into mature
information on the genomic characterization of myeloid neoplasms, myeloid cells.8,9

Submitted August 30, 2013; accepted October 8, 2013. Prepublished online as 2013 by The American Society of Hematology
Blood First Edition paper, October 17, 2013; DOI 10.1182/blood-2013-09-
381665.

BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25 4021


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4022 CAZZOLA et al BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25

Figure 1. Representative examples of morphologic abnormalities of myelodysplasia. May Grunwald Giemsa staining in all cases with the only exception of ring
sideroblasts (Perls staining). Magnification from 2003 to 10003, courtesy of Erica Travaglino.

In a landmark study, Walter et al10 used whole genome se- immature CD341CD382 and mature CD341CD381 progenitors.
quencing to identify somatic mutations in bone marrow samples from Although TET2 mutations were detected in only a small fraction of
patients with AML developing from MDS and then genotyped in CD341CD382 cells, they were present in a high proportion of
each patient a bone marrow sample obtained during the antecedent more mature progenitors. This suggests that the initial somatic
MDS phase. About 85% to 90% of unfractionated bone marrow cells TET2 mutation occurred in a CD341CD382 cell and was then
were found to be clonal in these patients, both in the MDS and in transmitted to its CD341CD381 progeny. A similar clonal archi-
AML phase, irrespective of the number of blasts. This study formally tecture has been more recently observed also in patients with chronic
proved that almost all cells of the bone marrow myeloid cell lines (ie, myelomonocytic leukemia (CMML).12
immature red cells, granulocytic/monocytic precursors, and mega- The occurrence in an immature hematopoietic stem cell of a
karyocytes) are clonally derived in MDS patients at any stage of the somatic mutation that provides survival and growth advantage (for
disease and not only after AML transformation.10 instance, lower propensity to apoptosis) leads to formation of a local
The clonal architecture of MDS has been elegantly studied by clone (Figure 2, step 1). For this clone to become fully dominant
Delhommeau et al11 following the identication of somatic TET2 in the whole body, the mutated stem cells must have additional
mutations. CD341 cells from MDS patients were fractionated into advantages. In adulthood, migration and trafcking of hematopoietic

Table 1. WHO classification of myeloid neoplasms


Major subgroups Conditions

MDS Refractory cytopenia with unilineage dysplasia, RARS, refractory cytopenia with multilineage dysplasia (RCMD),
refractory cytopenia with excess blasts (RAEB, type 1 and 2), MDS with isolated del(5q)
Myeloproliferative neoplasms (MPN) Chronic myeloid leukemia (CML, BCR-ABL1 positive), chronic neutrophilic leukemia (CNL), polycythemia vera,
essential thrombocythemia, primary myelofibrosis (PMF), chronic eosinophilic leukemia, mastocytosis
MDS/MPN CMML, atypical chronic myeloid leukemia (aCML, BCR-ABL1 negative), juvenile myelomonocytic leukemia (JMML),
RARS-T
AML Various subtypes based on blast morphology and/or underlying genetic lesion(s)

Information is from Swerdlow et al.1 The WHO classification of myeloid neoplasms also includes myeloid and lymphoid neoplasms with eosinophilia and abnormalities of
PDGFRA, PDGFRB, or FGFR1.
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BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25 MOLECULAR BASIS OF MYELODYSPLASIA 4023

Figure 2. Schematic representation of our current understanding of the pathophysiology of myelodysplasia. In this example, 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 as an anatomically separated bone marrow district to illustrate the
concept of mutated stem cell migration through peripheral blood. Bone marrow microphotographs: magnification from 6003, courtesy of Erica Travaglino.

stem cells are of crucial importance in maintaining homeostasis of the been shown to lead to granulo-monocytic differentiation skewing at the
hematopoietic system.13,14 Despite several investigations, the mecha- expense of erythroid and megakaryocytic differentiation.12
nisms by which neoplastic hematopoietic cells leave the primary site During the natural course of the disease, patients with MDS are at
and migrate to other bone marrow districts remain largely unclear.13 high risk of progressing to AML.1 The most likely interpretation is
Ultimately, however, mutated hematopoietic stem cells achieve full that the acquisition of additional driver mutations leads to formation of
clonal dominance in the bone marrow, and the vast majority of cir- subclones of hematopoietic cells with further impaired differentiation
culating mature cells derive from the dominant clone (Figure 2, step 2). and/or maturation capacity. The proportion of blast cells progressively
Once the myelodysplastic clone has become fully dominant in the increases over time, and overt AML eventually develops (Figure 2,
bone marrow, the disease may or may not become clinically apparent. step 4). This has been demonstrated by Walter et al10 in their study of
For instance, a somatic SF3B1 mutation appears to be able to cause the clonal architecture of secondary AML. In each of the 7 patients
a clinical phenotype per se,15,16 whereas a driver TET2 mutation can studied, in fact, progression to AML was characterized by the
determine clonal hematopoiesis without hematologic manifesta- persistence of the antecedent founding myelodysplastic clone and the
tions,17 suggesting that cooperating mutant genes might be required emergence of $1 subclone harboring new somatic mutations. Thus,
for phenotypic expression. Myelodysplastic hematopoiesis is charac- a secondary AML developing from MDS is not monoclonal in the
terized by excessive apoptosis of hematopoietic precursors, at least in strict sense but is instead a mosaic of several clones/genomes with
patients with low-risk disease.18 Ineffective hematopoiesis, ie, the different sets of somatic mutations, illustrating the concept of in-
premature intramedullary death of erythroblasts, immature granulocytes/ traclonal or intratumoral heterogeneity.10
monocytes, and megakaryocytes, is primarily responsible for the defec-
tive production of mature blood cells and peripheral blood cytopenia.
We must therefore assume that the somatic mutation responsible for gain
of function at the stem cell level involves loss-of-function at the hema- Recurrent chromosomal abnormalities are
topoietic precursor level (Figure 2, step 3). RARS associated with mostly secondary genetic events with
SF3B1 mutation represents an illustrative example of gain of function at established clinical relevance in MDS
the hematopoietic stem cell level combined with loss of function (ex-
cessive apoptosis of immature red cells) at the hematopoietic precursor Recurrent chromosomal abnormalities have been very important
level.16 In CMML, the early clonal dominance of TET2 mutations has thus far for diagnosis and prognostication of MDS. Regarding
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4024 CAZZOLA et al BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25

Table 2. MDS cytogenetic scoring system: prognostic relevance of cytogenetic abnormalities in 7012 patients included in the International
Working Group for Prognosis in MDS (IWG-PM) database
Prognostic cytogenetic Proportion of MDS Median overall Median time to 25%
subgroup Cytogenetic abnormalities patients, % survival, years AML evolution, years
Very good 2Y, del(11q) 4 5.4 Not reached
Good Normal karyotype, del(5q), del(12p), del(20q), 72 4.8 9.4
double abnormality including del(5q)
Intermediate del(7q), 18, 119, i(17q), any other single or 13 2.7 2.5
double independent clones
High 27, inv(3)/t(3q)/del(3q), double including 4 1.5 1.7
27/del(7q), complex: 3 abnormalities
Very high Complex karyotype: .3 abnormalities 7 0.7 0.7

Reproduced from Greenberg et al.26

diagnosis, the detection of a cytogenetic aberration in a patient with based genotyping to screen mutational hotspots in 111 genes in 439
peripheral cytopenia and bone marrow dysplasia provides an patients with MDS. In the last 2 years, studies of whole exome or whole
important marker of clonal proliferation. By contrast, the diagnosis genome sequencing led to the discovery of mutations in the RNA
of MDS may be difcult in patients with a normal karyotype or splicing machinery,15,31,32 of SETBP1 mutations in aCML,33 and of
noninformative cytogenetics.19 CSF3R mutations in CNL.34
Recurrent chromosomal abnormalities are detected in about half Recent studies have performed a systematic analysis of panels of
of patients with MDS,20 and the most common single cytogenetic known or putative genes relevant in myelodysplasia by coupling high-
aberrations include del(5q), trisomy 8, del(20q), and monosomy 7 or throughput sample handling with massive parallel sequencing2,35 or
del(7q).20-22 These are likely secondary genetic events, deriving from by combining deep sequencing with array-based genomic hybridiza-
the genome instability caused by the founding genetic mutation.5 The tion (Seishi Ogawa, Kyoto University, personal communication,
only exception to the rule known thus far is isolated del(5q), which August 9, 2013). The panels used included from 94 to 111 candidate
characterizes the 5q- syndrome: in fact, haploinsufciency for RPS14 genes, and in the 2 largest studies enrolling .700 patients, oncogenic
and miR-145, mapping to the common deleted region, represents the mutations were identied with condence in about half of the genes
pathophysiological basis of this MDS subtype.23-25 sequenced. Accounting for both gene mutations and chromosomal
With respect to the prognostic relevance of recurrent chromosomal aberrations, the overall frequency of genetic lesions range from 78%
abnormalities, in a recent collaborative study aimed to develop the to 90%. A list of the most common recurrently mutated genes in
revised International Prognostic Scoring System (IPSS-R) for MDS, patients with MDS or MDS/MPNs, based on studies published thus
patient databases from international institutions were coalesced to far, is reported in Table 3.
assemble a combined database including 7012 patients.26 Cytogenetic
abnormalities were categorized into 5 prognostic subgroups that were
shown to have signicant prognostic relevance with different median
survival and risk of evolution into AML (Table 2).
The 5-group cytogenetic risk classication reported in Table 2 Spliceosome mutations: their unexpected
was recently found to predict the outcome of allogeneic hemato- promotion of clonal proliferation and the
poietic stem cell transplantation in MDS patients.27 In particular, concept of genetic predestination
patients with a complex karyotype (very poor cytogenetic subgroup)
had a very poor outcome after transplantation. This was also true for Pre-mRNA splicing is catalyzed by the spliceosome, a macromolecule
monosomal karyotype, dened as the karyotype of patients who had composed of 5 small nuclear RNAs associated with proteins to form
$2 autosomal monosomies or 1 monosomy in combination with particles termed small nuclear ribonucleoproteins (snRNP).16 More
other structural abnormalities.27 than 50% of patients with myelodysplasia carry somatic mutations in
Thus, chromosomal aberrations will likely continue to have spliceosome genes encoding proteins involved in the 39 splice site
clinical relevance in MDS even in the era of genomic medicine. recognition and U2 snRNP function.2 Spliceosome mutations are
rarely found in childhood myeloid neoplasms, suggesting that they are
Because they basically consist in copy number changes, their
detection will likely be improved by array-based karyotyping28 typically acquired in the elderly.36
and/or by massive parallel sequencing itself.2 Mutations of the RNA splicing machinery are largely mutually
exclusive and are most often founding events. In fact, the mutant allele
burden is typically between 40% and 50%, indicating a dominant bone
marrow clone that is heterozygous for the mutation.37,38 Mutation
hotspots have been shown in the 3 most frequently mutated genes, ie,
Somatic gene mutations in myelodysplasia: SF3B1, SRSF2, and U2AF1, and almost all described mutations are
from studies of candidate genes to whole missense, with no evidence of nonsense or frameshift changes.15,31,32
genome sequencing Different patterns of missplicing associated with the above mutant
genes have been described.32,39,40 Altogether, the available evidence
Our understanding of the molecular basis of MDS has improved suggests that spliceosome mutations affecting the 39 splice site
dramatically in the last 4 years. The rst major breakthrough was the recognition and U2 snRNP function are likely to create novel protein
identication of somatic mutations of TET2 in patients with rearrange- isoforms that can drive clonal dominance of mutated hematopoietic
ments of chromosome 4q24, where the gene maps.11,29 Subsequently, stem cells.5 This conclusion was totally unpredictable since, as
Bejar et al30 used next-generation sequencing and mass spectrometry emphasized by Vogelstein et al,6 a spliceosome mutation was
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BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25 MOLECULAR BASIS OF MYELODYSPLASIA 4025

Table 3. Most common driver genes in patients with MDS and MDS/MPN
Biological pathways and Frequency, Timing of mutation Relationship between mutant gene and Prognostic or predictive relevance of
genes %* acquisition clinical phenotype mutant gene
RNA splicing
SF3B1 15-30% More often a founding Strictly associated with ring sideroblasts Associated with good overall survival and
mutation phenotype (RARS, RARS-T) low risk of leukemic evolution
SRSF2 10-20% More often a founding Associated with RCMD or RAEB, Associated with poor overall survival and
mutation co-mutated with TET2 in CMML high risk of leukemic evolution
U2AF1 ,10% More often a founding Mainly associated with RCMD or RAEB Associated with high risk of leukemic
mutation evolution
ZRSR2 ,10% More often a founding Not defined Not defined
mutation
DNA methylation
TET2 20-30% More often a founding Found in all MDS subtypes, high mutation No impact on overall survival, may predict
mutation frequency (50-60%) in CMML response to hypomethylating agents
DNMT3A ;10% More often a founding Found in all MDS subtypes, co-mutated with Associated with unfavorable clinical outcome
mutation SF3B1 in RARS (negative prognostic relevance mitigated
by SF3B1 co-mutation in RARS)
IDH1/IDH2 ;5% More often a founding Associated with RCMD or RAEB Associated with unfavorable clinical outcome
mutation
Chromatin modification
ASXL1 15-20% More often a subclonal Associated with RCMD or RAEB, high Associated with unfavorable clinical outcome
mutation mutation frequency (40%) in CMML in all myeloid neoplasms (MDS, MDS/
MPN, MPN)
EZH2 ;5% More often a subclonal Associated with RCMD or RAEB Associated with unfavorable clinical outcome
mutation in all myeloid neoplasms
Transcription
RUNX1 ;10% Typical subclonal mutation Associated with RCMD or RAEB Associated with unfavorable clinical outcome
BCOR ,5% Typical subclonal mutation Associated with RCMD or RAEB Associated with unfavorable clinical outcome
DNA repair control
TP53 ;5% Typical subclonal mutation Associated with advanced disease and Associated with poor overall survival and
complex karyotype, mutated in 20% of high risk of leukemic evolution, predicts
patients with MDS with del(5q) poor response to lenalidomide in MDS
with del(5q)
Cohesin
STAG2 ,10% More often a subclonal Associated with RCMD or RAEB. Mutated in Associated with unfavorable clinical outcome
mutation about 10% of patients with AML
RAS pathway
CBL ,5% More often a subclonal Found in different MDS subtypes, Not defined in MDS
mutation associated with JMML in children
NRAS/KRAS ,5% More often a subclonal Found in different MDS subtypes, Not defined in MDS
mutation associated with JMML in children
NF1 ,5% More often a subclonal Found in different MDS subtypes, Not defined in MDS
mutation associated with JMML in children
DNA replication
SETBP1 ,5% More often a subclonal Found in 25% of patients with aCML and in Associated with poor overall survival and
mutation subsets of patients with advanced MDS or high risk of leukemic evolution
CMML
Receptors
CSF3R ,1% Founding driver mutation in Strictly associated with CNL, found in Mutation type may predict response to
CNL a subset of patients with aCML specific inhibitors

*Approximate proportion of patients with MDS carrying the mutant gene reported in studies published so far.
Based on values for mutant allele burden or variant allele frequency.

expected to lead to a plethora of nonspecic cellular abnormalities with multilineage dysplasia and/or excess blasts and, at variance
rather than to promote clonal proliferation. with SF3B1 mutations, predict for high risk of leukemic evolution
The different spliceosome mutations are associated with different and poor survival.41-43 SRSF2 mutations have been detected in about
phenotypes and different clinical outcomes (Table 3).2,38 Somatic one fth of cases of AML transformed from MPN44 and, in particular,
SF3B1 mutations are found almost exclusively in patients with re- have been reported in ;40% to 50% of patients with CMML, where
fractory anemia with ring sideroblasts without or with thrombocy- they are frequently associated with TET2 mutations.12,31 Somatic
tosis (RARS and RARS-T, respectively), and this clearly suggests a mutations of U2AF1 have been reported in various MDS subtypes
causal relationship between mutation and ring sideroblast formation.16 and found to be predictive of a high risk of leukemic evolution and
In addition, the vast majority of patients with SF3B1 mutation shorter survival.32,41
have a good clinical outcome with a low propensity to AML The observation that spliceosome mutations are mainly
transformation.37 SRSF2 mutations are found mainly in patients founding mutations associated with different clinical outcomes
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4026 CAZZOLA et al BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25

has led Papaemmanuil et al2 to hypothesize that they give rise to outcome in all myeloid neoplasms.46,61,62,66 Of note, ASXL1
initiating clones with different genetic predestination. More mutation has been recently incorporated into a prognostic scoring
specically, the initial driver mutation would shape the future system for CMML as a negative prognostic factor.46 Similarly,
trajectory of clonal evolution through constraints on the repertoire of EZH2 mutations were found to be signicantly associated with
cooperating genetic lesions. The molecular mechanisms underlying a shorter overall in lower risk MDS, and their incorporation in
genetic predestination remain to be dened. a prognostic model improved risk stratication in these patients.56
Several studies, recently reviewed in Blood by Issa,45 have shown
that MDS patients not only carry epigenetic effector mutations but
also an abnormal epigenome. Some of these patients respond to
Somatic mutations in genes encoding epigenetic therapy, including hypomethylating drugs (azacitidine
epigenetic regulators and decitabine) and drugs that inhibit multiple histone deacetylases.45
However, although the efcacy of some of these treatments has been
In a recent review article in Blood, Issa45 described cellular differ- demonstrated in prospective clinical trials,67 we still lack reliable
entiation as an epigenetic process that requires specic and highly predictors of response to epigenetic therapy.
ordered DNA methylation and chromatin modication programs.
The disordered differentiation of MDS is often associated with
somatic mutations in genes that control DNA methylation (TET2,
DNMT3A, and IDH1/IDH2) or regulate chromatin modication Somatic mutations in other cellular pathways
(ASXL1 and EZH2).5,45
Somatic TET2 mutations were rst described in patients with Acquired mutations of transcription factors have been described
myeloid neoplasms in 2009.11,29 TET2 is mutated in 20% to 25% of not only in AML but also in MDS.4,30 Somatic mutations of
patients with MDS2,30 and in 50% to 60% of patients with CMML.46 RUNX1 are found in ;7% to 8% of all patients with MDS and are
In an elegant study, Busque et al47 found recurrent somatic TET2 generally associated with advanced disease, severe thrombocyto-
mutations in elderly females who had clonal hematopoiesis demon- penia, and poor clinical outcome.2,30,56
strated by X-chromosome inactivation skewing but no hematologic The gene TP53, located on chromosome 17p13.1, encodes p53,
phenotype. This and other observations support the notion that TET2 which coordinates transcription programs contributing to tumor
mutation can lead per se to increased hematopoietic stem cell self- suppression, and mutant p53 proteins have been identied in various
renewal and clonal myeloid proliferation.48,49 TET2 mutations are cancers.6,68 TP53 mutations are found in about 5% of patients with
frequently found in patients with normal karyotype, and therefore MDS, mainly in subjects with advanced disease, complex karyotype,
represent a useful marker of clonality in these subjects.30 In addition, abnormalities of chromosome 17, or deletions of chromosome 5 and
co-occurrence of TET2 and SRSF2 mutation is typically found in 7.30,55 MDS patients carrying TP53 mutation have an unfavorable
CMML.2 Thus far, no prognostic relevance in terms of overall clinical outcome and a high risk of leukemic evolution,2,30 and the
survival has been clearly dened,30,50 but recent studies suggest same is true for patients with MPN.69 In particular, TP53 mutated
that TET2 mutation may predict response to hypomethylating subclones may occur at an early disease stage in MDS with del(5q),
agents.51,52 Interestingly, TET2 mutations were found to be where they are associated with a lower response to lenalidomide and
associated with reduced overall survival among patients with an increased risk of progression to AML.70
intermediate-risk AML.53 The Ras superfamily includes small GTP-binding proteins
In a study based on massively parallel DNA sequencing, Ley involved in intracellular signal transduction. Several genes of this
et al 54 found that DNMT3A mutations are highly recurrent in superfamily have been found to be mutated in patients with
patients with de novo AML and are associated with a poor myelodysplasia, including NRAS, KRAS, NF1, PTPN11, and CBL.2
outcome. Somatic DNMT3A mutations have later been detected in Somatic or germ-line mutations of RAS pathway gene are present
;10% to 15% of patients with different subtypes of MDSs.35,55,56 in ;90% of patients with juvenile CMML,71 an MDS/MPN in
They are associated with unfavorable clinical outcome and more which secondary mutations of SETBP1 and JAK3 may cause
rapid progression to AML in patients with RCMD or RAEB55 but disease progression.72
not in those with RARS, likely because the co-occurrence of the SETBP1 encodes a protein that binds the SET nuclear oncogene
SF3B1 mutation mitigates the negative effect of DNMT3A involved in DNA replication. Although heterozygous de novo
mutation.56 As suggested by Papaemmanuil et al,2 this observation germ-line mutations in SETBP1 have been shown to be associated
may indicate that some genes may only be transforming in specic with the Schinzel-Giedion midface retraction syndrome,73 somatic
genomic contexts. mutations have been recently detected in patients with myeloid
Recurrent mutations in the isocitrate dehydrogenase genes IDH1 malignancies.33,74 In particular, SETBP1 mutations are found in
and IDH2 are found in AML and MDS.5,57 In AML, co-occurrence ;25% to 30% of patients with aCML.33,75 SETBP1 has a direct role
of NPM1 and IDH1 or IDH2 mutations is associated with a good in the transcriptional regulation of other genes,76 and SETBP1 mutations
clinical outcome.53 By contrast, IDH1 mutation has been found to be are more often genetic events associated with disease progression in
associated with a short leukemia-free survival in MDS.58 MDS.74,75,77
Two genes involved in chromatin modication and regulation CSF3R encodes the receptor for colony stimulating factor 3. The
are recurrently mutated in MDS: ASXL1, which interacts with the acquisition of nonsense mutations in this gene, resulting in the
polycomb-group repressive complex 1 and 2 (PRC1, PRC2),59,60 expression of truncated CSF3R protein, has been previously found to
and EZH2, which belongs to PRC2.2,30,61-65 In cellular and animal be associated with progression to MDS/AML in patients with severe
models, ASXL1 mutations have been shown to promote myeloid congenital neutropenia.78 Activating somatic mutations in CSF3R
transformation through loss of PRC2-mediated gene repression.60 have been recently detected in ;90% of patients with CNL and
ASXL1 mutations are common not only in MDS, but also in AML, in ;40% of those with aCML.34 This study also showed that
CMML, and PMF, and are generally associated with poor clinical distinguishing between these 2 disorders may be difcult using
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BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25 MOLECULAR BASIS OF MYELODYSPLASIA 4027

the WHO criteria, whereas follow-up investigations demon-


strated that CSF3R and SETBP1 mutations are not mutually Several driver genes may cause
exclusive.34 As pointed out by Gotlib et al,79 CNL and aCML are myelodysplasia, and genotype/phenotype
likely overlapping neoplasms: although the pathogenesis of the
former is mainly characterized by CSF3R mutation, that of aCML is
relationships have been defined
likely more multifactorial. In their recent article, Vogelstein et al6 concluded that there are
Cohesin is a highly conserved 4-subunit ring structure that ;140 genes whose intragenic mutations contribute cancer. Based
encircles sister chromatids during metaphase, allowing their co- on currently available data, the number of potential myelodys-
hesion, and also plays critical roles in transcriptional regulation and plasia driver genes is likely somewhat lower (50-60). However,
post-replicative DNA repair.80 Somatic mutations in STAG2, a gene reliable estimates can be provided only by whole genome sequencing
of the cohesin complex, have been found in ;6% of MDS patients.10 studies.10,35 Whole exome sequencing and candidate gene sequenc-
In a recent work, Kon et al81 detected mutations and deletions ing are clearly less informative and may not allow the identication of
involving various genes of the cohesin complex (STAG2, RAD21,
more rarely mutated genes.
SMC1A, and SMC3) in 8% of patients with MDS, 10% of those with
The patient cohorts studied thus far are heterogeneous, and all
CMML, and 12% of those with AML. A similar frequency was
studies are basically retrospective. Taking into account these limita-
previously reported in AML patients,3 suggesting that altered cohesin
tions, only 4 to 6 genes (SF3B1, TET2, SRSF2, ASXL1, DNMT3A, and
function plays a role in myeloid leukemogenesis.
RUNX1) have been found to be consistently mutated in $10% of MDS
The gene BCOR, located on chromosome Xp11.4, encodes
patients, whereas a long tail of about 40 to 50 additional genes are
a corepressor of BCL6, a POZ/zinc nger transcription repressor that
mutated in smaller subsets.2 In the study by Walter et al,35 the 2 most
is required for germinal center formation and may inuence apoptosis.
frequently mutated genes were TP53 and U2AF1: this likely reects the
Germ-line mutations of this gene are associated with oculofaciocar-
fact that their patient cohort included a low proportion of low risk and
diodental and Lenz microphthalmia syndromes.82 Inactivating somatic
a high proportion of high-risk MDS subtypes.
mutations of BCOR have been described in AML with normal
At presentation, most MDS patients have 2 or 3 oncogenic driver
karyotype83 and more recently in a subset of patients with MDS.2,84
mutations and hundreds of background or passenger mutations.
These are typical subclonal driver mutations, associated with a poor
Considering the variant allele frequency, some mutant genes, typically
clinical outcome.84
those of RNA splicing and DNA methylation, appear to be mainly
associated with the initial clonal proliferation, whereas others are
mainly involved in subclonal evolution (Table 3). However, the
temporal order of acquisition of driver mutations is not xed and varies
Familial MDSs and genetic predisposition to from subject to subject. Thus, the same mutant gene, eg, TET2, may be
acquisition of somatic mutations associated an early driver in some patients and a subclonal driver in others. Walter
et al35 observed that mutations in driver genes belonging to the same
with myelodysplasia: germ-line
biologic pathway tended not to co-occur, suggesting that a second
GATA2 mutations
mutation in the same pathway provides no additional growth advantage
For a deeper analysis of this subject, the reader is referred to a com- or is not even tolerated.
prehensive review article by Liew and Owen.85 Familial syndromes According to Vogelstein et al,6 most human cancers are caused
predisposing to MDS or AML include bone marrow failure inherited by 2 to 8 sequential genetic lesions that develop over the course of 20
disorders (Diamond-Blackfan, dyskeratosis congenita, severe congen- to 30 years. In their studies of whole genome sequencing, Welch
ital neutropenia), DNA repair deciency syndromes, Noonan syndrome et al3 estimated that as few as 2 key somatic mutations are needed to
and neurobromatosis I, Down syndrome, and familial platelet disorder cause the malignant founding clone and clinically manifest AML.
with propensity to myeloid malignancy (associated with germ-line The available evidence suggests that this may apply also to MDS: at
mutations of RUNX1 or CEBPA). variance with AML, however, the genetic lesions responsible for
More recently, germ-line mutations of GATA2 have been MDS likely occur sequentially over years, rather than over months
described in familial syndromes characterized by predisposition or weeks, at least in low-risk subtypes with long natural history of
to MDS and AML.86 In 3 families, subjects carrying the GATA2 disease, as is typically RARS.92
(C1061T) mutation had macrocytic anemia and developed MDS/ Unfortunately only few functional studies linking the various mutant
AML between the second and fth decade of life. Various germ- genes to a cellular or disease phenotype have been performed. In an
line mutations of GATA2 have been detected in patients with the animal model of conditional Tet2 loss, Tet2 haploinsufciency was
Emberger syndrome, characterized by primary lymphedema shown to lead to a disorder resembling human CMML.48 Another study
associated with a predisposition to AML. 87 Finally, several reported ndings suggesting that SF3B1 or Sf3b1 haploinsufciency
germ-line mutations in GATA2 have been reported to be associated leads to ring sideroblast formation in human cells or heterozygous
with the autosomal dominant and sporadic monocytopenia and knockout mice, respectively.93 The CSF3R (T618I) mutation has been
mycobacterial infection (MonoMAC) syndrome, which predis- shown to drive a lethal myeloproliferative disorder in a murine model.94
poses to myeloid malignancy.88-90 Typically these patients have Genotype/phenotype relationships have been dened in MDS,
a combination of severe monocytopenia with mild neutropenia and MDS/MPN, and related myeloid neoplasms. A tentative schematic
marginally reduced hemoglobin level, and progression to MDS/ representation of our current knowledge of these relationships is
AML typically occurs in the second or third decade of life.90 It reported in Figure 3. Although reliable conclusions will be made
should be noted that, although germ-line mutations of both RUNX1 possible only by prospective studies, this scheme provides a proof
or GATA2 may predispose to MDS,91 somatic mutations of the of concept of the potential feasibility of a molecular classication
same genes may represent mechanisms of disease progression in of MDS and related myeloid neoplasms. More specically, SF3B1
myeloid neoplasms.2 mutation appears to be strictly associated with refractory anemia
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4028 CAZZOLA et al BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25

Figure 3. Schematic representation of our current knowledge of genotype/phenotype relationships in MDS, MDS/MPN, and a related MPN like CNL. The SF3B1
mutation is strictly associated with RARS, whereas the combination of the SF3B1 mutation with subclonal driver mutations in JAK2 or MPL is associated with RARS-T. Thus
far, no conclusive genotype/phenotype relationship has been defined within refractory cytopenia with unilineage dysplasia. Various combinations of founding and subclonal
driver mutations can be found in RCMD and RAEB. CMML has a relatively well-defined molecular basis, involving primarily somatic mutations of TET2 and SRSF2:
comutation of these genes is almost invariably associated with CMML, whereas the ASXL1 mutation involves poor outcome. Within MDS/MPN, aCML is characterized by
various founding mutations plus a subclonal mutation of SETBP1. Activating mutations of CSF3R are strictly associated with CNL.

with ring sideroblasts with or without marked thrombocytosis,15,37 listed in Table 1. This is primarily true for AML,3,4,53 al-
the combination of SRSF2 and TET2 mutation with CMML,2 though, as underlined by Walter et al,35 some genes are over-
and an activating CSF3R mutation with CNL.34 Refractory represented in MDS compared with AML and vice versa.
anemia has no peculiar genotype thus far, raising the question Indeed, in the recent study by the Cancer Genome Atlas Re-
as to whether it should be considered as a separate entity. RCMD search Network, the 20 most recurrently mutated genes in
and refractory anemia with blast excess can be associated with AML were FLT3, NPM1, DNMT3A, IDH1/2, TET2, RUNX1,
different combinations of founding mutations, primarily in- TP53, N/KRAS, CEPBPA, WT1, PTPN11, KIT, U2AF1, SMC1A,
volving genes of RNA splicing (with the only exclusion of SMC3, PHF6, STAG2, and RAD21.4 Only half of these genes
SF3B1) or epigenetic regulation and subclonal driver mutations. are within the 20 most recurrently mutated ones in MDS
Interestingly, as shown in Table 4, a molecular classication appears (Table 3). Although the molecular pathophysiology of MDS is
to be already feasible in MDS/MPN, a group of myeloid neoplasms thus different from that of AML, some AML driver genes might
far dened on the basis of cumbersome clinical, hematologic, and behave as subclonal drivers in MDS and thereby drive leukemic
morphologic criteria.1 It is also apparent that specic driver genes are transformation.
responsible for the myeloproliferative component of the different MDS/ Somatic mutations of CBL, TET2, ASXL1, and IDH/IDH2
MPN, like JAK2 or MPL in RARS-T, SETBP1 in aCML, and CSF3R in have been detected in the advanced phase of chronic myeloid
CNL. We have previously shown that RARS-T develops from RARS leukemia.96 Several of the genes reported in Table 3 can also be
through the occurrence of a subclonal driver mutation in JAK2 or mutated in PMF, in combination with JAK2 (V617F) or MPL
MPL in the initial SF3B1 mutated clone.37,95 A similar evolutionary exon 10 mutations, and comutation has been shown to have a
process likely operates in CMML and aCML, which may develop negative impact on the clinical course of this MPN.66 Recently,
from a preexisting MDS through the acquisition of subclonal somatic mutations of TET2, SRSF2, ASXL1, CBL, and RUNX1
driver mutations that cause monocytosis and granulocytic have been detected in ;90% of patients with advanced mastocy-
leukocytosis, respectively. tosis, and overall survival was found to be signicantly shorter in
patients with additional mutations than in those carrying KIT
(D816V) only. 97
Myelodysplasia driver genes are recurrently Myelodysplasia driver genes may also interact with somatic
mutated also in other myeloid neoplasms mutations involving lymphoid cell lines, thus giving rise to
peculiar phenotypes. T-cell large granular lymphocytic leuke-
The driver genes whose mutations are responsible for MDS mia is characterized by clonal expansion of CD31 cytotoxic T
(Table 3) are frequently mutated also in other myeloid neoplasms lymphocytes and may be associated with autoimmune disorders
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BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25 MOLECULAR BASIS OF MYELODYSPLASIA 4029

Table 4. Somatic mutations that characterize the different types of MDS/MPN


Myeloid neoplasm according to the 2008 WHO
classification Main diagnostic/clinical feature(s) Most common mutant gene(s)
CMML (classified as MDS/MPN) Persistent peripheral blood monocytosis (.1 3 109/L). Co-occurrence of TET2 and SRSF2 mutations, or
combinations of ASXL1 mutations with other
mutant driver genes. ASXL1 mutation is
associated with poor overall survival and high
risk of progression to AML
aCML (classified as MDS/MPN) Peripheral leukocytosis ($13 3 109/L) with Combinations of founding mutations in various genes
dysgranulopoiesis and 10% or more circulating and subclonal mutations of SETBP1 or ASXL1.
immature granulocytes.
CNL (classified as MPN) Neutrophilic leukocytosis ($25 3 109/L) with less than 10% Activating somatic mutations of CSF3R (encoding
circulating immature granulocytes. the receptor for G-CSF) in the vast majority of
patients. Oncogenic lesions can be classified as
truncation or membrane proximal mutations,
involving different preferential downstream
kinase signaling.
JMML (classified as MDS/MPN) Persistent peripheral blood monocytosis (.1 3 109/L) in Somatic mutations of the RAS pathway (NRAS,
children. KRAS, NF1, PTPN11, and CBL). Heterozygous
germ-line CBL mutations may predispose to
JMML. Subclonal driver mutations of SETBP1
and JAK3 may cause disease progression.
RARS-T (classified as a provisional entity within Macrocytic anemia, ring sideroblasts, and thrombocytosis. Combinations of a founding somatic mutation of
MDS/MPN) SF3B1 and subclonal driver mutations of JAK2 or
MPL (and likely of other as-yet-unknown genes).

CNL has been included because of its overlapping features with aCML.

and immune-mediated cytopenias.98 The expansion of clonal shown to contribute to cytopenia in a subset of MDS patients,
T cells has been shown to be caused by somatic mutations of and these patients may benet from immunosuppressive treatment.101
STAT3 or STAT5b.99,100 Autoimmune processes have been Interestingly, a recent study describes the occurrence of STAT3

Figure 4. Current approach to diagnosis and prognostication of MDS and MDS/MPN and a hypothetical future approach based on massive parallel sequencing.
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4030 CAZZOLA et al BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25

Prognostic and predictive significance of


driver mutations and development of
molecular models for clinical decision making
As shown in Figure 5, the current WHO classication of MDS has
valuable prognostic relevance, with blast percentage and multi-
lineage dysplasia representing the most relevant parameters from
this point of view. However, the reproducibility of these latter
parameters is far from optimal,105 and there is a need for more robust
prognostic factors. The IPSS-R26 clearly represents a step forward
but does take into account only cytogenetic abnormalities, which are
secondary genetic events and not the driver lesions.
The denition of founding and subclonal driver mutations might
considerably improve prognostication of MDS and more generally
clinical decision-making in this eld. First, the identication of the
mutant gene responsible for the initial clone is relevant to clinical
outcome. For instance, ring sideroblasts may be found not only in
patients with a founding mutation in SF3B1, but also in those with an
initiating oncogenic lesion in SRSF2.31 However, the median
leukemia-free survival is .10 years in the former vs ,2 years in the
latter.2,37 Second, the detection of subclonal driver mutations
associated with small clones may allow early diagnosis of disease
progression, including evolution into AML. In chronic lymphocytic
leukemia, the presence of subclonal driver mutations adversely
impacts clinical outcome.106 Third, the number of driver mutations
Figure 5. Kaplan-Meier analysis of overall survival and leukemia-free survival of per patient represents an important prognostic factor per se. In the
1110 patients diagnosed with MDS at the Department of Hematology Oncology,
Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, between 1990 and 2012.
recent study by Papaemmanuil et al,2 the median leukemia-free
MDS patients are stratified according to the 2008 WHO classification categories. survival was .3 years in patients with 1 or 2 driver mutations vs ,2
Multilineage dysplasia and excess of blasts have a considerable impact on outcomes. years in patients with $3 driver mutations.
A few studies have already suggested that incorporation of
mutated T-cell clones in a subset of patients with MDS, suggesting somatic mutations into prognostic scoring systems can improve
that this may represent a molecular mechanism for the occurrence of prognostication of MDS.30,56 Solary and coworkers46 have pro-
autoimmune phenomena.102 posed a new prognostic score for CMML that includes not only age
and hematologic parameters, but also ASXL1 mutations status.
Under the aegis of the MDS Foundation, the International Working
Relevance of gene mutations in the diagnostic Group for Prognosis in MDS has started a research project aimed to
approach to myelodysplasia: toward develop a prognostic scoring system (IPSS-Mol) that includes
a molecular classification of clinical, hematologic, and molecular parameters. Our preliminary
evidence suggests that parameters such as hemoglobin level, blast
myeloid neoplasms
count, and high-risk cytogenetic abnormalities will continue to
The current diagnostic approach to MDS includes peripheral blood retain strong independent prognostic value (M.C., L.M., and M.G.D.P.,
and bone marrow morphology to evaluate abnormalities of peripheral unpublished data, August 8, 2013). Additional parameters that may
blood cells and hematopoietic precursors (Figure 1), bone marrow signicantly contribute to a rened risk assessment of MDS include
biopsy to assess marrow cellularity, brosis, and topography,103 and gene expression proling-based signatures.107
cytogenetics to identify nonrandom chromosomal abnormalities Finally, characterization of the patients genome may guide ther-
(Table 2).104 apeutic programs, and its inclusion in prospective clinical trials is
Massive parallel sequencing has the potential of dramatically therefore of crucial importance. TET2 mutations might be associated
improving our approach to diagnosis of MDS as illustrated in with response to hypomethylating agents,52 whereas U2AF1 muta-
Figure 4. The price of whole genome sequencing is expected to drop tions would independently predict for poor outcome after allogeneic
below $1000 in the next years, whereas that of targeted gene stem cell transplantation.108 There is considerable therapeutic potential
sequencing2 should be denitely lower. Deep sequencing may allow for epigenetic-targeted therapies in AML,109 and this may be also true
the simultaneous detection of both somatic gene mutations and copy in MDS. Several drugs that target the spliceosome are being in-
number changes, the cytogenetic abnormalities typical of MDS, in vestigated for potential use in various malignancies,110 whereas drugs
a single assay.2 Although whole genome sequencing is clearly more targeting oncogenic Ras signaling111 might be useful in many myeloid
informative, massive parallel sequencing of a panel of myeloid genes is neoplasms. Case reports suggest that ruxolitinib may be effective in
more feasible in a clinical laboratory. The genes to be sequenced may CNL associated with CSF3R mutation34 and in chronic eosinophilic
include ;50 to 60 myelodysplasia driver genes,2,35 genes associated leukemia associated with a PCM1-JAK2 fusion gene.112 More
with inherited disorders that predispose to MDS, and a reasonable generally, the identication of the biologic pathways that are
number of germ-line single nucleotide polymorphisms that allow activated by mutation might allow personalized treatment in the
reliable detection of copy number changes from sequencing data.2 individual patient with myelodysplasia. It should also be considered
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BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25 MOLECULAR BASIS OF MYELODYSPLASIA 4031

that characterization of the patients genome before and after Clonal dominance: A condition in which most cells of a tissue belong to a clone. In
treatment may allow a correct assessment of response, in particular, myelodysplastic syndromes, about 85-90% of bone marrow cells are clonally
the impact of treatment on clonal pattern of hematopoiesis. derived.
Subclonal evolution or expansion. The process by which the founding malignant
For the above expectations to be realized, functional studies of
clone generates subclones through the acquisition of additional driver mutations.
the mutant genes and prospective clinical trials that include the
In some patients, the order of genetic changes, i.e., the temporal order of
molecular characterization of the patients genome are now needed. acquisition of driver mutations, can be inferred by means of massive parallel
sequencing though calculation of the mutant allele burden or variant allele
frequency (VAF).
Intraclonal (intratumoral) heterogeneity. A founding malignant clone that has
Acknowledgments
undergone subclonal evolution is no longer monoclonal in the strict sense, but is
instead a mosaic of several clones/genomes with different sets of somatic
The authors thank the many colleagues of the Chronic Myeloid
mutations.
Disorders Working Group of the International Cancer Genome
Mutation: A change of the nucleotide sequence of the genome.
Consortium and those of the Associazione Italiana per la Ricerca Germline mutation: A mutation that is inherited though a germ cell (oocyte or
sul Cancro (AIRC) Gruppo Italiano Malattie Mieloproliferative spermatozoon) at the time of conception, and is therefore present in all cells of
(AGIMM). a developed body.
The studies on the genetic basis of myeloid neoplasms conducted Somatic mutation: A mutation that occurs in a non-germ cell of a body after
at the Department of Hematology Oncology, Fondazione Istituto di conception (the ancient Greek somatikos means of the body).
Ricovero e Cura a Carattere Scientico Policlinico San Matteo, and Nonsynonymous mutation: A nucleotide mutation that alters the amino acid
the Department of Molecular Medicine, University of Pavia, were sequence of a protein.
Driver mutation: A mutation that causes a selective advantage in a cell
supported by grants from AIRC, Fondazione Cariplo, Fondo per gli
with capacity for self-renewal, leading to formation of a clone of mutated
Investimenti della Ricerca di Base (FIRB, project RBAP11CZLK),
cells.
and Ministero dellIstruzione, dellUniversita e della Ricerca
2 Founding or initiating driver mutation: A driver mutation that gives rise to the
(PRIN 2010-2011) (to M.C.) and Fondazione Berlucchi (to M.G.D.P.). initial clone of a malignancy.
In particular, M.C. acknowledges funding from the AIRC Special 2 Subclonal or cooperating driver mutation: A driver mutation that occurs in
Program Molecular Clinical Oncology 5 per mille (project 1005). a cell of an already established clone and generates a subclone carrying both the
founding and the newly acquired mutation.
Background or passenger mutation: A mutation that occurs in a tissue before
neoplastic transformation and has no pathophysiological significance. In
Authorship most tissues, including hematopoietic stem cells, the number of passenger
mutations is a function of age. Whole genome sequencing studies have
Contribution: M.C. conceived this review article, analyzed the shown that in hematopoietic stem cells, their number may range from about
100 to 1000, depending on age. When an initiating driver mutation gives rise
literature, wrote the manuscript, and prepared the illustrations; and
to a malignant clone (neoplastic transformation), all background or
M.G.D.P. and L.M. analyzed the literature and contributed to the
passenger mutations present at that time are captured and carried forward.
manuscript preparation. Additional passengers mutations can be captured during subclonal
Conict-of-interest disclosure: The authors declare no compet- evolution.
ing nancial interests. Mutant allele burden or VAF: The relative proportion of a mutant or variant
Correspondence: Mario Cazzola, Department of Hematology allele (i.e., the allele carrying a somatic mutation) in a tissue or tumor
Oncology, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, sample. The mutant allele burden can be estimated using various
Italy; e-mail: mario.cazzola@unipv.it. approaches: i) by means of allele-specific quantitative PCR [e.g., the
procedure employed for estimating the proportion of JAK2 (V617F)-mutant
alleles in granulocytes from a patient with polycythemia vera]; or ii) more
directly by assessing variant and wild-type reads using next-generation
sequencing. A mutant allele burden or VAF of about 50% in regions of diploid
Appendix: Definitions for basic terms used in DNA content in a homogeneous cell population (e.g., circulating
studies of the genetic basis of granulocytes in myeloid neoplasms) suggest a fully clonal population of cells
myeloid neoplasms that are heterozygous for the mutation. In myeloid neoplasms, the mutant
allele burden in a bone marrow sample is most commonly between 40 and
Clone: A group of cells that derive from a common parent cell and share its genome. 50% due to the presence of non-myeloid cells.
Malignant clone: A clone generated by the occurrence in the parent cell of Variant-allele clusters: Group of mutation with similar VAF, potentially belonging to
a somatic mutation that alters the cell biology and function. the same clone or subclone.
Founding or initiating clone: A malignant clone generated by a founding somatic Whole exome sequencing: A procedure that allows to sequence all the coding
mutation. regions (exomes) of a genome.
Subclone: A clone generated by the occurrence of an additional driver mutation in Whole genome sequencing: A procedure that allows to sequence the coding and
a cell of an already established clone. non coding regions of a genome.
Clonal architecture: Clonal composition of a neoplasm based on its clonal origin For deeper insight into these concepts, the reader is referred to recent articles by
and subclonal evolution. Ley and colleagues3,4 and Vogelstein et al.6
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4032 CAZZOLA et al BLOOD, 12 DECEMBER 2013 x VOLUME 122, NUMBER 25

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2013 122: 4021-4034


doi:10.1182/blood-2013-09-381665 originally published
online October 17, 2013

The genetic basis of myelodysplasia and its clinical relevance


Mario Cazzola, Matteo G. Della Porta and Luca Malcovati

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