Myelodysplastic Syndrome

RECENT ADVANCES IN HEMATOLOGY RESEARCH
MYELODYSPLASTIC SYNDROMES
(MDS)
RISK FACTORS, TREATMENT
AND PROGNOSIS
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RECENT ADVANCES IN
HEMATOLOGY RESEARCH
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RECENT ADVANCES IN HEMATOLOGY RESEARCH
(MDS)
RISK FACTORS, TREATMENT

AND PROGNOSIS
DEANNA RODGERS
EDITOR
New York
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CONTENTS
Preface vii
Chapter 1 From Risk Factors to Prognosis in Myelodysplasic Syndromes:
The Role of Genetic Variance 1
Ana Bela Sarmento-Ribeiro, Ana Cristina Gonalves,
Andr Barbosa Ribeiro, Emlia Nobre Barata Roxo Corteso
and Jos Manuel Nascimento Costa
Chapter 2 The Inflammatory and Autoimmune Nature of
Myelodysplastic Syndromes 27
Ota Fuchs
Chapter 3 Pathogenesis of 5q- Syndrome 51
Ota Fuchs
Chapter 4 Genetic Mutations Identified in Myelodysplastic Syndromes and
Their Significance 87
Ota Fuchs
Chapter 5 Risk Factors and Prognostic Scoring Systems in Argentinean
Patients with MDS: A Multicentric Study 115
Carolina B. Belli, Jacqueline Gonzalez, Virna Barcal,
Marcela de Dios Soler, Marcelo Iastrebner
and Mara Gabriela Flores
Chapter 6 Epigenetics and Epigenetic Therapy of Myelodysplastic Syndromes 141
Ota Fuchs
Chapter 7 Introduction of Mild Oral Chemotherapy for Elderly Patients with
Higher-Risk Myelodysplastic Syndromes 175
Akira Horikoshi
Chapter 8 Lenalidomide Treatment in Lower Risk
Ota Fuchs
vi Contents
Chapter 9 The Old and the New-Integrating Prognostic Models and

Mutational Advances with Epigenetic and Cellular Therapies for
Uma Borate and Antonio Di Stasi
Chapter 10 Entropy Evaluation of Bone Marrow Biopsies in
Giorgio Bianciardi and Pietro Luzi
Index 251
PREFACE
Myelodysplastic syndrome (MDS) is a family of clonal haematopoietic stem cells

disorders characterized by dysplasia, ineffective hematopoiesis and susceptibility to
transformation to Acute Myeloblastic Leukaemia (AML) that are shown to be strikingly
refractory to current therapeutic modalities. The first chapter of this book provides a detailed
review of the risk factors, treatment options and prognosis of MDS. Chapter two studies the
inflammatory and autoimmune nature of MDS. Chapter three discusses the pathogenesis of
5q-syndrome. Chapter four examines the genetic mutations identified in MDS and their
significance. Chapter five reviews different prognostic factors and stratifications of risk in
Argentinean patients with MDS. Chapter six discusses epigenetics and epigenetic therapy.
Chapter seven introduces mild oral chemotherapy treatments for elderly patients with a
higher-risk myelodyspastic syndrome. Chapter eight discusses lenalidomide treatment in
lower risk myelodysplastic syndromes. Chapter nine analyzes the old and new-integrating
prognostic models and mutational advances with epigenetic and cellular therapies for MDS.
The last chapter studies the entropy evaluation of bone marrow biopsies in MDS.
In: Myelodysplastic Syndromes (MDS) ISBN: 978-1-63484-329-4
Editor: Deanna Rodgers 2016 Nova Science Publishers, Inc.
Chapter 1
FROM RISK FACTORS TO PROGNOSIS IN

MYELODYSPLASIC SYNDROMES:
THE ROLE OF GENETIC VARIANCE
Ana Bela Sarmento-Ribeiro1,2,3,4,, MD, PhD,

Ana Cristina Gonalves1,2,4, MSc, Andr Barbosa Ribeiro1,3, MD,
Emlia Nobre Barata Roxo Corteso1,2,3, MD, PhD,
and Jos Manuel Nascimento Costa2,5,6, MD, PhD
1
Oncobiology and Hematology Lab, University Clinic of Haematology and Applied
Molecular Biology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal
2
Center of Investigation in Environment, Genetics and Oncobiology (CIMAGO),
University of Coimbra, Coimbra, Portugal
3
Clinical Hematology Service, Centro Hospitalar
Universitrio de Coimbra (CHUC), Coimbra, Portugal
4
Center for Neuroscience and Cell Biology and Institute for Biomedical Imaging and Life
Sciences (CNC.IBILI), University of Coimbra, Coimbra, Portugal
5
University Clinic of Oncology, Faculty of Medicine,
University of Coimbra, Coimbra, Portugal
6
Clinical Oncology Service, Centro Hospitalar
Universitrio de Coimbra (CHUC), Coimbra, Portugal
ABSTRACT
Myelodysplastic syndrome (MDS) is a family of clonal haematopoietic stem cells
disorders characterized by dysplasia, ineffective hematopoiesis and susceptibility to
transformation to Acute Myeloblastic Leukaemia (AML) that are shown to be strikingly
refractory to current therapeutic modalities.
Ana Bela Sarmento-Ribeiro, MD, PhD, Adress: Faculty of Medicine, University of Coimbra, Azinhaga de Sta
Comba-Celas, 3000-548 Coimbra, Portugal, E-mail: absarmento@fmed. uc.pt Tm: 00351917835675.
2 A. B. Sarmento-Ribeiro, A. C. Gonalves, A. Barbosa Ribeiro et al.
Mechanisms of this complex disease include pluripotent stem cell injury with
multistep pathogenesis and deranged haematopoietic proliferation, differentiation, and
apoptosis.
Genetic background, namely single nucleotide polymorphisms (SNP), and epigenetic
abnormalities, including genes involved in cell cycle regulation, oxidative stress and
apoptosis, may affect the individual risk of MDS development and progression to AML.
Furthermore, dietary factors such as folate, vitamin B12 and methionine mediate one
carbon metabolism interfering with the supply of methyl groups, and therefore, the
biochemical pathways of methylation processes. Polymorphisms in key genes of one-
carbon metabolism, as well as in nutrient transporters, can also influence DNA
methylation.
The etiology and the latency time of most cases of primary MDS remain unknown.
The fact that the age of presentation is late may indicate that the aging process has a role
this entity. A substantial proportion of these complex diseases arise in the setting of
exposures to environmental or occupational toxins, including cytotoxic therapy for a prior
malignancy or other disorder. In fact, in a minority of cases, previous exposure to
chemotherapy or ionizing radiation, are identified as risk factors (SMD secondary or
related therapy). Benzene has been reported as one of environmental toxicants often
associated with the development of MDS. The existence of polymorphisms of certain
genes involved in the metabolism of environmental factors has been related to the high
susceptibility for the occurrence of MDS. Furthermore, some authors have reported an
association with autoimmune disease, to precede or coincide with the diagnosis of MDS.
The International Prognostic Score System (IPSS) is a prognostic rating system for
MDS patients classified according to WHO criteria. It is based on percentage of blasts in
the marrow, cytogenetic pattern and the number of cytopenias. It is been useful to
evaluate the survival and the risk of progression for acute leukemia, facilitating
therapeutic clinical decision. However, other prognostic system have been developed as
IPSS-R, marrow fibrosis and LDH levels, among others.
Until now MDS is a therapeutic challenge because the only curative treatment is
bone marrow transplantation. But, the majority of these patients are of advanced age and
because of that more susceptible to the adverse side effects of chemotherapies. Targeted
therapies could be an alternative option.
For the majority of elderly patients, supportive treatment is the treatment of choice.
For the treatment of high-risk MDS is indicated the hypomethylating drug, azacytidine.
However other drugs may be used as lenalidomide, an immunomodulatory and
antiangiogenic drug. Another therapeutic approach, also used in aplastic anemia is
isantithymocyte globulin.
MDS remain an inadequately characterized disorder and novel strategies for studying
disease pathogenesis and progression continue to shape our understanding and approach
to diagnosing and treating. Understanding the factors that contribute to MDS
development may facilitate the development of strategies for disease prevention and
control.
DEFINITION AND EPIDEMIOLOGY

Myelodysplastic syndromes (MDS) are a family of clonal hematopoietic stem cell
malignancies characterized by a hypercellular bone marrow with ineffective hematopoiesis
and dysplasia, resulting in peripheral blood cytopenias and high susceptibility to
transformation into Acute Myeloblastic Leukemia (AML). The term MDS arose in 1982
when the French-American-British co-operative group recognized this heterogeneous group
From Risk factors to Prognosis in Myelodysplasic Syndromes 3
of malignancies, currently included in the World Health Organization (WHO) Classification

of Hematological Malignancies.
MDS are the most common hematological neoplasias in the elderly, with a median age of
70 years, and may arise de novo, probably due to the prolonged effect of leukemogenic agents
or may be secondary to previous radiation or chemotherapy exposure. In fact, excepting those
secondary to previous therapy, MDS are rare before the age of 50 years.
However, it may occur at any age, including in the early childhood (estimated incidence
of 1 in a million per year between the age of 5 months to 15-year-old) in which most cases are
associated to hereditary diseases predisposing to the development of MDS, such as Down
Syndrome, Bloom Syndrome, Schwachman-Diamond Syndrome and Fanconi Anemia,
usually presenting with different features from those of adulthood MDS. On the other hand,
the MDS subtypes Refractory Anemia with Ring Sideroblasts (RARS) and the 5q-Syndrome
are rarely seen in children, as opposed to chromosome 7 aberrations, much more frequent in
children (up to 30% of MDS in children) and only present in about 10% of MDS adulthood.
The real incidence of MDS is difficult to establish for several reasons, in particular the
limited number of statistical studies and the existence of patients with mild cytopenias,
potentially MDS, that dont undergo further investigation. European studies reveal the
incidence rate worldwide to be of 3 to 5 cases in 100 000 individuals per year in the general
population, increasing to 20 to 50 cases in 100 000 individuals per year after the age of 70. In
the United States of America 15 000 new cases are diagnosed each year, placing MDS at least
as common as Chronic Lymphocytic Leukemia, the most common leukemia in the western
world (9). Despite the initial increase in the incidence of MDS probably due to increased
diagnostic accuracy and the tendency to investigate patients with cytopenias, the current
incidence rate is remaining stable. MDS is slightly more common in the male gender with a
male: female ratio of 1.4:1.
ETIOLOGY AND RISK FACTORS

The majority of cases of primary MDS are of unknown etiology and neither is known
about the latency time. The late-onset of the disease may indicate that marrow aging may play
a role in these entities. Mitochondrial-mediated oxidative stress has been implicated in the
marrow aging process and despite mitochondrial alterations are fairly well described in the
RARS subtype its relationship to the aging process hasnt been possible. On the other hand,
some families have been described to have more than one member to develop MDS but there
is no evidence that these diseases may have a hereditary basis.
A minority of cases (15%) develops after previous exposure to chemotherapy or ionizing
radiation and these cases are classified as therapy-related MDS (t-MDS) or secondary MDS,
together with therapy-related AML (t-MDS/t-AML) in the WHO classification of AML and
precursor lesions. In these cases the latency time varies from 1 to 41 years in those previously
exposed to radiation and from 1 to 10 years after exposure to alkylating agents. The most
common subtypes of t-MDS are RARS and Chronic Myelomonocytic Leukemia (CMML).
These patients also have a lower survival rate when compared to those with primary MDS - 7
months vs. 32 months, respectively. Chromosomal aberrations are qualitatively similar to de
novo MDS but are present in different proportions, as well as genetic mutations (Table 1).
Until now more than 60 mutated genes have been identified in MDS patients. The most
common mutations detected in these patients occur in genes involved in RNA splicing
(PRPF40B, SF1, SF3A1, SF3B1, SRSF2, U2AF1, U2AF35, U2AF65, and ZRSR2), DNA
methylation (TET2, DNMT3A, and IDH1/IDH2), chromatin modification (ASXL1 and EZH2),
as well as transcription (ETV6, RUNX1, BCOR, NPM1, and TP53) and cell cycle regulation
(CDKN2A and PTEN). Moreover, some MDS display mutations in genes that encode signal
transduction proteins, such as BRAF, CBL, GNAS, JAK2, PTPN11, NRAS, KRAS, and NF1
genes.
Table 1. Mutations in SMD
Gene Frequency in MDS (%) MDS associated risk
DNMT3A 8 Poor
IDH1/2 4-11 Unclear
TET2 19-26 Neutral
EZH2 6 Poor
ASXL1 14 Poor
SF3B1 80 (RARS); 6 (MDS without RS) Good
SRSF2 2 (RARS); 12 (MDS without RS) Poor
U2AF35 12 (MDS without RS) Unclear
RUNX1 9 Poor
TP53 8 Very poor
NRAS 4 Poor
JAK2 3 Neutral
ETV6 3 Poor
CBL 2 Poor
NPM1 2 Unclear
KRAS 1 Poor
RS - ring sideroblasts; RARS - refractory anemia with ring sideroblasts; MDS - myelodysplastic
syndrome. (Adapted from Bejar R., 2014; Larsson C. A., 2013; Pedersen- Bjegaard et al., 2007.)
Ionizing radiation is a well known leukemogenic agent whose effects are dependent on
dosing and duration of exposure. It is known to cause DNA breaks resulting in chromosomal
deletions or translocations. A study regarding hematological neoplasias among atomic bomb
survivors shows the association of AML1 (acute myeloid leukemia 1) gene mutations with
MDS and/or AML.
The most frequent cytostatic drugs associated to MDS are Alkylating Agents and
Topoisomerase II Inhibitors, frequently used in lymphomas and solid tumors treatment. The
risk increases with age and is proportional to duration of exposure, the maximum incidence
being of about five to years after exposure with a time interval varying from 1 to 10 years.
These drugs can cause DNA damage as well as to the DNA repair systems, leading to loss of
chromosomal integrity. Besides, in patients with Lymphoma, Multiple Myeloma or solid
neoplasias who underwent autologous bone marrow transplant the risk of developing MDS
related to the conditioning regimen varies from 1 to 12%, most happening 5 years after the
transplant and the risk factors include age and the conditioning regimen. Alkylating agents
may cause deletion, monosomy or loss of the long arm of chromosomes 5 or 7 as a
consequence of the grater susceptibility to centromeric breaks following exposure to these
drugs.
There are some cases of MDS in patients previously treated for other myeloid
hematological neoplasias, particularly acute promyelocytic leukemia due to its high survival
rate. The existence of other marrow failure diseases such as Fanconi Anemia or Aplastic
Anemia may increase the predisposition to develop MDS.
Recently, a new theory has stated that polymorphisms, namely single nucleotide
polymorphisms (SNP), in genes that code proteins related to the metabolization of
environmental toxics may confer greater susceptibility to develop MDS. Some of these
enzymes are related to carcinogenic metabolism (gluthathion-S-transferase), oxidative stress
metabolism (superoxide dismutase - SOD, catalase and glutathione peroxidase - GPx) and
DNA repair (RAD51). Our results show that polymorphisms in SOD2 (rsrs4880) and GPX1
(rs1050450) genes, which encode antioxidant enzymes, increased the susceptibility of MDS
development, supporting a possible role of oxidative stress in MDS etiology. Moreover, we
also found that OGG1 (rs1052133) and NEIL1 (rs5745920) gene variants, that encode DNA
repair glycosylases, confer susceptibility to MDS development. This association may explain
the presence of chromosomal breaks, DNA double-strand breaks and elevated levels of 8-
hydroxy-2-deoxyguanosine (8-OHdG) observed in MDS patients.
Benzene is one of the environmental toxics usually associated to MDS development. The
major sources of daily low concentration benzene exposure are cigarette smoke and gasoline.
Results in vitro support that benzene in low concentrations is cytotoxic to hematopoietic cells
however there isnt strong epidemiological evidence to support this. On the other hand, it is
undoubted that high concentration of benzene produces marrow toxicity, usually bone
marrow aplasia, which can evolve to myelodysplasia and/or AML. The reduced activity of the
enzyme NADPH quinone oxidoreductase, which inactivates benzoquinone, a highly toxic
benzene active metabolite, is associated to an increase in the risk of developing leukemia.
The main differences between t-MDS and de novo MDS are an early age of onset, high
incidence of progression to acute leukemia, more severe cytopenias, more severe marrow
dysplasia (in particular, trilineage dysplasia), decreased marrow cellularity with fibrosis, high
incidence of clonal cytogenetic changes and therapeutic resistance. Globally, t-MDS has a
poorer prognosis.
Some authors have reported an association to autoimmune disorders, either preceding or
concomitant to MDS diagnosis, documented in several epidemiological studies. Some suggest
up to 10% of MDS are related to the immune system. Particularly, severe myelodysplasia,
sustained by MDS may occur after the onset of several autoimmune disorders such as Sweet
Syndrome, Bullous Pemphigoid and several forms of vasculitis and rheumatic disorders,
including Polychondritis, Polyneuropathy and Inflammatory Bowel Disease. The remission of
both the cutaneous disease and the related MDS may occur simultaneously with targeted
therapy.
THF
SHMT
dTMP
Pyrimidine 5,10-methylene
synthesis THF Methionine
DHFR
TYMS MTR
dUMP
MTHFR SAM
Homocysteine
DHF
DNMTs
SAH DNA
5-methyl THF methylation
RFC hFR
Serum folate
Figure 1. Diagram of folate and one-carbon metabolism. The one-carbon transfer reactions are mainly supported by folate. Dietary folates are hydrolyzed to
monoglutamates and then absorbed via reduced folate carrier (encoded by SLC19A1 gene) or by passive diffusion at high concentrations. The 5-
methyltetrahydrofolate (5 methyl THF) donates its one-carbon moiety to methylate homocysteine to methionine, yielding tetrahydrofolate (THF). Methionine,
from homocysteine or diet, is converted to S adenosylmethionine (SAM) via methyltransferases (DNMTs). The THF obtains one-carbon moiety from the amino
acid serine via serine hydroxymethyltransferase (SHMT) catalysis, yielding 5,10-Methylenetetrahydrofolate (5,10 methylene THF), which is an important
common substrate to methylation pathway via methylenetetrahydrofolate reductase (MTHFR) or to nucleic acid synthesis pathways via thymidylate synthase
(TYMS; pyrimidine synthesis) with production of dihydrofolate (DHF). The DHF is then converted into THF via dihydrofolate reductase (DHFR). dTMP,
deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate. (Adapted from Cornelia M. et al., 2003.)
Accumulating epidemiological evidence has suggested that folate intake is associated

with a decreased risk of some types of cancer, such as colon and pancreas, indicating that
folate is associated with cancer risk by interfering with DNA methylation and DNA synthesis.
In fact, the interaction of the epigenome with the environment, including nutrition, can
change patterns of gene expression. Nutrients can modify DNA methylation by altering the
expression of critical genes associated with physiologic and/or pathologic processes including
carcinogenesis. One-carbon metabolism pathway can not only influence DNA methylation
but also it synthesis and repair, critical steps in carcinogenesis. Dietary factors involved in
this pathway include folate and methionine, which can influence the endogenous supply of
methyl groups, as well as vitamin B12 - a relevant cofactor in this pathway (Figure 1).
Moreover, vitamin B12 and alcohol intake can modify the biological response to inadequate
dietary folate by interfering with folate absorption and folate-metabolizing enzymes. S-
adenosylmethionine (SAM) as well as S-adenosylhomocysteine (SAH) are metabolites from
one-carbon pathway that can affect DNA methylation. SAM is the methyl donor for
methylation reactions, and SAH have high-affinity to the catalytic region of most SAM-
dependent methyltransferases, inhibiting their function. Some studies suggest that SAH levels
are negatively correlated with global DNA methylation. This observation suggests that
SAM/SAH ratio could be used as a predictor of methylation status; however, this hypothesis
is not yet confirmed.
While the clinical and biological aspects of hematopoietic neoplasias are well
documented, little is known about the factors that contribute to the individuals susceptibility
to MDS. Recent studies have provided evidence that common genetic variations could
account for several leukemias and could also influence disease outcome; although results still
controversial.
Genetic background, such as SNP, and epigenetic abnormalities may affect the individual
risk of MDS development and progression to acute leukemia. Our previous results show that
superoxide dismutase polymorphisms may be associated with a high risk for MDS. Moreover,
functional polymorphisms in enzymes from one-carbon metabolism pathway have been
associated with various malignancies, including acute lymphoblastic leukemia; however only
one study has been reported for MDS. In a previous study, we observed that allele C from
MTHFR polymorphisms (C677T, rs1801133) and the AC genotype from the same gene
(A1298C, rs1801131) significantly increase 1,55-fold and 2,01-fold the risk of MDS
development, respectively.
Only a few studies examined the association of DNA methylation with dietary and
genetic factors related to one-carbon metabolism, but none studied this association in MDS.
Our previous results showed a tendency for low folate and high vitamin B12 levels in MDS
patients with methylated tumor suppressor genes, especially with methylated P15 and P16.
Moreover, we observed lower folate levels and higher methylation frequency in patients with
wild type homozygous genotype of MTHFR polymorphisms (rs1801131 and rs1801133).
Furthermore, we found that in MDS patients variants in CBS (844 Ins68), MTHFR
(rs1801131), DNMT1 (rs759920), and DNMT3B (rs2424908) genes influence the localized
and global methylation status in a genotype-dependent manner. The CC DNMT1 genotype
was associated with a decreased number of methylated TSG and 5-mC levels. On the
contrary, the TT DNMT3B genotype and homozygous insertion of the CBS gene were
associated with an increased number of methylated TSG.
PATHOPHYSIOLOGY
The pathophysiology of MDS appears to be very complex but it is still largely unknown.
Besides marrow failure and peripheral cytopenias which are common in all MDS subtypes,
the clonal proliferation of hematopoietic progenitor cells associated with inherited or acquired
genetic mutations and/or epigenetically gene silencing can also be present.
Therefore, the natural history of MDS is highly variable, reflecting the wide variety of
genetic and epigenetic mutations that can occur. MDS development and progression to acute
leukemia occurs stepwise involving several mechanisms both hereditary and environmental,
targeting the hematopoietic stem cell, leading to changes in cell functioning, the arise of
clonal proliferation and consequent evolution to acute leukemia. This malignant clone
exhibits genome instability, proliferation advantage, dysplasia and cell dysfunction, such as
increased local secretion of inhibitory cytokines, innefective hematopoiesis and
differentiation changes.
One of MDS paradoxes is the coexistence of peripheral cytopenias with hyperplastic
bone marrow. The mutated clone exhibits an accelerated proliferation, especially in the
myeloid lineage, but that is quickly balanced by an increased apoptosis. Cell death may be
initiated by activated T lymphocytes (attempting to eliminate the malignant clone), increased
cell death protein secretion (FAS/FAS-ligand) and/or pro-inflammatory cytokines (tumor
necrosis factor - TNF- - and interleukin 6 - IL-6), increased expression of BCL-2
superfamily proapoptotic proteins (BAX, BAD, BAK) and/or by stromal changes, in
particular, deficiency in hematopoietic growth factors.
Despite multiple attempts to explain the molecular mechanisms involved in MDS, little is
known regarding the pathogenesis of the promoter event or of the early stages of the disease.
Signal transduction pathways have been implicated, particularly involving the RAS proteins.
On the other hand, the inactivation of the tumor suppressor gene p53 has been reported in 5 to
10% of MDS, especially in advanced stages or in MDS with unstable karyotypes, implying
that such mutations may play a role in leukemic progression.
Several studies have provided evidence of increased apoptosis in the early stages or a
decreased cell death with disease progression. Although the increased programmed cell death
may occur as an initial attempt to control or compensatory response to cell proliferation
deregulation, it may also be a pathophysiological consequence of the epigenetic changes
underlying the biology of these diseases (Figure 2).
One of the mechanisms involved in apoptotic cell death may be related to oxidative
stress, particularly to production of oxygen free radicals and mitochondrial dysfunction. The
persistent oxidative stress may also cause adaptive responses in the neoplastic cells providing
apoptotic and therapeutic resistance. Some studies report increased expression or activity of
thiol-based antioxidants, as well as, antioxidant enzymes such as SOD, GPx and catalase in
some tumor tissues.
The increased expression and release of TRAIL (TNF-related apoptosis inducing ligand),
also known as Apo2 ligand (Apo2L), probably alters marrow erythropoiesis contributing to
anemia, the main feature of MDS. The exact mechanism by which TRAIL eliminates cells is
unknown but may related to differential expression of the apoptotic TRAIL receptors and/or
of its anti-apoptotic receptors or the differential expression or functional defects in the
apoptotic cytoplasmic inhibitors such as interleukin-1 alpha converting enzyme, FAS-
associated death domain (FADD), cellular FADD-like interleukin-1 beta-converting enzyme

inhibitor protein or other apoptotic inhibitors such as survivin.
Several studies suggested an association of oxidative stress with MDS, and excessive
reactive oxygen species (ROS) production, oxidative DNA damage, and/or deficient DNA
damage repair have been frequently observed in this malignancy. The oxidative stress occurs
when an overproduction of ROS and/or a deficiency of enzymatic and non-enzymatic
antioxidants are established. The persistence of oxidative stress has been implicated in
malignant cell proliferation and chemotherapy susceptibility in hematological neoplasms.
Chronic exposure to ROS leads to genomic instability, involving single strand breaks (SSBs),
double strand breaks (DSBs), DNA bases modifications, DNA cross-links, and epigenetic
modifications. These abnormalities influence various cellular processes, such as silencing or
induction of transcription, induction of signal transduction pathways, replication errors and
genomic instability, all of which are associated with carcinogenesis and with MDS
pathogenesis. MDS patients display increased levels of oxidants (such as hydrogen peroxide
and superoxide anion), DNA damage (such as 8-OHdG) and lipid damage (such as
malonyldialdehyde - MDA), as well as decreased levels of antioxidants (such as reduced
gluthatione - GSH) and DNA repair activity (such as lower activity and expression of OGG1).
Oxidative Genotoxic
stress agents
ROS
Figure 2. Molecular and cellular mechanisms involved in MDS. MDS originates probably in a
hematopoietic stem cell genetically transformed by genotoxic agents or endogenous oxidative stress
mediated by mitochondria. Additional genetic and epigenetic alterations and abnormal immune
response contribute to phenotypic diversity and susceptibility the leukaemic transformation. (Adapted
from A. B. Sarmento Ribeiro et al., 2015).
Aberrant methylation patterns are another common mechanism involved in human

neoplasms, especially those of the hematopoietic system, and several genes may be involved
such as p15, an important gene in cell cycle regulation and G1 to S phase transition. This
gene codes an inhibitory protein of CDK-1 (cyclin-dependent kinase 1), frequently

inactivated in MDS by aberrant 5CpG islands methylation and has been associated with bad
prognosis, given the risk of progression to AML (50). In a study with 26 patients, the results
suggest that methylation may be an early mechanism in MDS pathogenesis.
The methyl groups necessary for methylation reaction may derivate from dietary products
such as folate and vitamin B12 (51), however the relationship between ingestion/folate status
and DNA methylation remains controversial.
Functional polymorphisms in key genes may also influence DNA methylation, in
particular, polymorphisms related to the methylenetetrahydrofolate reductase (MTHFR). At
least two polymorphisms have been described C677T and A1298C, and MTHFR genotype
may influence the methylation status as referred.
DIAGNOSIS, CLASSIFICATION AND PROGNOSTIC FACTORS

Significant clinical variability is a keystone in MDS, reflecting the diversity and
complexity of the underlying genetic defects and the type and severity of cytopenias.
About 20% of patients with primary MDS are initially asymptomatic and the diagnostic
suspicion is based on routine Complete Blood Count (CBC). Most patients, however, present
with clinical symptoms of marrow failure, mainly signs and symptoms related to anemia
(80%), such as weakness and fatigue. Less frequently (20%) patients present with infectious
or hemorrhagic complications, the first characterized by recurrent bacterial infections (mainly
bacterial pneumonia or cutaneous infections if the absolute neutrophil count (ANC) is below
1,0 G/L) and the latter by easy or spontaneous bruising. Rarely, fever unrelated to an infection
may be present.
Other patients present with systemic symptoms or features related to autoimmune
diseases such as arthralgias (joint pains), possibly related to the association with some rare
immune-based disorders, such as acute neutrophilic dermatosis (Sweet Syndrome), pyoderma
gangrenosum, cutaneous vasculitis or relapsing polychondritis.
To establish the diagnosis of MDS both bone marrow examination (cytogenetics, aspirate
and trephine biopsy) and peripheral blood smear are essential, showing morphological
features of the disease and may exclude other diseases that may present with cytopenias.
Over the years, there have been several diagnostic criteria, most recently those of the
WHO (2008), and several prognostic scoring systems, the most widely used being the
International Prognostic Scoring System (IPSS).
The National Comprehensive Cancer Network (NCCN) has recently recommended that
the initial approach to a patient with suspected MDS should include clinical history, physical
examination, CBC including differential white cell count and reticulocyte count, bone
marrow aspirate, trephine biopsy with iron staining, erythropoietin serum levels, iron
metabolism assessment and cytogenetic alterations.
The peripheral blood and the bone marrow smears should be criteriously examined
looking for dysplasia, percentage of blasts, monocytes and ring sideroblasts. The blast count
may include myeloblasts, monoblasts and megakaryoblasts. However, poor sample quality is
an often obstacle to the correct diagnosis. The identification of CD34 + cells may be of
diagnostic and prognostic significance as multiparametric flow cytometry can detect

abnormal myeloid maturation.
Bone marrow aspirate is typical, exhibiting features of hypercellular marrow with
dysplasia in more than 10% of a cell lineage; atypical megakaryocytes (small monolobated or
bilobated megakaryocytes), erythroid hyperplasia with maturative asynchronism and altered
myeloid maturation with increased number of blasts or ring sideroblasts. However, in 15 to
20% of patients the bone marrow is hypocellular, similar to aplastic anemia. The finding of
clinical and morphological features of dysplasia with the presence of a clonal cytogenetic
alteration improves the diagnostic accuracy. However, cytogenetic changes are only present
in 40-60% of patients and in even lower percentage in low risk subtypes.
The latest WHO classification of MDS(2008) includes 7 subtypes of MDS and 5
subtypes of Myelodysplastic/Myeloproliferative neoplasms (neoplasms with features of both
myelodysplastic syndromes and myeloproliferative neoplasms) (Table 2).
The International Prognostic Score System (IPSS) is a score system used to evaluate the
prognosis of patients labeled under the WHO criteria (2008). It is based on the percentage of
marrow blasts, cytogenetic abnormalities and peripheral cytopenias being useful to predict
survival and risk of progression to acute leukemia, thus influencing treatment options (Table
3). However, it has several limitations, particularly the fact that it is based on untreated
patients with de novo MDS and excludes t-MDS and CMML with a leukocyte count greater
than 12 G/L. This can also be stated regarding the WPSS (55), another prognosis score, but
this one is dynamic, including transfusion dependency as an independent factor of poor
outcome (Table 3). Greenberg and coworkers presented the IPSS-R (Table 4), based on a
multicentric international study, which includes 5 cytogenetic subgroups, instead of 3, leading
to a total of 5 risk groups (56). The major changes include incorporation of additional less
common karyotype abnormalities, in which prognostic relevance was delineated from a larger
cohort of patients. R-IPSS retains emphasis on the prognostic value of myeloblast percentage
and recognizes prognostic importance of the magnitude of each lineage cytopenia. To
overcome some IPSS and WPSS limitations, the Global M.D. Anderson Scoring System
(MDAS) was developed. This model incorporates patient features such as age and
performance status, as well as anemia, transfusion burden, karyotype, and severity of
thrombocytopenia (Table 6). The Global MDAS was able to refine the prognostic value
among each of the IPSS categories, identifying patients with lower or higher risk features.
This prognosis system allows the evaluation of all MDS patients at any time during the course
of their disease without need of WHO evaluation.
In the everyday clinical practice, patients are divided in two groups, low-risk patients
(including the low-risk and int-1 IPSS groups) and high-risk patients (including the int-2 and
high-risk IPSS groups), reflecting two different therapeutic strategies.
Bone marrow fibrosis grades 2 and 3 is also related to a poor outcome and point
mutations in the TP53, EZH2, ETV6, RUNX1, NRAS and ASXL1 genes have all been
associated with a poor outcome, as well as spliceosome mutations in SRSF2and U2AF1,
despite SF3B1 mutations being associated with greater survival (Table 5). The clinical
significance of the mutational pattern analysis is not yet fully established and cannot yet be
recommended in MDS. Age is also an important indicator of poor prognosis, being the loss of
years of life expectancy greater in younger adults than in the older ones. However, age-related
factors, especially non-haematological comorbidities, affect survival and must be taken into
account in decisions on intensive treatments. The high levels of serum ferritin and lactate
dehydrogenase as well as marrow cells features evaluated by flow cytometry are other factors
with diagnostic and prognostic value not included in any prognostic systems. In a recent
study, we identify the GSH, an antioxidant molecule, as a diagnostic biomarker for MDS.
Furthermore, the increase in erythropoietin levels in MDS patients can be, by itself, a poor
prognostic factor, related with progression to acute leukemia and decreased overall survival.
TREATMENT
Age, Performance status, existing comorbidities, MDS subtype and IPSS risk
stratification influence the therapeutic strategy adopted for each patient (Table 6).
As the majority of patients are elderly, Best Supportive Care (BSC) is the best strategy
for most and its purpose is to maintain the best quality of life. Red Blood Cell (RBC)
transfusions are frequently necessary to compensate for symptomatic anemia and antibiotics
are also commonly used to treat bacterial infections. In time, iron overload becomes an issue
in patients undergoing frequent RBC transfusions and iron chelators are recommended after
the 20th RBC transfusion or when serum ferritin levels exceed 1000 ng/mL. Deferoxamine
has traditionally been used subcutaneously or intravenously. Deferesirox has recently been
approved in transfusion-related iron overload, in a dose of 20 mg/Kg/day given orally but
requires liver and kidney function monitoring.
Platelet transfusions are only used in patients with active or severe bleeding and/or with
severe thrombocytopenia.
The administration of exogenous growth factors may be of some benefit in some patients.
Erythropoietin (EPO) may restore the hemoglobin levels in 20-40% of patients, especially in
RBC-transfusion-independent patients and EPO serum levels lower than 200 U/L.
Granulocyte-Colony Stimulating Factor (G-CSF) in association with EPO may increase the
odds of erythroid response.
In high-risk MDS patients, Azacytidine is an option that should be considered. Not only
does it present cytotoxic activity but this hypometilating agent may also reactivate tumor
suppressor or cell cycle regulator genes that were inactive by hypermethylation of regulatory
regions. A randomized controlled trial compared this drug, in a dose of 75 mg/sqm/day
subcutaneously, for 7 days every 28 days, with best supportive care in RBC-transfusion-
dependent patients and severe peripheral cytopenias. After 4 months of treatment, 60% of
patients treated with azacytidine achieved clinical improvement (defined as a reduction of
50% in transfusion needs) compared to 5% in the BSC treatment arm. Besides, 7% of patients
treated with azacytidine achieved complete remission and the risk of leukemic transformation
or death was significantly reduced.
Decitabine is another hypomethylating agent currently approved for MDS treatment. It is
administered intravenously, in a dose of 15mg/sqm/day for 3 days every 6 weeks and induces
a response in 20% of MDS patients but may cause severe and late-onset myelosuppression.
Thalidomide has been studied in MDS for its immunomodulator properties as well as its
antiangiogenic effect. This drug decreases the production of pro-apoptotic cytokines and
improves anemia in about 20% of patients. However, the side effects (including fatigue,
dizziness, drowsiness, constipation, edema, peripheral neuropathy) often limit its use.
Table 2. WHO Classification of MDS
Name Abbreviation Peripheral blood: key Bone marrow. Key features Frequency (%)
features
Refractory cytopenias with
unilineage dysplasia (RCDU):
Refractory anemia RA Anemia; <1% peripheral Unilineage erythroid dysplasia 10-20%

blood blasts (in >10% of cells); <5% blasts
Refractory neutropenia RN Neutropenia; <1% Unilineage granulocytic <1%

peripheral blood blasts dysplasia; <5% blasts
Refractory RT Thrombocytopenia; <1% Unilineage megakaryocytic <1%

thrombocytopenia blasts dysplasia; <5% blasts
Refractory anemia with ring RARS Anemia; no blasts Unilineage erythroid 3-11%
sideroblasts dysplasia; 15% of erythroid
precursors are ring
sideroblasts; 5% blasts
Refractory cytopenias with RCMD Cytopenia(s); <1% blasts; Multilineage dysplasia ring 30%
multilineage dysplasia no Auer rods sideroblasts; <5% blasts; no
Auer rods
Refractory anemia with RAEB-1 Cytopenia(s); <5% blasts; Unilineage or multilineage 40%*
excess blasts, type 1 no Auer rods dysplasia; 5%-9% blasts; no
Auer rods
Refractory anemia with RAEB-2 Cytopenia(s); 5%-19% Unilineage or multilineage

excess blasts, type 2 blasts; Auer rods dysplasia; 10%-19% blasts;
Auer rods
Myelodysplastic syndrome Del(5q) Anemia; normal or high Isolated 5q31 chromosome <5%
(MDS) associated with platelet count; <1% deletion; anemia,
isolated del(5q) blasts hypolobated megakaryocytes
Childhood MDS, including RCC Pancytopenia <5% marrow blasts for RCC; <1%
refractory cytopenia of marrow usually hypocellular
childhood (provisional)
MDS, unclassifiable MDS-U Cytopenias; 1% blasts Does not fit other categories; ?
precursors are ring
sideroblasts; 5% blasts
Refractory cytopenias with RCMD Cytopenia(s); <1% blasts; Multilineage dysplasia ring 30%
multilineage dysplasia no Auer rods sideroblasts; <5% blasts; no
Auer rods
Refractory anemia with RAEB-1 Cytopenia(s); <5% blasts; Unilineage or multilineage 40%*
excess blasts, type 1 no Auer rods dysplasia; 5%-9% blasts; no
Auer rods
Table 2. (Continued)
Refractory anemia with RAEB-2 Cytopenia(s); 5%-19% Unilineage or multilineage
excess blasts, type 2 blasts; Auer rods dysplasia; 10%-19% blasts;
Auer rods
Myelodysplastic syndrome Del(5q) Anemia; normal or high Isolated 5q31 chromosome <5%
(MDS) associated with platelet count; <1% deletion; anemia,
isolated del(5q) blasts hypolobated megakaryocytes
Childhood MDS, including RCC Pancytopenia <5% marrow blasts for RCC; <1%
refractory cytopenia of marrow usually hypocellular
childhood (provisional)
MDS, unclassifiable MDS-U Cytopenias; 1% blasts Does not fit other categories; ?
dysplasia; <5% blasts; if no
dysplasia, MDS-associated
karyotype
Note: If peripheral blood blasts are 2%-4%, the diagnosis is RAEB-1 even if marrow blasts are < 5%. If Auer rods are present, the WHO considers the diagnosis
RAEB-2 if the blast proportion is < 20% (even if < 10%) or acute myeloid leukemia (AML) if 20% blasts. Cases of RCUD, RARS, or RCMD where the
peripheral blood blasts are exactly 1% should be considered MDS-U, according to the WHO. For all subtypes, peripheral blood monocytes are < 1 109/L.
Bicytopenia may be observed in RCUD subtypes, but pancytopenia with unilineage marrow dysplasia should be classified as MDS-U. Therapy-related MDS (t-
MDS), whether due to alkylating agents, topoisomerase II inhibitors, or radiation, is classified together with therapy-related AML (t-MDS/t-AML) in the WHO
classification of AML and precursor lesions. The listing in this table excludes MDS/myeloproliferative neoplasm overlap categories, such as chronic
myelomonocytic leukemia, juvenile myelomonocytic leukemia, and the provisional entity RARS with thrombocytosis.
*
This 40% figure represents the proportion of patients with RAEB-1 or RAEB-2, collectively. (Adapted from Swerdlow S. H. et al., eds. 2008.)
Table 3. International Prognostic Score System (IPSS) and risk stratification
Category Score (sum all three subscores for overall IPSS score)
0 (best) 0.5 1 1.5 2.0 (worst)
Prognostic factor
Marrow blasts (%) <5 5-10 - 11-20 21-30*
Karyotype Good: normal, isolated Intermediate: all Poor: abnormal - -

Y, isolated del(5q), or karyotypes not defined chromosome 7 or a
isolated del(20q) as good or poor complex karyotype (3
anomalies)
Peripheral blood 0 or 1 2 or 3 - - -
cytopenias
Total score Risk stratification Medium Overal survival (years) LMA rate progression (%)
0 Low 5,7 19
0,5 a 1 Intermediate-1 (INT-1) 3,5 30
1,5 a 2 Intermediate-2 (INT-2) 1,2 33
2,5 High 0,4 45
*
No longer considered myelodysplastic syndrome (redefined as acute myeloid leukemia by World Health Organization in 2001).
IPSS definition of peripheral blood cytopenias: hemoglobin < 10 g/dL; absolute neutrophil count < 1800/L; and platelet count < 100000/L.
Scoring system: A point value from 0 to 2.0 is determined for each of the three prognostic factors and the three values are summed to obtain the total IPSS
score. (From Greenberg P. et al., 1997.)
Table 4. IPSS-R and risk stratification
Category Score (sum all three subscores for overall IPSS score)
Prognostic
factor 0 (best) 0.5 1 1.5 2.0 3 4(worst)
Marrow blasts 2 - >2 - <5 - 5 - 10 >10
(%)
Karyotype Very good: - Good: - Intermediate: Poor: -7, Very poor:
Y; del (11q) normal, del(7q), +8, Inv(3)/t(3q)/d complex
isolated +19, inv(7q), el(3q), - karyotype
del(5q), all 7/del(7q), (>3
del(12p), karyotypes complex anomalies)
del(20q) + 1 not defined as karyotype (3
additional good or poor anomalies)
Hemoglobin, 10 - 8 - 10 <8 - - -
g/dL
Platelets, x10 9 /L 100 50 - <100 < 50
ANC, x10 9 /L 0.8 < 0.8
Total score Median Overal survival

Risk stratification LMA progression (25%)
(for age 70 years) (years)
1.5 Very low 8.7 Not reached

> 1.5 - 3 Low 5.3 10.8
> 3 - 4.5 Intermediate 3.0 4.0
> 4.5 - 6 High 1.6 1.4
>6 Very high 0.8 0.7
(Adapted from Greenberg P. L. et al., 2012; Bejar R., 2013).
Table 5. Prognostic implications of gene mutations in patients with MDS
Gene Prognosis of patients carrying mutation vs. wild type
DNMT3Aa Worse OS (P = 0.005), worse EFS (P = 0.009), higher rate of transformation to AML (P = 0.007)
TET2 a No impact on survival, but in other studies is an independent prognostic factor for OS [HR, 5.2 (95% CI, 1.616.3); P = 0.005]
ASXL1 Worse OS [HR, 1.38 (95% CI, 1.001.89); P = 0.006]
EZH2 a Worse OS [HR, 2.13 (95% CI, 1.363.33); P = 0.001]
SF3B1 Favorable prognosis, longer EFS [HR, 0.1 (95% CI, 0.00.7); P = 0.02]
SRSF2 a Shorter OS [median, 17 vs. 39% at 5 y (HR, 1.76; 95% CI, 1.03.1); P = 0.049]; shorter 5-y DFS [median, 39 vs. 69% (HR, 2.5;
95% CI, 1.225.1); P = 0.012]
U2AF1 a More rapid transformation to AML [HR, 2.53 (95% CI, 0.97.13); P = 0.079]; no impact on OS [HR, 1.49 (95% CI, 0.544.1);
P = 0.44]
CI, confidence interval; DFS, disease-free survival; EFS, event-free survival; OS, overall survival.
a
Prognostic value remains controversial and is not currently used in clinical practice. (Adapted from A. B. Sarmento Ribeiro et al., 2015.)
Lenalidomide is a similar compound to thalidomide but it is safer and is not associated

with neurotoxicity. It is approved by the FDA for MDS treatment in patients with 5q deletion
karyotype. A phase II clinical trial with lenalidomide, in a dose of 10 mg/day for 21 or 28
days, in del5q, low or intermediate risk, RBC-transfusion-dependent patients revealed that
67% of patients achieved transfusion-independency and that 74% of patients achieved a
cytogenetic response, with 44% of complete responses. These results were sustained in time
with chronic administration of the drug.
Another therapeutic option, also used in Aplastic Anemia, is antithymocyte globulin
which can induce a response (mainly RBC-transfusion-independency) in about a third of
patients with the RA subtype.
Lastly, AML-like chemotherapy regimens allow complete remissions in 40 to 60% of
selected MDS patients. In the elderly the therapy-related mortality is at least as high as
conservative therapy and those who achieve a remission dont usually sustain it for long. On
the other hand, high risk patients for leukemic transformation should be considered for
allogeneic transplant considering that standard chemotherapy is not an option as a curative
treatment. Despite the fact that allogeneic transplant might be the only curative option, most
patients are ineligible for such treatment due to the advanced age. The recurrence rate is
higher among patients with the RAEB and RAEB-t subtypes (50-70%) than in RA or RARS
(< 5%) and as such nonmyeloablative Allogeneic Stem-Cell Transplantation may be a
potential therapeutic approach in such patients.
Table 6. MDS treatment options
Best supportive care: transfusions, antibiotics
Hematopoietic growth factors: erythropoietin, darbepoetin,
Transcriptional modifying therapy

- Hypomethylating agents: 5-azacytidine,* decitabine*
- Histone deacetylase inhibitors
Immunomodulatory agents: lenalidomide,* ATG, CsA, thalidomide
Low-dose chemotherapy (low-dose cytarabine)
Intensive (AML-like) chemotherapy [such as cytarabine (7 days) + an anthracycline (3 days)]
Allogeneic blood (PPSCs) or marrow cell transplantation
*
FDA approved for the treatment of MDS.
Only available on clinical trial; see www.clinicaltrials.gov for a listing of available investigation
agents.
G-CSF - granulocyte-colony stimulating factor, ATG - anti-thymocyte globulin, CsA - cyclosporine A,
PPSCs - pluripotent stem cells.
PROGNOSIS
In many MDS patients the disease remains stable for several years without significant
progression of the anemia or other symptoms. A small proportion of patients develop
progressive marrow failure, severe cytopenias and morbidity due to infection or bleeding.
Iron overload is common and some patients may develop hemosiderosis. The HLA-A3
serotype is significantly higher in patients developing iron overload. However, its frequency
is similar to that found in hereditary hemochromatosis, implying that the combination of a
genetic predisposition with sideroblastic anemia enables iron overload in such patients. The
iron chelating therapy may improve anemia and the side effects of tissue iron overload.
The most important variables affecting survival and progression to acute leukemia are the
percentage of blasts and cytogenetic abnormalities. The number of peripheral cytopenias is of
a secondary prognostic significance.
As aforementioned, the IPSS allows an estimate of the clinical course of the disease.
However, despite the usefulness of such scoring systems, the outcomes vary considerably
among MDS patients with similar scores and although the cytogenetic changes are considered
the most important independent prognostic factor, it is limited by the cytogenetic diversity
inherent in these diseases. The addition of additional parameters could be useful to enhance
the prognostic value of the IPSS.
In fact, the use of the LDH serum values as a prognostic factor has been demonstrated to
improve the prognostic significance of the IPSS and the serum 2M levels at the date of the
diagnosis is correlated to overall survival and the risk of progression to AML, especially in
high-risk MDS.
The relationship between prognosis and DNA methylation has been studied either for a
single gene or for several genes. Although there is strong evidence that epigenetic mutations
may contribute to leukemogenesis and the biological behaviour of the disease, the impact of
gene methylation as a factor of prognostic significance or in disease progression is not yet
fully established.
Moreover, oxidative stress also influences MDS prognosis. We have reported that
patients with high intracellular peroxides and superoxide levels in erythroid precursors or
granulocytes, with high superoxide/peroxides ratio, as well as with low GSH levels had a
shorter overall survival.
Finally, genetic polymorphisms in genes associated with DNA methylation (DNMT3A),
folato metabolism (MTRR) and DNA repair (NEIL1 and OGG1) can also influence the AML
transformation rate and the survival of MDS patients.
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Chapter 2
THE INFLAMMATORY AND AUTOIMMUNE NATURE

OF MYELODYSPLASTIC SYNDROMES
Ota Fuchs*
Institute of Hematology and Blood Transfusion,
Prague, Czech Republic
ABSTRACT
Very early in the study of myelodysplastic syndromes (MDS), clinicians described
frequent association of MDS with rheumatoid arthritis. Both pathologies were
concomitantly diagnosed in 27% of cases. Other acute and chronic inflammation and
autoimmune disorders associated with MDS are inflammatory bowel disease, diverse
types of vasculitis, autoimmune anemias, several rheumatic and skin disorders and
certain thyroid disorders. Recent clinical and molecular studies of MDS showed the
contribution of abnormal activation of innate immune signals and associated
inflammation to the pathogenesis of MDS. The presence of abnormal levels of cytokines,
chemokines and growth factors (tumor necrosis factor alpha /TNF-/, interferon gamma
/IFN-/, transforming growth factor beta, myeloid growth factors /G-CSF and GM-CSF/,
interleukin (IL)-6, IL-8) in the peripheral blood and in bone marrow of MDS patients has
been described. These levels depend on analyzed MDS subtypes. High-risk MDS have
poor response to immunosuppressive therapy. Further evidence that pro-apoptotic
signaling is strongly associated with lower risk MDS is that genes involved in apoptosis
are most significantly upregulated in CD34+ cells from RA versus controls and RAEB.
Toll-like receptors (TLR) participate in the pathogenesis of chronic polyarthritis.
Engagement of TLRs by their specific ligands leads to the activation of transcription
factors that cooperatively regulate the expression of INFs and pro-inflammatory
cytokines and chemokines. TLRs and multiple downstream signaling mediators have
been shown to be overexpressed in MDS. NF-B transcription factors are activated in
response to inflammatory cytokines, pathogenic antigens, oxidative stress, DNA damage
and the activation of pattern recognition receptors. NF-B regulates the expression of a
number of inflammatory cytokines and chemokines including TNF-, IL-6 or IL-8,
*
Corresponding author: Ota Fuchs, PhD. Tel: +420 221977313; Fax: +420 221977370; E- mail:
Ota.Fuchs@uhkt.cz.
28 Ota Fuchs
inducible enzymes, adhesion molecules and proteins regulating immune responses, but
also anti-apoptotic proteins and proliferative factors. Therefore, NF-B signaling
influences the survival of MDS progenitors and is correlated with the progression of
MDS. Mesenchymal stem cells (MSCs) are primitive, non-hematopoietic stem cells that
give rise to all of the various types of stromal cells that form bone marrow
microenvironment (niche). The immunosuppressive capacity of MSCs is decreased in
cells from low risk MDS. MDS-derived MSCs and bone marrow stromal cells are
determinants of the fate of hematopoietic progenitors and have an important role in
pathogenesis of MDS. Myeloid-derived suppressor cells (MDSCs) are inflammatory and
immunosuppressive effectors localized to the bone marrow that express the immune
receptor CD33. MDS patients have increased numbers of MDSCs and they induce
defects in myeloid and erythroid differentiation. MDSCs reduce T-cell proliferation and
functionality in MDS patients. These effects are mediated by CD33, for which the
inflammatory molecule S100A9 is a specific ligand. The blockade of TLR2-mediated
signaling in MDS 34+ cells with a specific inhibitor of MyD88 increased the number of
erythroid colonies and the expression of erythroid marker genes in lower risk MDS.
IRAK1 or TRAF6 inhibition was also used as anti-innate immune therapies in MDS.
Keywords: MDS, innate immune signaling, inflammatory signaling, cytokines, chemokines,

Toll-like receptors, NF-B, microenvironment, anti-innate immune therapies
INTRODUCTION
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic
stem cell (HSC) disorders characterized by ineffective hematopoiesis, peripheral cytopenias,
frequent karyotypic abnormalities and risk of transformation to acute myeloid leukemia
(AML) [1-10]. The first MDS classification, the French-American-British (FAB)
classification, published 33 years ago allowed scientific research of this disease [11]. FAB
group system was modified and further defined to recognize and classify distinct sub-
categories of MDS based on genetic features.
Chronic myelomonocytic leukemia (CMML) has long been recognized as a distinct
clinico-pathological entity with features of both myelodysplastic and myeloproliferative
syndromes resulting in different clinical presentations [12, 13]. FAB group system classified
CMML as part of MDS, given the morphologic evidence of dysplastic hematopoiesis. The
FAB Group later proposed a reclassification of CMML patients into two subtypes based on
white blood cell (WBC) count at diagnosis [14]. Patients with WBC counts of < 13x109/L
were considered to have myelodysplastic CMML (MD-CMML) and those with > 13x109/L
were considered to have myeloproliferative CMML (MP-CMML). However, the two groups
have overlapping features. Voglova et al. [15] analyzed 69 patients with CMML, 31 (45%)
classified as MD-CMML and 38 (55%) classified as MP-CMML. Cytogenetic abnormalities
were more frequent among MP-CMML patients. The median overall survival (OS) was
significantly longer in the MD-CMML patients than in the MP-CMML group (30 vs. 16
months, respectively; p value < 0.01) and there was no significant difference in leukemic
transformation. Over the course of disease, WBC count in 24 MD-CMML patients increased
to more than 13x109/L. Therefore, MD-CMML and MP-CMML should be considered as
different stages of the same disease. World Health Organization (WHO) in 2002 recognized
CMML as a distinct entity and moved it to a new category called MDS/MPD [16]. WHO
The Inflammatory and Autoimmune Nature 29
classification differentiates CMML-1 and CMML-2 according to blast procentages and more
recently also CMML-0 with less than 5% medullary blasts [17].
Both, WHO classification and criteria for MDS are shown in Table 1 [18]. Using the
International Prognostic Scoring System (IPSS), MDS is classified into low, intermediate-1,
intermediate-2 and high-risk for progression towards AML [19]. Despite increasing insight
into the tumor biology of MDS, the etiology of these syndromes remains undetermined.
However, there is increasing evidence that cytopenia in MDS may, at least in part, be due to
lymphocyte-mediated myelosuppression suggesting that dysregulated immune mechanisms
may be involved in the pathogenesis of MDS [20]. This hypothesis is supported by frequent
association of MDS and CMML with clinical manifestations of autoimmune disorders (AD)
and inflammatory response of the immune system [12, 20, 21-53]. Epidemiologic studies
demonstrated that AD patients (suffering from rheumatoid arthritis, Sjgren syndrome, lupus
erythematosus, seronegative arthritis, panarteritis nodosa, autoimmune hemolysis and
pernicious anemia) have a higher risk to develop MDS or AML compared to general
population [45].
Aberrant immunity, including abnormal immune cells and molecules, contributes to the
development of MDS. Various immune molecules, including interferon- (IFN-), tumour
necrosis factor- (TNF-) and interleukins (ILs), produced by antigen-presenting cells
(APCs) and T lymphocytes generate a cytokine milieu that can lead to the destruction of
HSCs. The excessive apoptosis was largely cytokine mediated with a number of
proinflammatory and proapoptotic cytokines such as TNF-, transforming growth factor beta
(TGF-), and interleukin 1 (IL1) being overexpressed in the marrows of MDS patients.
Stem/progenitor cells are impaired by these factors and exhibit marked deficiencies in
proliferation and differentiation, high levels of apoptosis and dysfunctional responses to
growth factor stimulation [42, 54-56]. Overexpression of immune-related genes is widely
reported in MDS [52]. Hyperactivation of innate immune/Toll-like receptor (TLR) signaling
was described in MDS [50, 53, 57-60]. The innate immunity system is a conserved host
defence mechamism that detects and eliminates pathogens [61, 62]. Signals are mediated via
downstream signaling mediators and eventually lead to activation of key intracellular
molecular effectors such as transcription factor NF-B and mitogen-activated protein kinases
(MAPK). The resulting immune responses, including release of inflammatory cytokines,
cause elimination of pathogens. Innate immunity responses are mediated by phagocytes such
as macrophages and dendritic cells. However, TLR on hematopoietic progenitor cells
stimulate innate immune system replenishment [63, 64] and may be involved in the
pathogenesis of MDS [50, 53, 65-69].
MDS are characterized not only by abnormal HSCs and immune system defects but also
by changes in the hematopoietic microenvironment (niche) [70-74]. The pathogenesis of
MDS likely depends on the interaction between aberrant hematopoietic cells and their
microenvironment. Chronic immune stimulation in combination with senescence dependent
changes was observed in both, hematopoietic stem/progenitor cells (HSPC) and niche and
seems to be critical to the pathogenesis of the disease. Inflammatory processes are regulatory
stimulus promoting the proliferation and apoptotic death of hematopoietic progenitors in
MDS. Immune system dysregulation, as a key driver of the pathological evolution of MDS,
includes cytokine milieu abnormalities and inflammatory alterations in natural killer cells,
T cells, and myeloid-derived suppressor cells (MDSC).
30 Ota Fuchs
A detailed understanding of these mechanisms, which contribute to the pathogenesis of

MDS, may help to find and to define novel targets for diagnosis and therapy in this disease.
Table 1. World Health Organization MDS Classification

and Criteria (2008)
Classification Blood findings Bone marrow findings

Refractory cytopenia with Unicytopenia or bicytopenia Unilineage dysplasia: >10% cells
unilineage of dysplasia (RCUD) No or rare blasts (<1%) in one myeloid lineage
Refractory anemia (RA), <5% blasts; <15% of erythroid
refractory neutropenia (RN), precursors are ring sideroblasts
refractory thrombocytopenia (RT)
Refractory anemia with ring Anemia > 15% of erythroid precursors are
sideroblasts(RARS) No blasts ring sideroblasts Erythroid
dysplasia only<5% blasts
Refractory cytopenia with Cytopenia(s) Dysplasia in >10% of the cells in
multilineage dysplasia (RCMD) No or rare blasts (<1%) two or more myeloid lineages
No Auer rods <5% blasts in marrow

No Auer rods 15% ring
<1x109/L monocytes sideroblasts
Refractory anemia with excess Cytopenia(s) <5% blasts Unilineage or multilineage
blasts-1(RAEB-1) No Auer rods dysplasia; 5%-9% blasts
<1 x 109/L monocytes No Auer rods
Refractory anemia with excess Cytopenia(s) Unilineage or multilineage
blasts-2(RAEB-2) 5%-19% blasts dysplasia; 10%-19% blasts
Auer rods Auer rods
<1 x 109/L monocytes
Myelodysplastic syndrome - Cytopenia Unequivocal dysplasia in <10% of
unclassified (MDS-U) <1% blasts cells in one or more myeloid cell
lines when accompanied by
acytogenetic abnormality
considered as presumptive
evidence for a diagnosis of MDS
<5% blasts
MDS associated with isolated Anemia Normal to increased

del(5q) Usually normal or increased megakaryocytes with hypolobated
platelet count nuclei <5% blasts
No or rare blasts (<1%) Isolated del(5q) cytogenetic
abnormality
No Auer rods
Adapted according Nybakken and Bagg [17].

THE ASSOCIATION BETWEEN AUTOIMMUNE DISEASES

AND MYELODYSPLASTIC SYNDROMES
Immune and autoimmune biological anomalies have also been reported in MDS, such as
the presence of antinuclear antibodies, antimitochondrial autoantibodies, antineutrophil
cytoplasmic autoantibodies (ANCA), autoantibodies rheumatoid factor, and cryoglobulins
[32, 75, 76]. Approximately 10-30% of patients with MDS or CMML are associated with AD
(Table 2), the most frequent being vasculitis, seronegative polyarthritis and specific skin
lesions (Sweets syndrome, pyoderma gangrenosum) [21, 23, 26, 43, 76-84]. Distribution of
AD among MDS subtypes is controversial [45]. It seems that is more involved in RAEB,
where in one study 86% MDS patients is associated with AD and 52% MDS patients is
without AD. In a series of 235 MDS patients, 46 (19.6%) patients displayed features of AD
[85]. In this study, distribution of MDS subtypes was similar between MDS cohorts with and
without AD. MDS patients with AD are mostly male (up to 70%) and of older age (mean 78-
83% years), which may be somewhat different from AD observed in general population, that
predominate in females and in younger patients. Apart from trisomy 8 in patients with
Behcets disease, frequently associated with MDS [81-83], there seems to be no significant
association between karyotype and MDS associated with AD [45]. Distribution of MDS
subtypes with del(5q) and del(7q)/ monosomy 7 are similar in association with AD or without
AD. Association of MDS with vasculitis may independently predict adverse outcome [86].
Other AD do not influence outcome of MDS patients when associated with MDS [45].
Table 2. Autoimmune diseases associated with MDS
Type of autoimmune manifestation in MDS Examples

Systemic vasculitis Giant-cell arteritis
Aortitis
Medium- and small sized vessel
vasculitis
Isolated autoimmune disorders Cutaneous vasculitis
Polyarthritis
Polyneuropathy
Classical connective tissue disorders Systemic lupus erythematosus
Raynauds disease
Polymyalgia rheumatic
Autoimmune hematological disorders Autoimmune hemolytic anemia
Immune thrombocytopenia
Asymptomatic immunological serological Positive antineutrophil antibody
abnormalities Positive rheumatoid factor
Adapted according Oostvogels et al. [87].
32 Ota Fuchs
A causal relationship is yet to be established between MDS and autoimmunity. The exact
mechanism(s) underlying MDS and AD are not well established and are supposed to be
related to immune dysfunction induced by MDS or by immunosuppressive therapy. Immune
deregulation and synthesis of autoantibodies due to abnormalities in T and B cells with
production of cytokines, defective macrophage clearance and neutrophil function, with
subsequent prolonged circulation of immune complexes and activation of inflammatory
mediators, reduced CD4 count, immature natural killer cells and impaired function of
monocytes and dendritic cells with abnormal antigen presentation are observed. All these
features may result from abnormal stimulation by dysplastic bone marrow stem cells [32, 88-
90]. Treatment options include treating both diseases concomitantly with 5-azacitidine
[91, 92].
T-CELL AND B-CELL ABNORMALITIES IN MDS

The existence of functionally polarized human T cell responses based on their profile of
cytokine secretion in both the CD4+ T helper (Th) and the CD8+ T cytotoxic cell subset has
been established. Human Th1 and Th2 cells not only produce a different set of cytokines but
also exhibit distinct functional properties. Deviation of type I and type II T cells and its
negative effect on hematopoiesis in MDS in vitro was described [93]. In MDS, autologous T
lymphocytes suppress both erythroid (CFU-E) and granulocytic (CFU-GM) progenitor cell
growth in vitro [94, 95]. CD8+ cells mediated this inhibition of hematopoiesis through the
major histocompatibility complex (MHC) class I molecules on target marrow cells. Removal
of T cells from bone marrow often enhances colony formation [96, 97]. Successful treatment
with anti-thymocyte globulin (ATG) eliminates or reduces the myelosuppressive effect of
these autologous T cells [98]. The decline of CD8+ cells in high-risk MDS is related to the
expression of the negative co-stimulatory T-cell receptor programmed death-1 (PD-1) and its
ligand PDL-1. Higher levels of PD-1/PDL-1 in bone marrow cells are associated with
resistance to therapy and with a poorer prognosis. It has recently been shown that T cell
expression of the immunoinhibitory receptor PD-1 is regulated by DNA methylation. The
hypomethylating agents (azacitidine and decitabine; HMAs) induced PD-1 expression on T
cells in the MDS microenvironment, thereby likely hampering the immune response against
the MDS blasts. Combination therapy using HMAs with a PD-1 pathway inhibitor can solve
this problem.
Oligoclonal expansion of T cells in MDS was reported using flow cytometry and
spectratyping [99-101]. Cukrova et al. [38] questioned the auto-reactivity of T-cells in MDS
and found a defective in vitro cytotoxicity of these cells. The frequencies of activated T-cells
were not related to characteristics of MDS patients [102]. However, the absolute lymphocyte
count at diagnosis showed the adverse prognostic impact in a large cohort of MDS patients
suggesting an influence of the host immunity on the disease in MDS patients [103].
More than 50% of early stage MDS patients have anti-erythroid autoantibodies in their
bone marrow cultures. These autoantibodies are mainly directed against autologous
erythroblasts and correlated with increased apoptosis [35].
THE INVOLVEMENT OF REGULATORY T-CELLS (TREG)

IN THE MDS PATHOGENESIS
Treg are known to influence both the autoimmunity and tumour progression [104].
Kordasti et al. [105] evaluated the absolute number of both CD4+ and CD8+ Treg in the
peripheral blood of 52 MDS patients. A significant correlation was shown between increased
number of CD4+ Treg and several markers of disease aggressiveness (number of blasts in
bone marrow, disease progression). None of these correlations was found for CD8+ Treg
[105]. Kotsiniadis et al. [106] confirmed the effect of Treg on antitumour immunity in course
of MDS. They found different Treg pattern in early and late stage of MDS. Treg were
impaired in function and also in bone marrow homing in early stage MDS. However, Treg
retained their function in late stage disease but were expanded [106]. In a retrospective study,
Mailloux et al. [107] investigated the phenotypic features of Treg subsets including naive,
cental memory and effector memory cells, in association with MDS progression. They found
a significant shift from a central memory phenotype toward an effector phenotype connected
with a higher percentage of abnormal bone marrow myeloblasts. The analysis of effector
memory Tregs using flow cytometry may be a simple and useful method to predict an early
immune escape in MDS patients [108]. Moreover, Treg were significantly reduced in MDS
patients responding to treatment. The recruitment of CD8+ cytotoxic T-cells, the degree of
dyserythropoiesis and the need for erythropoietin treatment were inversely related with the
levels of Treg in the bone marrow of MDS patients [109].
NATURAL KILLER CELLS IN MDS

Natural killer (NK) cells interact with clonal cells. NK cytolytic function against different
tumour targets was reduced in MDS patients in relation with increased risk of MDS, higher
IPSS, abnormal karyotype and excess of blasts [110]. The percentage of NK cells was similar
in MDS patients and in healthy controls. However, NK cells of MDS patients expressed
increased levels of granzyme B and were mediators of cytotoxicity against dysplastic
hematopoietic precursors [111]. Marcondes et al. [112] explored the relationship between NK
function and IL-32 expression. NK cells from 48 MDS patients displayed impaired NK
function utilizing distinct receptor-ligand interactions compared to healthy controls [111].
Reduced NK function showed global defects in NK receptor signaling. NK cell receptor
proteins G2D (NKG2D), DNAX-accessory molecule 1 (DNAM-1) and natural cytotoxicity
receptor NKp30 are involved in activation of NK cells. CD226 (Cluster of Differentiation
226), PTA1 (platelet and T cell activation antigen 1) or DNAM-1 is a protein that in humans
is encoded by the CD226 gene which is located on chromosome 18q22.3. Reduced expression
of all these activating receptors were associated with impaired NK function in MDS [111,
113, 114]. NK cells from MDS patients fail to exhibit appropriate effector response. Based on
low levels of IL-32 in CMML and high levels in MDS but suppressed cytotoxicity function in
both diseases, IL-32 levels did not correlate with cytotoxic function. Abnormal stroma in
MDS may play an inhibitory role in NK cell differentiation and development. Loss of
immunosurveilanced may then lead to the accumulation of cells with DNA damage in the
34 Ota Fuchs
intermediate stages of MDS progression. NK cells become more dysfunctional as MDS

progresses. However, the direct cause and effect are unknown.
ABNORMAL LEVELS OF INFLAMMATORY CYTOKINES AND

CHEMOKINES IN THE PEREIPHERAL BLOOD AND
BONE MARROW OF MDS PATIENTS
A variety of immune molecules, including IFN-, TNF- and ILs produced by antigen-
presenting cells (APCs) and T lymphocytes generate a cytokine milieu that can lead to
destruction of HSCs. The secretion of TNF- and and other related cytokines, such as IFN-
or IL-6, is higher in low-risk MDS, whereas these and other cytokines are down-regulated in
high-risk cases [50]. The overproduction of IFN-, TNF- and ILs is hypothesised to
conribute to the pathogenesis of MDS. Elevated amount of these secreted factors impair
stem/progenitor cells that exhibit marked deficiencies in proliferation and differentiation, high
levels of apoptosis and dysfunctional responses to growth factor stimulation [42, 115-123].
IL-17 enhances the production of IFN- and TNF- by bone marrow T lymphocytes from
patients with lower risk MDS and may be involved in the pathogenesis of lower risk MDS
[124].
Increased rates of intramedullary apoptosis are the main cause of the cytopenias in MDS.
Apoptosis is initiated by the death receptor Fas and its specific ligand (Fas-L), which is
overexpressed and correlates with the rate of apoptosis in MDS [125-131]. Fas/Apo-1 (CD95)
and Fas-L are measured by flow cytometry, quantitative PCR of cDNA generated from
mRNA and immunohistochemistry. TNF- related apoptosis inducing ligand (TRAIL) is
a member of the TNF family, which controls apoptosis by binding to agonistic receptors
TRAIL-R1 and TRAIL-R2 and decoy receptors TRAIL-R3 and TRAIL-R4. TRAIL is present
in normal marrow in negligible amounts, but is constitutively expressed in MDS marrow
[119]. Fas-associated death-domain-like interleukin-1-converting enzyme inhibitory protein
(FLIP) is important in controlling apoptosis in normal cells. Isoforms of FLIP are products of
alternative mRNA splicing and have pro-apoptotic or anti-apoptotic properties. In early MDS,
the anti-apoptotic isoform of FLIP downregulated and apoptosis is higher than in MDS with
excess blasts, where resistance to apoptosis was described [132]. TNF- receptor TRAIL-R2,
which transmits cytoprotective signals via transcription factor NF-B is also increased in late
MDS and exert anti-apoptotic signal through regulation of bcl-2 and bcl-xL [133, 134].
However, various cytokines, such as TGF-, IFN- and TNF- itself, activate the p38
mitogen-activated protein kinase (MAPK) downstream signaling pathway in hematopoietic
stem and progenitor cells, that increases apoptotic signaling in MDS bone marrow cells [135-
140].
TOLL-LIKE RECEPTOR SIGNALING AND

ITS ACTIVATION IN MDS
The innate immune system is an evolutionarily conserved defense mechanism against
pathogens which is implicated in the pathogenesis of MDS [65-68]. The toll-like receptor
(TLR) family (10 different TLRs in humans) plays a major role in the initial detection and
subsequent elimination of foreign pathogens. This process is achieved through activation of
intracellular signaling pathways, such as NF-B and MAPK, which initiate a coordinated set
of responses. Wei et al. [141] performed a genome-wide chromatin immunoprecipitation
(CHIP) followed by sequencing (Seq) analysis of H3K4me3 in MDS. This analysis identified
multiple genes marked by increased H3K4me3 in bone marrow CD34+ cells. A large majority
of the genes identified are known to be involved in TLR-mediated innate immunity signaling
and NF-B activation [141]. These authors showed in the same study that the histone
H3K27me3 demethylase JMJD3/ KDM6B containing Jumonji domain 3 (Jmjd3) is
significantly overexpressed in MDS bone marrow CD34+ cells and has an important role in
the regulation of expression of genes involved in innate immunity. Thus JMJD3 demethylase
is capable to remove the trimethyl group from histone H3 lysine 27 [142].
Gene expression and mutational analysis of eight human TLRs were performed in a large
cohort of MDS [143]. TLR1, TLR2 and TLR6 are significantly overexpressed in MDS bone
marrow CD34+ cells. TLR1 and TLR6 are known to form functional heterodimers with
TLR2. Deep genetic sequencing identified a rare genetic variant of TLR2 (F217S) present in
11% of bone marrow mononuclear cells of patients with MDS where is associated with NF-
B activation. The level of this variant is in MDS is significantly higher than in normal
population. Inhibition of TLR2 in cultured MDS bone marrow CD34+ cells from patients with
lower risk of MDS results in increased formation of erythroid colonies. TLR2-mediated
innate immune signaling has a role in pathophysiology of MDS and its targeting may have
therapeutic potential.
Velegraki et al. [144] demonstrated increased expression of a wide panel of genes
involved in TLR4 signaling in bone marrow mononuclear cells. A gene expression
microarray showed that TRAF6 is overexpressed in MDS CD34+ cells in comparison with
healthy controls [145]. Furthermore, DNA arrays revealed the amplification of the TRAF6
locus (chromosome 11p12) and the TIRAP locus (chromosome 11q24.2) in MDS [146, 147].
TLR4 is the receptor for lipopolysaccharide (LPS), which induces the release of critical
proinflammatory cytokines that are necessary to activate potent immune responses [148].
LPS/TLR4 signaling has been intensively studied in the past years. Two pathways diverge
downstream of TLR4, the myeloid differentiation primary response gene 88 (MyD88)-
dependent and independent pathways, resulting in the expression of inflamatory cytokines of
IFN-inducible genes. The MyD88-dependent pathway mediates a rapid and acute response,
whereas MyD88-independent pathway is responsible for delayed response. MyD88 is a toll-
interleukin 1 receptor (TIR) containing adaptor protein that forms a complex on TIR domain
of TLR4 (TIRAP). MyD88 recruits IL-1 receptor associated kinase 4 (IRAK4), which then
recruits IRAK1, resulting in subsequent autophosphorylation and disassociation from the
TLR4-MyD88 complex. This complex then binds to TNF receptor-associated factor (TRAF),
key effector of the innate immune signaling complex. E3 ubiquitin ligase TRAF6 plays a key
role in downstream activation of NF-B. MyD88 is overexpressed in bone marrow
progenitors of MDS and is associated with risk stratification and patient survival [60].
Rhyasen et al. [149, 150] demonstrated IRAK1 upregulation in MDS bone marrow
mononuclear cells and showed that targeting of IRAK1 is a therapeutic approach for MDS.
36 Ota Fuchs
BONE MARROW NICHE AND ITS INVOLVEMENT IN MDS

Hematopoietic stem and progenitor cells reside within the bone marrow niche, which is
cellular and molecular microenvironment, which maintains and regulates stem cell self-
renewal, differentiation and proliferation. Mesenchymal stem cells (MSCs) are primitive,
non-hematopoietic stem cells that give rise to all of the various types of stromal cells that
form bone marrow microenvironment [50, 70-74, 151-160]. MSCs have important roles in
hematopoiesis and immune regulation.
Several studies have indicated that impaired MSCs propagate MDS [50, 70-74, 151-160].
Among the MSC impairments is altered expression of Aurora kinase genes (AURK) with an
important role in the regulation of G2/M phase of cell cycle, centrosomes and cytokinesis.
The expression of AURK is highly upregulated in MSCs in MDS patients. Dysregulated
expression of AURK leads to increased number of centrosomes, gain or loss of chromosomes
causing cell death of normal cells and survival of malignant cells.
Hematopoietic stem cells and mesenchymal stem cells undergo changes in response to
induction factors like TNF-, Fas and TGF- in the bone marrow niche of MDS. However,
these stem cells do not originate from the same neoplastic clone and often harbor different
chromosomal aberrations, suggesting distinct genetic origin of MDS niche [161].
MDS MSCs release higher amount of IL-6 than normal MSCs [162]. Il6 is secreted by
macrophages in MDS bone marrow and induces apoptosis in hematopoietic cells [162]. MDS
MSCs inhibit T-cell proliferation in vitro and suppress the immune system in vivo. MSCs
inhibit the proliferation of T-cells in normal healthy controls through secretion of TGF- and
hepatocyte growth factor (HGF). However, in MDS this secretion is decreased and MSCs
may increase the proliferation of T-cells, thereby reducing immunosuppression, which results
in increased apoptosis of MDS cells.
CXCL12, a member of the CXC family of chemokines, also known as stromal cell
derived factor 1 (SDF1), is thgough to have an important role in cell migration in and out of
the bone marrow microenvironment. It is produced by bone marrow stromal cells, including
endothelial cells and fibroblasts [152]. CXCL12 expression is lower in normal bone marrow
than in MDS bone marrow [71, 163]. Upregulated CXCL12 expression increases homing
signaling for CXCR4 expressing hematopoietic cells, resuting in their hyperproliferation.
This increased CXCL12 may be the reason for hypercellular bone marrow in MDS. CXCR4
high-expression group of MDS patients had a shorter overall survival time and shorter
relapse-free survival time compared with those of the low-expression group [164, 165]. There
are positive correlations between CXCL12 and apoptosis in the low-grade MDS. For the
high-grade MDS, there were positive correlations between CXCR4 and VEGF, and between
CXCL12 concentration and bone marrow microvessel density (MVD). The apoptosis is one
of the hallmarks for low-grade MDS and the angiogenesis for high-grade MDS. A refined
understanding of the roles that CXCL12/CXCR4 axis and its correlation with angiogenesis
and apoptosis play in MDS will fuel the development of therapies that can be targeted to the
CXCL12/CXCR4 axis.
Perhaps the most striking evidence that bone marrow MSCs may play an important role
in the induction of MDS is based on a study in mice, where selective deletion in
osteoprogenitors of Dicer1, a RNaseIII endonuclease, essential for miRNA biogenesis and
RNA processing, resulted in development of myelodysplasia and secondary leukemia [166].

Dicer1 was not deleted in hematopoietic stem cells.
As stromal cells in the endosteal niche, osteoblasts have important regulatory role in
MDS bone marrow microenvironment. Osteoblasts regulate the maturation and proliferation
of osteoclasts that are involved in hematopoietic stem cells (HSCs) support. Strong adhesion
between HSCs and osteoblasts maintains HSCs in bone marrow. In response to stress,
infection and bleeding, HSCs migrate to vascular niche, resulting in their proliferation and
differentiation. Within MDS bone marrow niche, malignant HSCs are found both in vascular
niche and endosteal niche.
INDUCTION OF MYELODYSPLASIA BY MYELOID-DERIVED

SUPPRESSOR CELLS
Immature myeloid-derived suppressor cells (MDSCs), known to accumulate in tumor-
bearing mice and cancer patients, are site-specific inflammatory and T cell
immunosuppressive effector cells that contribute to cancer progression [167-171]. Their
suppressive activity is in part driven by inflammation-associated signaling molecules, such as
the danger-associated molecular pattern (DAMP) heterodimer S100A8/S100A9 (also known
as myeloid-related protein 8 /MRP8/ and MRP14, respectively), which interact with several
innate immune receptors that are involved in the biology of MDSCs activation [46, 74, 172,
173].
Human MDSCs lack most markers of mature immune cells (LIN-, HLA-DR-) but possess
CD33, the prototypical member of sialic acid-binding Ig-like super-family of lectins (Siglec)
[168, 174]. CD33 possesses an immunoreceptor tyrosine-based inhibition motif (ITIM) that is
associated with immune suppression [174]. LIN-, HLA-DR-CD33+ MDSCs specifically
accumulate in the bone marrow of MDS patients and impair hematopoiesis through a
mechanism that involves S100A9 as an endogenous ligand for CD33-initiated signaling.
Since S100A8/S100A9 proteins activation is through the NF-B signaling pathway, drugs
that target this pathway may reduce MDSCs levels and be useful therapeutic agents in
conjunction with active immunotherapy in MDS patients.
ANTI-INNATE IMMUNE THERAPIES IN MDS

Recent findings regarding innate immune and inflammatory signals in MDS have been
exploited for the development of novel therapeutic strategies in MDS. Preclinical studies with
the specific inhibition of the activity or expression of TLR2 and its downstream effectors in
primary MDS bone marrow cells by shRNA significantly improved differentiation, induced
apoptosis and impaired their clonal generation potential, particularly in cells from lower risk
MDS patients [50]. Preclinical studies using MyD88 inhibition by inhibitory peptide, IL-8
inhibition by neutralizing antibody and IRAK1 inhibition by RNAint or specific inhibitor
molecule were described [60, 149]. Using a physics-based computational approach, Nimbus
and their co-founding partner, Schrdinger Inc., uncovered the first truly selective small
molecule IRAK4 inhibitors. The three Nimbus novel compounds, ND-346, ND-2110 and
38 Ota Fuchs
ND-2158 demonstrated high selectivity against a panel of 334 kinases, and potent in vitro
inhibition of cytokine production in cells and whole blood.
SCIO-469 is a small-molecule p38 mitogen-activated protein (MAP) kinase inhibitor
developed by Scios Inc as a potential oral therapy for inflammatory disorders. Preclinical
studies with SCIO-469 in MDS were described [137]. ARRY-614, a potent, small-molecule
dual p38/Tie2 inhibitor, developed by Biopharma, is being studied (Phase 1 study) in patients
with International Prognostic Scoring System (IPSS) low and intermediate-1 risk MDS. In an
initial dose-escalation study, using a powder-in-capsule formulation of ARRY-614, multi-
lineage activity was observed. The most promising effects were seen in patients with
thrombocytopenia and neutropenia, with transfusion independence frequently observed in
platelet transfusion-dependent patients. ARRY-614 decreased the presence of phosphorylated
p38 MAPK in bone marrow and reduced bone marrow apoptosis in most MDS patients while
efficiently decreasing the levels of some inflammatory factors and erythropoietin in patients'
plasma [175]. OPN-305 is the first humanized IgG4 monoclonal antibody against TLR2 and
is studied in phase II clinical trial to improve erytroid differentiation of MDS bone marrow
CD34+ cells.
Curis in collaboration with Aurigene designed an orally bioavailable small molecule,
which binds with high affinity to PD-L1 and disrupts the interaction between PD-L1 and PD1
receptors on T cells. Preliminary results generated by Aurigene demonstrate that in in vitro
studies, such small molecule PD-L1 antagonists can induce effective T cell proliferation and
IFN- production by T cells that are specifically suppressed by PD-L1 in culture. In addition,
such small molecules also appear to have effects similar to anti-PD1 antibodies in in vivo
tumor models, including IFN- production and inhibition of tumor growth. The anti-tumor
effect of the oral PD-L1 antagonist is similar to that seen with a known anti-PD1 antibody in
this mouse model.
Sotatercept (formerly called ACE-011) is an investigational protein therapeutic that
increases red blood cell (RBC) levels by targeting molecules in the TGF- superfamily.
Acceleron is developing sotatercept in collaboration with Celgene Corporation for the
treatment of anemia in rare blood diseases, including MDS.Sotatercept inhibits osteoclasts
and promotes osteoblast survival in MDS bone marrow microenvironment.
An oral small molecule inhibitor of TGF- receptor I kinase, LY-2157299, galunisertib,
is also being tested in a phase II trial in low and intermediate-1 risk MDS [176]. Inhibitor of
IDO1 is an inhibitor of the enzyme indoleamine 2,3-dioxygenase (IDO). This inhibitor is
proposed for the treatment of malignant diseases and has been used in phase II INCB024360
study for patients with MDS [177].
CONCLUSION AND PERSPECTIVES

Great progress has been made in recent years in understanding the role of innate immune
deregulation in the MDS pathogenesis. Constitutively activated innate immune and
inflammatory pathways affect directly hematopoiesis, lead to altered cytokine secretion and
impact T-cell immunity. All these biological effects contribute to the development and
progression of MDS. Innate immune deregulation could be induced by cellular stresses
asociated with senescent changes, genomic instability and other genetic and epigenetic
abnormalities that occur in hematopoietic cells with aging, but could be also initiated by
abnormal cellular interactions in the bone marrow environment (niche). However, it is
necessary to identify the endogenous ligands responsible for Toll-like receptors activation and
the conditions that contribute to their release. This information will help to develop new
effective therapeutic approaches.
ACKNOWLEDGMENTS
This work was supported by the grant NT/13836 from the Ministry of Health of the
Czech Republic and by project for conceptual development of research organisation No
00023736 from the Ministry of Health of the Czech Republic.
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[173] Ehrchen, J.M.; Sunderktter, C.; Foell, D. et al. The endogenous Toll-like receptor 4
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[174] Crocker, P.R.; Paulson, J.C.;Varki A. Siglecs and their roles in the immune system. Nat
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Chapter 3
PATHOGENESIS OF 5q- SYNDROME
Ota Fuchs
ABSTRACT
Interstitial deletion of a segment of the long arm of chromosome 5q [del(5q)] is one
of the most frequent karyotypic abnormalities described in de novo myelodysplastic
syndrome (MDS), occuring in approximately 15% of MDS patients. Patients with
5q- syndrome are the distinct subgroup of MDS with isolated del(5q) and have a del(5q)
as the sole karyotypic abnormality. They are characterized by macrocytic anemia, a
normal or elevated platelet count, unilobular megakaryocytes, and low risk of progression
to acute myeloid leukemia (AML) compared with other types of MDS. The long arm of
chromosome 5 contains two distinct commonly deleted regions (CDRs).
Haploinsufficiency of genes located in CDR of 5q31.2 is typical for progression of MDS
to AML and cooperates frequently with TP53 loss. The more distal CDR lies in 5q32-q33
and contains 40 protein coding genes and genes coding micro RNAs (miR-143, and miR-
145). In addition, miR-146a is located very near of this CDR. The deletion of this region
is minimally sufficient for the 5q- syndrome. In 5q- syndrome one allele is deleted that
accounts for haploinsufficiency of genes in deleted region. The decreased expression of
the ribosomal protein S14 (RPS14) gene in the 5q- syndrome causes impaired
erythropoiesis, the profound macrocytic anemia and a block in the processing of the 18S
ribosomal RNA and in the formation of the 40S ribosomal subunit. Accumulation of
unconsumed ribosomal proteins as a result of impaired ribosome biogenesis leads to
ribosomal stress. Free ribosomal proteins, particularly RPL11, bind to and sequester the
human homolog of the E3 ubiquitin ligase MDM2 (mouse double minute 2 protein), the
major enzyme for p53 ubiquitination and degradation in proteasomes. The mechanism of
erythroid failure appears to involve induction of p53 and up-regulation of the p53
pathway by ribosomal stress. Expression of miR-145 and miR-146a is lower in 5q-
syndrome compared with MDS patients with intact chromosome 5. Two key regulators of
the innate immune response, Toll-interleukin-1 receptor domain-containing adaptor
Ota.Fuchs@uhkt.cz.
52 Ota Fuchs
protein (TIRAP) and tumor necrosis factor receptor-associated factor-6 (TRAF6) are
targeted by miR-145 and miR-146a, respectively. These miRNAs target corresponding
genes through partial base pairing to the 3-UTR of the target genes. Both these targets
were aberrantly upregulated in 5q- syndrome. Down-regulation of the miRNAs (miR-145
and miR-146a) led to an up-regulation of interleukin-6 (IL-6) that was dependent on
TRAF6. Elevated IL-6 was found also in patients with 5q- syndrome. TIRAP is known to
lie upstream of TRAF6 in innate immune signaling. Knockdown of miR-145 and miR-
146a together or enforced expression of TRAF6 in mouse hematopoietic stem/progenitor
cells resulted in thrombocytosis, mild neutropenia and megakaryocytic dysplasia, features
typical for 5q- syndrome. Patients with 5q- syndrome have decreased expression of miR-
145 and increased expression of Fli-1 (Friend leukemia virus integration 1), a critical
transcriptional regulator of megakaryocyte differentiation. Overexpression of miR-145 or
inhibition of Fli-1 in CD34+ cells decreases megakaryocyte production, while inhibition
of miR-145 or overexpression of Fli-1 has the reciprocal effect. These findings have been
validated in vivo using transgenic mice. Moreover, the combined loss of miR-145 and
RPS14 cooperate to alter erythroid-megakaryocytic differentiation in a manner similar to
the 5q- syndrome. Taken together, these findings demonstrated for the first time that
coordinate deletion of a microRNA and a protein-coding gene contributes to the
phenotype of a human malignancy, the 5q- syndrome. Recently, missense mutations in
casein kinase 1 (CSNK1A1) have been described in 7.2% of MDS patients with del(5q).
This gene is located on 5q32 in CDR. Mutant casein kinase 1 enhanced -catenin
expression but did not activate p53. Casein kinase 1 is a serine/threonine kinase and is
after lenalidomide treatment a target of the cullin 4-containing E3 ubiquitin ligase
(CRL4cereblon) with cereblon as substrate receptor. Without lenalidomide, cereblon itself
and other component of CRL4cereblon complex, DNA damage binding protein 1 (DDB1)
are endogenous targets of this E3 ubiquitin ligase. Immunomodulatory drug lenalidomide
is successfully used in the treatment of 5q- syndrome. The significance of cereblon in 5q-
syndrome pathogenesis is currently studied. Isolated deletion of 5q is associated with a
favorable prognosis in MDS. However, the prognosis is less favorable, if mutation in
TP53 co-occurs in 5q- syndrome patients. The 5q- syndrome shares some characteristics
with another ribosomopathies (Diamond Blackfan anemia, Schwachman-Diamond
syndrome, and Treacher Collins syndrome).
Keywords: myelodysplastic syndromes, 5q- syndrome, RPS14, ribosomopathies, p53, innate

immunity, transcription factor Fli-1, miR-145, casein kinase 1, haploinsufficiency,
cereblon, ubiquitin ligase, lenalidomide
INTRODUCTION
Approximately 15% of patients with MDS have abnormalities of chromosome 5 [1].
These abnormalities include interstitial deletion of a segment of the long arm of chromosome
5q [del(5q)], monosomy, and unbalanced translocations. Patients with 5q- syndrome have a
del(5q) as the sole karyotypic abnormality and less than 5% of medullary blasts.
5q- syndrome as MDS category was defined by the World Health Organization (WHO) [2]
and it is characterized by refractory macrocytic anemia with dyserythropoiesis, transfusion-
dependence, normal to elevated platelet counts, hypolobated and non-lobated
megakaryocytes, female preponderance, a favourable prognosis and low risk of progression to
AML compared with other types of MDS [3-10]. Many research groups analysed
Pathogenesis of 5q- Syndrome 53
chromosome 5q deletions in patients with 5q- syndrome. We will shortly describe these
studies from historical point of view and not for relevance in the pathogenesis.
Deletion of interferon-regulatory-factor-1 gene (IRF1) mapped to chromosome 5q31 was
detected [11]. IRF1 is a putative tumor suppressor and a transcriptional activator of interferon
and interferon-stimulated genes. IRF1 dosage experiments demonstrated that 2 patients with
5q- syndrome retained both copies of this gene [12]. Thus, IRF1 maps outside the common
deleted segment of the 5q- chromosome and the same result was obtained in the case of EGR1
(epidermal growth receptor 1) [13, 14].
Molecular mapping and fluorescent in situ hybridization techniques defined the region of
gene loss in two patients with the 5q- syndrome and uncharacteristically small 5q deletions
(5q31-q33) [14]. The allelic loss of 10 genes localized to 5q23-qter [centromere-CSF2
(colony stimulating factor 2 /granulocyte-macrophage/)-EGR1 (early growth response1)-
FGFA (fibroblast growth factor acidic)-GRL (glucocorticoid receptor)-ADRB2 (-2
adrenergic receptor)-CSF1R (colony stimulating factor 1)-SPARC (secreted protein, acidic,
cysteine-rich/osteonectin/BM-40)-GLUH1(human glutamate receptor 1)-NKSF1 (NK cell
stimulating factor chain 1)-FLT4 (Fms-related tyrosine kinase 4)-telomere] was investigated
in peripheral blood cell fractions. Gene dosage experiments demonstrated that CSF2, EGR1,
NKSF1, and FLT4 were retained on the 5q- chromosome in both patients and that FGFA was
retained in one patient, thus placing these genes outside the critical region. GRL, ADRB2,
CSF1R, SPARC, and GLUH1 were shown to be deleted in both patients. The proximal
breakpoint is localized between EGR1 and FGFA in one patient and between FGFA and
ADRB2 in the other, and the distal breakpoint is localized between GLUH1 and NKSF1 in
both patients [14]. Pulsed-field gel electrophoresis was used to map the 5q deletion
breakpoints, and breakpoint-specific fragments were detected with FGFA probe in the
granulocyte but not the lymphocyte fraction of one patient. This study has established the
critical region of gene loss of the 5q- chromosome in the 5q- syndrome, giving the location
for a putative tumor-suppressor gene in the 5.6-Mb region between FGFA and NKSF1 [14].
Boultwood et al. [15] characterised the commonly deleted region (CDR) in a study
involving sixteen 5q- syndrome patients to a 1.5 Mb interval located on 5q32-33 between
D5S413 marker and GLRA1 (glycine receptor subunit -1). This region contains PDE6A
(phosphodiesterase 6A), CSF1R, CD74 (CD74 /Cluster of Differentiation 74/ molecule, major
histocompatibility complex, class II invariant chain), TCOF1 (Treacher-Collins-Franceschetti
syndrome 1), ANXA6 (annexin A6), SPARC, FAT2 (FAT tummor suppressor homolog 2, also
known as cadherin family member 8 or multiple epidermal growth factor-like domains
protein 1) genes. Advanced MDS and AML had a large del(5q) which contained both, 5q31
and 5q32-33 CDRs [16, 17].
It was shown that MDS arises from the transformation of a multipotent hematopoietic
stem cell (HSC) or myeloid-committed progenitor cell [18, 19]. These data suggest that the
gene or genes that are inactivated in the 5q- syndrome will be expressed in normal
hematopoietic stem and progenitor cells. The expression of all 40 genes assigned to the CDR
by means of the Ensembl program was thus examined in normal human bone marrow CD34+
cells by means of RT-PCR. Of the 40 genes in the CDR, 33 were expressed in CD34+ cells
and, therefore, represented candidate genes since they were expressed within the
HSC/progenitor cell compartment [15].
Cytogenetic mapping of commonly deleted regions (CDRs) centered on 5q31 and 5q32-
q33 identified candidate tumor-suppressor genes, including the ribosomal subunit RPS14, the
54 Ota Fuchs
transcription factor Egr1/Krox20 and the cytoskeletal remodeling protein, alpha-catenin.

Although each acts as a tumor suppressor, alone or in combination, no molecular mechanism
accounts for how defects in individual 5q candidates may act as a lesion driving MDS or
contributing to malignant progression in MPN (myeloproliferative neoplasms). One candidate
gene that resides between the conventional del(5q)/5q- MDS-associated CDRs is DIAPH1
(5q31.3). DIAPH1 encodes the mammalian Diaphanous-related formin, mDia1. mDia1 has
critical roles in actin remodeling in cell division and in response to adhesive and migratory
stimuli. Eisenmann et al. [20] examined evidence, with a focus on mouse gene-targeting
experiments, that mDia1 acts as a node in a tumor-suppressor network that involves multiple
5q gene products. The network has the potential to sense dynamic changes in actin assembly.
At the root of the network is a transcriptional response mechanism mediated by the MADS-
box transcription factor, serum response factor (SRF), its actin-binding myocardin family
coactivator (MAL), and the SRF-target 5q gene, EGR1, which regulate the expression of
PTEN and p53-family tumor-suppressor proteins. The authors suggest that the network
provides a homeostatic mechanism balancing HPC/HSC growth control and differentiation
decisions in response to microenvironment and other external stimuli [20].
5q- MDS patients are lower-risk MDS patients. One of the initial approaches for lower-
risk MDS patients is to optimize production of the remaining normally functioning stem cells
in patients bone marrow through the use of growth factors or erythropoiesis-stimulating
agents (ESAs). Another treatment approach is to block the effects of proapoptotic,
proinflammatory cytokines with the immunomodulatory drug lenalidomide.
THE POSSIBLE ROLE OF CANDIDATE GENES

FROM CDR IN 5q- SYNDROME
All the 40 genes within the CDR were sequenced and no mutations were found [21]. This
finding suggests that 5q- syndrome may be a one-hit cancer in contrast to the two-hit
hypothesis for the mechanism of cancer development described by Alfred Knudson [22].
Knudson's two-hit model of tumour suppressor genes supposes that two mutations are
required to cause a tumour, one occurring in each of the two alleles of the gene. Many reports
described candidate tumour suppressors that do not conform to this standard definition,
including haploinsufficient genes requiring inactivation of only one allele, and genes
inactivated not by mutation but rather epigenetic hypermethylation [23]. Thus, a dosage effect
as the result of the loss of a single allele of a gene (haploinsufficiency) may be responsible for
the 5q- syndrome. Haploinsufficiency of multiple genes mapping to the CDR at 5q32-33 may
contribute to the pathogenesis of 5q- syndrome [24-27]. Haploinsufficiency may be a driving
force in cancer [28].
Schneider et al. has recently shown that CSNK1A1 plays a central role in the pathogenesis
of del(5q) MDS [29]. They found mutations in CSNK1A1 using whole-exome sequencing in 2
of 19 del(5q) MDS patients. Further CSNK1A1 mutation was found in an additional cohort of
22 MDS cases with isolated del(5q) using next-generation targeted sequencing giving an
overall frequency 7.3% [29]. Further two papers described CSNK1A1 mutations in MDS
patients with deletion of chromosome 5q using a larger cohort of del(5q) MDS patients [30,
31]. CSNK1A1 is the first gene from the common deleted region 5q32-q33 in del(5q) where
mutation was detected.
Boultwood et al. [21] compared the transcriptome of the CD34+ cells in a group of 10
patients with the 5q- syndrome using the Affymetrix Gene Chip U133 Plus 2.0 array platform
with the transcriptome of CD34+ cells from 16 healthy control subjects and 14 patients with
refractory anemia and a normal karyotype. The majority of the genes assigned to the CDR of
the 5q- syndrome at 5q31-q32 showed a reduction in expression levels in patients with the 5q-
syndrome, consistent with the loss of one allele. Further candidate genes showing
haploinsufficiency in the 5q- syndrome included the tumour suppressor gene SPARC and
RPS14, a component of the 40S ribosomal subunit. Two genes mapping to the CDR, RBM22
(RNA-binding motif 22) and CSNK1A1, showed more than 50% reduction in gene
expression, consistent with the downregulation of the remaining allele. These results were
confirmed by Pellagatti et al. [32]. RBM22 plays a role in splicing and nuclear translocation
of the calcium binding protein ALG2 (apoptosis-linked gene 2) and causes deregulation of
apoptosis [33].
Casein kinase 1 (CK1 or CSNK1A1), a serine/threonine protein kinase, regulates the
Hedgehog signal pathway which governs cell growth in cooperation with Wnt signaling [34-
36]. CK1 is encoded by the CSNK1A1 gene, which is located in the more distal del(5q)
common deleted region 5q32-q33, and is expressed at haploinsufficient level in del(5q) MDS
[15, 21, 32].
Role of SPARC in 5q- Syndrome
Lehmann et al. [37] further studied the hematopoietic system in SPARC-null mice. These
mice showed significantly lower platelet counts compared to wild-type animals. Although
hemoglobin, hematocrit and mean corpuscular volume (MCV) were lower in mice lacking
SPARC, differences were not statistically significant. SPARC-null mice showed a
significantly impaired ability to form erythroid burst-forming units (BFU-E). However, no
significant differences were found in the formation of erythroid colony-forming units (CFU-
E), granulocyte/monocyte colony-forming units (CFU-GM) or megakaryocyte colony-
forming units (CFU-Mk) in these animals. These authors concluded that many of the genes
within the CDR associated with the 5q- syndrome exhibit significantly decreased expression
and that SPARC, as a potential tumor suppressor gene, may play some but not the key role in
the pathogenesis of this disease.
Haploinsufficiency of SPARC is implicated in the progression of the 5q- syndrome.
However, the role of SPARC in other subtypes of MDS is not fully understood, particularly in
the del(5q) type with a complex karyotype, which has a high risk to transform into AML.
SPARC silencing inhibited the growth of AML transformed from MDS by activating p53-
induced apoptosis and cell cycle arrest [38]. These data indicate that SPARC acts as an
oncogene in transformed MDS/AML and is a potential therapeutic target in MDS/AML [38].
SPARC overexpression is associated with adverse outcome in cytogenetically normal AML
patients and promotes aggressive leukemia growth in murine models of AML [39].
56 Ota Fuchs
RPS14 and Ribosomopathies
Ebert et al. [40] used an RNA-mediated interference (RNAi)-based approach to discovery

of the 5q- disease gene. They found that partial loss of function of the ribosomal subunit
protein RPS14 phenocopies the disease in normal haematopoietic progenitor cells. The forced
expression of RPS14 with a lentiviral cDNA expression vector rescued the disease phenotype
in patient-derived bone marrow cells. In addition, they identified a block in the processing of
pre-ribosomal RNA in RPS14-deficient cells. This block was functionally equivalent to the
defect in Diamond-Blackfan anemia, linking the molecular pathophysiology of the 5q-
syndrome to a congenital syndrome causing bone marrow failure. These results indicate that
the 5q- syndrome is caused by a defect in ribosomal protein function and suggest that RNAi
screening is an effective strategy for identifying causal haploinsufficiency disease genes.
Multiple different RPS14 shRNAs decreased the ratio of erythroid to megakaryocytic cells
produced in vitro. These RPS14 shRNAs decreased also the ratio of erythroid to myeloid cells
and caused the apoptosis of differentiating erythroid cells. The decreased level of RPS14 gene
expression in 5q- syndrome is not caused by the aberrant methylation of the RPS14 gene
promoter [41]. The 5q- syndrome belongs to ribosomopathies that are disorders in which
genetic abnormalities cause impaired ribosome biogenesis and function [25].
A murine model was developed in which haploinsufficiency of the Cd74-Nid67 (CD74
antigen gene - nerve growth factor-induced differentiation clone 67 gene) region containing
the RPS14 gene on mouse chromosome 18 maps CDR of the human 5q- syndrome and
contains 8 known genes [42]. This was achieved using Cre-loxP recombination to delete this
region. An Lmo2-Cre transgene was used to restrict the deletion to the hematopoietic
compartment. Two genes (Ndst1/glucosaminyl N-deacetylase/N-sulfotransferase 1 gene/ and
Cd74) from this segment on mouse chromosome 18 have been excluded as candidate genes.
RPS14 was the major candidate gene in relation to the phenotype [42]. Mice with one deleted
allele displayed hypocellular bone marrow as a consequence of a defect in hematopoetic
progenitor function that could be rescued by p53 inactivation. This murine model also
displayed a macrocytic anemia with dysplastic features in the bone marrow and
monolobulated megakaryocytes, common features of 5q- syndrome. These changes correlated
with an increase in a population of highly p53 positive cells in the bone marrow and with
elevated apoptosis (see paragraph The importance of p53 in the molecular mechanism of
5q- syndrome and Figure 1).
Inherited mutations for several ribosomal protein genes (RPS19, RPS27A, RPS26, RPS24,
RPS17, RPS15, RPS10, RPS7, RPL5, RPL11, RPL35a and RPL36) cause Diamond-Blackfan
anemia (DBA, OMIM#205900) [48-58]. Josephs [59] and Diamond and Blackfan [60]
reported the first cases of this rare disease almost exclusively affecting the erythroid lineage.
DBA is characterized by a moderate to severe anemia with normal neutrophil and platelet
counts and a marked reduction in number of red cell precursors in an otherwise normocellular
bone marrow. Several other bone marrow failure syndromes are also associated with defects
in ribosome biogenesis and function [25, 50].
Pelagatti et al. [61] investigated the expression profiles of a large group of ribosomal- and
translation-related genes in the CD34+ cells isolated from bone marrow samples of 15 MDS
patients with 5q- syndrome, 18 MDS patients with refractory anemia and a normal karyotype,
and 17 healthy controls
Figure 1. Ribosomal stress in 5q- syndrome. On the top is schematically shown normal erythroid cell
with normal ribosome biogenesis and p53 levels. On the bottom is erythroid cell where p53 is activated
by ribosomal stress secondary to RPS14 haploinsufficiency. Ribosomal proteins RPL5, 11, 23 and
RPS27L bind HDM2 and activate p53. The ribosomal protein S27-like (RPS27L) is induced by p53
activating signals and promotes apoptosis [43, 44]. To date, the role of oxidative stress in MDS has not
been fully elucidated [45-47].
Human genome U133 Plus 2.0 arrays (Affymetrix) covering over 47 000 transcripts
representing 39 000 human genes were used in this study. The expression data for selected
genes were validated using real-time quantitative PCR with predeveloped TaqMan assays
(Applied Biosystems). The expression profiles of of 579 probe sets for genes coding
ribosomal proteins (229 for large 80S ribosomal subunit /RPL/ and 176 for small 40S
ribosomal subunit /RPS/) and for genes coding translation-related factors (149 for eukaryotic
translation initiation factors /EIF/ and 25 for eukaryotic translation elongation factors /EEF/)
were analysed [61]. 55 genes were differentially expressed. 49 from these 55 genes (89%)
showed lower expression in the 5q- syndrome patient group in comparison with MDS patients
with refractory anemia and a normal karyotype, and in comparison with healthy controls.
These data were compared with data about the defective expression of genes for ribosomal
58 Ota Fuchs
proteins and translation-related factors in DBA published by Gazda et al. [62]. Three genes
(RPL28, RPS14 and EEF1D) expression were found downregulated in both diseases (MDS
5q- syndrom and DBA) [61, 62]. In addition, the expression of two pro-apoptotic genes,
TNFRSF10B (tumor necrosis factor receptor superfamily, member 10b gene) and BAX
(BCL2-associated X protein gene), was upregulated in both diseases. Pelagatti et al. [61]
suggested that the deregulation observed in ribosomal gene expression and translation-related
gene expression in the CD34+ cells isolated from bone marrow samples of MDS patients with
5q- syndrome are secondary to RPS14 haploinsufficiency. These data and similar data of
Sridhar et al. [63] support the hypothesis that the 5q- syndrome belongs to ribosomopathies,
disorders of impaired ribosomal biogenesis.
The Role of miR-145 and miR-146a in the 5q- Syndrome
In mammalian cells, miRNAs (microRNAs) are the most abundant family of small non-
coding RNAs that regulate mRNA translation through the RNA interference pathway or
target specific mRNA for degradation [64]. In general, it appears that the major function of
miRNAs is in development, differentiation and homoeostasis, which is indicated by studies
showing aberrant miRNA expression during the development of cancer.
Starczynowski et al. [65] examined expression of microRNAs (miRNAs) encoded on
chromosome 5q as a possible cause of haploinsufficiency. They showed that deletion of
chromosome 5q correlates with loss of two miRNAs that are abundant in hematopoietic
stem/progenitor cells (HSPCs), miR-145 and miR-146a. The miR-145 gene maps within the
CDR of the 5q- syndrome [21] and the miR-146a gene lies adjacent to the distal boundary of
the CDR and is also deleted in most patients with 5q- syndrome. Starczynowski et al. [65]
identified Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) and tumor
necrosis factor receptor-associated factor-6 (TRAF6) as respective targets of these miRNAs.
These miRNAs target corresponding genes through partial base pairing to the 3-UTR of the
target genes. Both these targets were aberrantly upregulated in 5q- syndrome [65]. Down-
regulation of the miRNAs (miR-145 and miR-146a) led to an up-regulation of IL-6 that was
dependent on TRAF6. Elevated IL-6 was found also in patients with 5q- syndrome [66]. IL-6
down regulates p53 expression and activity by stimulating ribosome biogenesis [67]. TIRAP
is known to lie upstream of TRAF6 in innate immune signaling. Knockdown of miR-145 and
miR-146a together or enforced expression of TRAF6 in mouse HSPCs resulted in
thrombocytosis, mild neutropenia and megakaryocytic dysplasia. A subset of mice
transplanted with TRAF6-expressing marrow progressed either to marrow failure or acute
myeloid leukemia. Thus, inappropriate activation of innate immune signals in HSPCs
phenocopies several clinical features of 5q- syndrome. Starczynowski et al. have recently
showed that loss of two miRNAs, miR-145 and miR-146a, results in leukemia in a mouse
model [68].
Patients with 5q- syndrome have decreased expression of miR-145 and increased
expression of Fli-1 [69]. Overexpression of miR-145 or inhibition of Fli-1 in CD34+ cells
decreases megakaryocyte production, while inhibition of miR-145 or overexpression of Fli-1
has the reciprocal effect. These findings have been validated in vivo using transgenic mice.
Moreover, the combined loss of miR-145 and RPS14 cooperate to alter erythroid-
megakaryocytic differentiation in a manner similar to the 5q- syndrome. Taken together, these
findings demonstrated for the first time that coordinate deletion of a microRNA and a protein-
coding gene contributes to the phenotype of the 5q- syndrome [69].
Other research groups found normal expression levels of miR-145 in most CD34+ cells
isolated from bone marrow samples of MDS patients with 5q- syndrome [70, 71].
Remarkably, the guardians of the genome p53, p73, and p63 play a role in control of most of
the known tumor suppressor miRNAs [72, 73]. Thus, it is possible that one allele of miR-145
is lost in 5q- syndrome but the second allele of miR-145 is overexpressed after p53 or related
genes activation. A putative tumor suppressing miRNA, miR-145, has been shown to be
decreased in various human cancers [74, 75], and it decreases the apoptosis and proliferation
rate of colorectal cancer cells and B-cell lymphoma cell lines [76, 77].
Zhang et al. [78] demonstrated that miR-145 targets a putative binding site in the 3-UTR
of the Friend leukemia virus integration 1 (Fli-1) gene, and that miR-145 abundance is
inversely related with Fli-1 expression in colon cancer tissues. Some other targets of miR-145
are important regulators of cell apoptosis and proliferation, such as c-Myc and IRS-1 [69, 79].
IRS-1, a docking protein for both the type 1 insulin-like growth factor receptor and the insulin
receptor, delivers anti-apoptotic and anti-differentiation signals. MiR-145 also down-regulates
the proto-oncogene c-Myc, whose aberrant expression is associated with aggressive and
poorly differentiated tumors. Zhang et al. [80] demonstrated that DNA fragmentation factor
45 (DFF45) expression is controlled at the translational level by miR-145, using
bioinformatic and proteomic techniques. DFF45 is caspase-3 or caspase-7 substrate that must
be cleaved before apoptotic DNA fragmentation can proceed [81, 82].
Changes in the expression of miR-146a have been implicated in both the development of
multiple cancers and in the negative regulation of inflammation induced via the innate
immune response. Furthermore, miR-146a expression is driven by the transcription factor
NF-B (nuclear factor kappaB), which has been implicated as an important causal link
between inflammation and carcinogenesis. Williams et al. [83] and Li et al. [84] reviewed the
evidence for a role of miR-146a in innate immunity and cancer and assessed whether changes
in miR-146a might link these two biological responses.
The role of miR-146a in hematopoiesis was investigated by using retroviral infection and
overexpression of miR-146a in mouse hematopoietic stem/progenitor cells, followed by bone
marrow transplantations [85]. miR-146a is mainly expressed in primitive hematopoietic stem
cells and T lymphocytes. Overexpression of miR-146a in hematopoietic stem cells, followed
by bone marrow transplantation, resulted in a transient myeloid expansion, decreased
erythropoiesis, and impaired lymphopoiesis in select anatomical locations. Enforced
expression of miR-146a also impaired bone marrow reconstitution in recipient mice and
reduced survival of hematopoietic stem cells. These results indicate that miR-146a, a
lipopolysaccharide (LPS)-induced miRNA, regulates multiple aspects of hematopoietic
differentiation and survival. Furthermore, the consequences of miR-146a expression in
hematopoietic cells mimics some of the reported effects with acute LPS exposure.
Third gene for miRNA in the CDR is miR-143 [69]. Cordes et al. [86] and Boettger et al.
[87] proposed that miR-145 and miR-143 regulate smooth muscle cells (SMC) fate and
plasticity and play the important role in cardiovascular development. MDS patients with 5q
deletion were characterized by decreased levels of miR-143 and miR-378 mapped within the
commonly deleted region at 5q32 [88].
60 Ota Fuchs
The Importance of p53 in the Molecular Mechanism of 5q- Syndrome
Dysregulation of ribosome biogenesis through insufficiency of ribosomal subunits has

been demonstrated to activate a p53 checkpoint and regulate developmental pathways [25,
89-93]. The p53 pathway provides a surveillance mechanism for translation in protein
synthesis and for genome integrity.
Activation of p53 by Ribosome Dysfunction in Model Systems
Activation of p53 stimulates proteasome-dependent truncation of eIF-4E-binding

protein 1 (4E-BP1) [94]. The translational inhibitor protein 4E-BP1 regulates the availability
of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds
the 5' cap structure present on all cellular mRNAs. Its ability to associate with initiation
factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex
during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive
manner by 4E-BP1. Phosphorylation of 4E-BP1 decreases the affinity of this protein for
eIF4E, thus favouring the binding of eIF4G and enhancing translation. The truncated protein
4E-BP1 is almost completely unphosphorylated and exerts a long-term inhibitory effect on
the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the
growth-inhibitory and pro-apoptotic effects of p53.
The murine double minute 2 protein (MDM2) and human analogue (HDM2) function as
a link between ribosome biogenesis and the p53 pathway. MDM2 or HDM2 proteins function
in two major ways: 1) as an E3 ubiquitin ligase that recognizes the N-terminal trans-activation
domain (TAD) of the p53 tumor suppressor and 2) as an inhibitor of p53 transcriptional
activation [95-97]. Having a short half-life, p53 is normally maintained at low levels in
unstressed mammalian cells by continuous ubiquitination and subsequent degradation by the
26S proteasome. This is primarily due to the interaction of p53 with the RING-finger
ubiquitin E3 ligases, MDM2 or HDM2. MDM2 interacts with a variety of ribosomal proteins,
including RPL5, RPL11, RPL23, RPL26 and RPS7 [98-106]. These interactions, which
typically involve the acidic domain and sometimes the adjacent zinc finger of MDM2,
interfere with the inhibitory functions of this region of MDM2 and contribute to p53
activation. As first exemplified for RPL11 [99], these interactions increase when ribosome
biogenesis is disrupted, in a situation termed ribosomal biogenesis stress or nucleolar
stress [107, 108]. Such stress can be induced by drugs that inhibit RNA polymerase I, e.g.,
low levels of the chemotherapeutic agents, actinomycin D [99, 101], 5-fluorouracil [97], or
other growth inhibitory conditions such as serum starvation and contact inhibition [101].
Mechanistically, ribosomal stress causes translocation of free ribosomal proteins from the
nucleolus to the nucleoplasm [101, 110], where they bind MDM2 [101]. The increased
binding of ribosomal proteins to MDM2 augments cellular p53 activity, leading to growth
arrest and coupling deficient protein synthesis with cessation of cell proliferation.
However, it is not clear whether ribosomal protein regulation of MDM2 is specific to
some, but not all ribosomal proteins. Sun et al. [111] showed that RPL29 and RPL30, two
ribosomal proteins from the 60 S ribosomal subunit, do not bind to MDM2 and do not inhibit
MDM2-mediated p53 suppression, indicating that the ribosomal protein regulation of the
MDM2-p53 feedback loop is specific. Interestingly, direct perturbation of the 60 S ribosomal
biogenesis by knocking down either RPL29 or RPL30 drastically induced the level and
activity of p53, leading to p53-depedent cell cycle arrest. This p53 activation was drastically
inhibited by knockdown of RPL11 or RPL5. Consistently, knockdown of RPL29 or RPL30
enhanced the interaction of MDM2 with RPL11 and RPL5 and markedly inhibited MDM2-
mediated p53 ubiquitination, suggesting that direct perturbation of 60 S ribosomal biogenesis
activates p53 via RPL11- and RPL5-mediated MDM2 suppression. Mechanistically,
knockdown of RPL30 or RPL29 significantly increased the NEDDylation and nuclear
retention of RPL11. Knocking down endogenous NEDD8 suppressed p53 activation induced
by knockdown of RPL30. These results demonstrate that NEDDylation of RPL11 plays a
critical role in mediating p53 activation in response to perturbation of ribosomal biogenesis.
Deletion of the 40S RPS6 gene in mouse liver prevents hepatocytes from re-entering the
cell cycle after partial hepatectomy. This response leads to an increase in p53, which is
recapitulated in culture by RPS6-siRNA treatment and rescued by the simultaneous depletion
of p53 [112]. However, disruption of biogenesis of 40S ribosomes had no effect on nucleolar
integrity, although p53 induction was mediated by RPL11, leading to the finding that the cell
selectively upregulates the translation of mRNAs with a polypyrimidine tract at their 5'-
transcriptional start site (5'-TOP mRNAs), including that encoding RPL11, on impairment of
40S ribosome biogenesis. For ribosomal protein (RP) genes the start of transcription is rigidly
controlled to maintain the 5'-TOP signal on the messenger RNA. Increased 5'-TOP mRNA
translation takes place despite continued 60S ribosome biogenesis and a decrease in global
translation. Thus, in proliferative human disorders involving hypomorphic mutations in 40S
ribosomal proteins, specific targeting of RPL11 upregulation would spare other stress
pathways that mediate the potential benefits of p53 induction.
Activation of p53 and up-Regulation of the p53 Pathway in 5q- Syndrome
Results of several laboratories (described in paragraph Activation of p53 by ribosome

dysfunction in model systems) showed that up-regulation of the p53 pathway is a common
response to haploinsufficiency of ribosomal proteins. Pellagatti et al. [113] carried out the
analysis of the p53 pathway in the 5q- syndrome. They used Affymetrix arrays [61]. Ten
genes in the p53 pathway (FAS /tumor necrosis factor receptor superfamily, member 6 gene/,
CD82 /cluster of differentiation 82 gene/, WIG1/wild type p53-induced gene 1 gene/, CASP3
/cysteine-aspartic acid protease, caspase 3 gene/, SESN3 /sestrin 3 gene/, TNFRSF10B,
MDM4 / murine double minute 4 gene/, BAX, DDB2 /damage-specific DNA binding protein 2
gene/ and BID2 /BH3 interacting domain death agonist 2 gene/) were significantly
deregulated. All these genes with exception of MDM4 (negative regulator of p53, structurally
related to MDM2 [114]) were expresed at higher levels in CD34+ cells from 5q- syndrome
patients in comparison with healthy controls. MDM4 is expressed at lower level in 5q-
syndrome patients. Further up-regulated genes in 5q- syndrome (RPS27L /ribosomal protein
S27-like gene/, PHLDA3 /pleckstrin homology-like domain, family A, member 3 gene/,
BCL11B /B-cell lymphoma/leukemia 11B gene/, FDXR /ferredoxin reductase gene/) are p53
targets [21, 61]. The strong p53 expression was confirmed not oly on the mRNA level but
also on the protein level by an immunohistochemical analysis. Activation of p53 as a result of
ribosomal stress seems to play an important role in the 5q- syndrome. Up-regulation of the
62 Ota Fuchs
p53 pathway offers a new potential therapeutic target in the human 5q-syndrome. This
therapy is possible only if the critical tumor suppressor function of p53 will be preserved.
The Relation between p53 and miR145
The tumor suppressor p53 is a master gene regulator capable of regulating numerous
genes. Since p53 plays a critical role in suppression of cell growth and proliferation, it is
considered as a complementary opposite factor to the oncoprotein c-Myc.
Figure 2. Schematic diagram of the development of the 5q- syndrome. The roles of the
haploinsufficiency of RPS14, miR145 and miR146 and possible progression to AML by adjunct
cytogenetic abnormalities are shown. Fli1 upregulation stimulates transcriptionally HDM2 [118] and
inhibits partially p53 activation caused by ribosomal stress resulting from RPS14 haploinsufficiency.
The oncoprotein c-Myc can counteract the p53s action to a great degree. The cell has
developed sophisticated mechanisms during evolution to balance the effect of both these
factors. It has been known for a long time that there is a negative correlation between p53 and
c-Myc. Although previous report [115] suggested that this might involve transcriptional
repression the precise mechanism of p53-mediated repression of c-Myc is not fully

understood. Sachdeva et al. [116] have recently demonstrated that p53 induces miR-145 and
subsequently represses c-Myc (Figure 2). Thus, identification of miR-145 (described in
paragraph The role of miR-145 and miR-146a in the 5q- syndrome as a new player in this
p53 regulatory network) provides at least one aspect of how the cell balances the effects of
p53 and c-Myc [117].
ROLE OF FURTHER GENES IN 5q- SYNDROME

The Role of Genes Positioned on 5q Chromosome
The murine model for the 5q- syndrome with haploinsufficiency of the Cd74-Nid67
region (described in the paragraph RPS14 and ribosomopathies) showed that other gene in
this region may play an additional role. Dctn4 gene encodes Dynactin 4, a subunit of the
Dynactin complex. The dynactin complex binds cargo, such as vesicles and organelles, to
cytoplasmic dynein for retrograde microtubule-mediated trafficking [119]. Retroviral
expression in megakaryocytes of dynamitin (p50), which disrupts dynactin-dynein function,
inhibits proplatelet elongation [120]. Patel et al. [121] concluded that while continuous
polymerization of microtubules is necessary to support the enlarging proplatelet mass, the
sliding of overlapping microtubules is a vital component of proplatelet elongation. Thus, the
inhibition of proplatelet elongation might reduce platelet formation and lead to
thrombocytopenia [121]. The further gene Rbm22 was described in paragraph 2. RBM22
belongs to the SLT11 family; it has been reported to be involved in pre-splicesome assembly
and to interact with the Ca2+-signaling protein ALG-2 [122]. Slt11p, a splicing factor in yeast,
is required for spliceosome assembly. RBM22 is under-expressed in 5q- syndrome and
deregulates probably apoptosis [33].
In addition to the minimal CDR on chromosome 5q33.1 in 5q- syndrome, a second,
proximal CDR on chromosome 5q31.2 contains genes EGR1, CTNNA1 (gene encoding alpha-
catenin) and HSPA9 (gene encoding mortalin, GRP75, PBP74, MTHSP75). Loss of this CDR
contibutes to a more aggressive MDS or AML phenotype. The gene CTNNA1 is expressed at
a much lower level in leukemia-initiating stem cells from individuals with AML or MDS with
a 5q deletion than in individuals with MDS or AML lacking a 5q deletion or in normal
hematopoietic stem cells. Analysis of HL-60 cells, a myeloid leukemia line with deletion of
the 5q31 region, showed that the CTNNA1 promoter of the retained allele is suppressed by
both methylation and histone deacetylation [123]. Restoration of CTNNA1 expression in HL-
60 cells resulted in reduced proliferation and apoptotic cell death. Thus, loss of expression of
the alpha-catenin tumor suppressor in hematopoietic stem cells may provide a growth
advantage that contributes to human MDS or AML with del(5q) [123].
In 146 AML cases, methylation of CTNNA1 was frequent, and more common in AML
patients with 5q deletion (31%) than those without 5q deletion (14%), whereas no
methylation of other 5q genes was observed [124]. In 31 MDS cases, CTNNA1 methylation
was only found in high-risk MDS (RAEB2), but not in low-risk MDS, indicating that
CTNNA1 methylation might be important in the transformation of MDS to AML. CTNNA1
expression was lowest in AML/MDS patients with CTNNA1 methylation, although reduced
64 Ota Fuchs
expression was found in some patients without promoter methylation. Repressive chromatin
marks (H3K27me3) at the promoter were identified in CTNNA1-repressed AML cell lines
and primary leukemias, with the most repressive state correlating with DNA methylation.
These results suggested progressive, acquired epigenetic inactivation at CTNNA1, including
histone modifications and promoter CpG methylation, as a component of leukemia
progression in patients with both 5q- and non-5q- myeloid malignancies [124]. Thus,
CTNNA1 is an essential tumor suppressor gene involved in leukemia stem cell transformation.
Restoration of the CTNNA1 expression represents a potential therapeutic strategy to eliminate
human leukemia-initiating cells (LICs) expressing low levels of CTNNA1 without adversely
affecting normal stem cells. There is PTEN (phosphatase and tensin homolog) - C/EBP
(CCAAT enhancer binding protein alpha) CTNNA1 axis to control myeloid development
and transformation and its targeting might be important in LICs elimination [125].
Joslin et al. [126] studied homozygous and heterozygous EGR1 knock-out mice under
normal physiologic conditions and showed no differences in comparison with wild-type mice.
However, when they treated these EGR1 knock-out mice with N-ethyl-nitrosourea (ENU),
mice developed T-cell lymphomas and a myeloproliferative disease. Thus, the data suggests
that heterozygous loss of EGR1 contributes to myeloid disease in mice by cooperating with
unidentified, ENU-induced mutations [126]. These findings also raised the possibility that
decreased expression of multiple del(5q) genes might be necessary for disease initiation, and
highlighted the need to develop whole-genome approaches to identify additional cooperating
mutations in MDS and AML.
Mortalin (HSPA9), a heat uninducible member of the heat shock protein (HSP) 70 family
of chaperones, was first identified as human mitochondrial hsp 75 [127], having important
roles in stress response, glucose regulation, cell proliferation, differentiation, and
tumorigenesis. Mortalin is part of the translocase of the inner mitochondrial membrane
complex [128] and HSPA9 knockdown in primary human hematopoietic cells and in a murine
bone marrow transplantation model using lentiviral mediated gene silencing significantly
delayed the maturation of erythroid precursors [129]. Mortalin is also located in the
cytoplasm where it can bind tumor suppressor p53 and prevent its translocation into the
nucleus [130-134]. HSPA9 knockdown causes p53 to redistribute from the cytoplasm to the
nucleus resulting in cell cycle arrest and apoptosis [129]. Mortalin also binds p53 and MPS1
at the centrosome during cell cycle progression [135-137]. MPS1 is a dual-specificity kinase
required for spindle pole body (SPB) duplication and spindle checkpoint function. HSPA9
knockdown results in a reduction of centrosome re-duplication which is necessary for normal
cell division and chromosomal stability and preferentially affects erythroid cells because
mortalin was identified as a mediator of erythropoietin signaling [138]. Reduced levels of
HSPA9 attenuate signal transducer and activator of transcription 5 (STAT5) activation in
mouse B cells [139].
Apc, a negative regulator of the canonical Wnt signaling pathway, is a bona-fide tumor
suppressor whose loss of function results in intestinal polyposis. APC is located in a
commonly deleted region on human chromosome 5q, proximal to the 5q31.2 minimally
deleted region, suggesting that haploinsufficiency of APC contributes to the MDS phenotype.
Analysis of the hematopoietic system of mice with the Apc(min) allele that results in a
premature stop codon and loss of function showed no abnormality in steady state
hematopoiesis [140]. Bone marrow derived from Apc(min) mice showed enhanced
repopulation potential, indicating a cell intrinsic gain of function in the long-term
hematopoietic stem cell (HSC) population. However, Apc(min) bone marrow was unable to
repopulate secondary recipients because of loss of the quiescent HSC population. Apc(min)
mice developed a MDS/myeloproliferative phenotype [140]. Wnt activation through
haploinsufficiency of Apc causes insidious loss of HSC function that is only evident in serial
transplantation strategies. These data provide a cautionary note for HSC-expansion strategies
through Wnt pathway activation, provide evidence that cell extrinsic factors can contribute to
the development of myeloid disease, and indicate that loss of function of APC may contribute
to the phenotype observed in patients with MDS and del(5q).
Other genes localized on chromosome 5q31.2 (MATR3, FAM53C, ETF1, JMJD1B and
KLHL3) showed reduced expression in MDS patients with deletions spanning chromosome
5q31.2 [141].
The gene for nucleophosmin (NPM1) localized on chromosome 5q35.1 is deleted in
many cases of 5q- MDS. Targeted knockout of one allele of NPM1 leads to genetic instability
[142-144]. NPM1 heterozygous mice develop both myeloid and lymphoid malignancies. The
bone marrow of these mice was hypercellular with evidence of dysplasia of the erythroid and
megakaryocytic lineages. Immature erythroid cells accumulate in this bone marrow. The
NPM1 haploinsufficiency in patients with large 5q deletions contributes to the phenotype of
MDS. Moreover, NPM1 is the most commonly mutated gene in patients with normal
karyotype AML [145-147].
The Role of Genes Not Positioned on Chromosome 5
We studied the role of the levels of mRNA for transcriptions factors Fli-1 (Friend
leukemia virus integration 1) and EKLF (erythroid Krppel-like factor, also named KLF1) in
mononuclear cells isolated from bone marrow and peripheral blood of MDS patients with 5q-
syndrome in comparison with patients with low risk MDS without 5q chromosome
abnormality and with healthy controls [148, 149]. We found increased Fli1 mRNA levels and
decreased EKLF mRNA levels in 5q- syndrome. The decreased expression of miR-145 might
be responsible for increased Fli-1 levels in 5q- syndrome (see paragraph The role of miR-
145 and miR-146a in the 5q- syndrome and Figure 2). We suggest that increased Fli-1
expression in mononuclear cells of MDS patients with 5q- syndrome is further stimulated by
the increased Fli-1 level and by the decreased EKLF level. Kuvardina et al. found thatrunt-
related transcription factor (RUNX1) inhibits erythroid differentiation of murine
megakaryocytic/erythroid progenitors and primary human CD34+ progenitor cells [150].
RUNX1 represses the erythroid expression program by epigenetic repression of the erythroid
master regulator KLF1. RUNX1 shifts the balance between KLF1 and Fli-1 in the direction of
Fli-1.
Fli-1 is a member of the Ets family of transcription factors. Fli-1 was originally identified
as a proto-oncogene in Friend virus-induced erythroleukemia in mice [151]. The founding
member of this family, the oncogene v-ets, was discovered in the genome of avian leukosis
virus E26. Ets transcription factors bind to DNA elements containing the consensus sequence
GGA(A/T). Fli-1 is preferentially expressed in hematopoietic cells and in vascular endothelial
cells. Pulse-field gel analysis has localized the Fli-1gene within 240 kb of the Ets-1 locus on
mouse chromosome 9 and on human chromosome 11q23, suggesting that these two ets genes
66 Ota Fuchs
arose by gene duplication from a common ancestral gene [152, 153]. Human Fli-1 contains
nine exons which extend over approximately 120 kb [154].
Fli-1 plays an important role during the normal development of megakaryocytes. Fli-1
knockout mice produce small, undifferentiated megakaryocytic progenitors with abnormal
features. Fli-1 represses the transcription of EKLF gene [155] and activates the promoters of
several megakaryocyte-specific genes (glycoprotein IX and thrombopoietin receptor genes
promoters) [156, 157]. Fli-1 gene promoter is upregulated by Fli-1 and other Ets factors
(Ets1, Ets2 and Elf1) but is inhibited by Tel [158]. Transcription factor PU.1 (also known as
Spi-1) is also a positive regulator of the Fli1 gene [159]. On the other hand, PU.1 inhibits
transcription factor GATA-1 function and erythroid differentiation by blocking GATA-1
DNA binding [160, 161]. Both, PU.1 and Fli-1 directly activate common target genes
involved in ribosome biogenesis [162]. One of the consequences of miR145 and miR146a
depletion or TIRAP and TRAF6 activation in the innate immune system pathway is
overexpression of IL-6 which activates Fli-1 gene expression [65, 85, 163-165].
EKLF (erythroid Krppel-like factor, also named KLF1) is a zinc finger transcription
factor that plays a prominent role during erythroid development [166-168]. Human gene
EKLF is positioned on chromosome 19p13.12-13 [169]. Commitment towards
megakaryocyte versus erythroid blood cell lineages occurs in the megakaryocyte-erythroid
progenitor (MEP). EKLF restricts megakaryocytic differentiation to the benefit of
erythrocytic differentiation and this effect is mediated by the inhibition of Fli-1 recruitment to
megakaryocytic and Fli-1 genes promoters [154, 170 - 172]. A marked increase in the number
of circulating platelets in EKLF null mouse was found [173].
Therapy of 5q- Syndrome
MDS with deletion of the long arm of chromosome 5 (del5q) belong to the lower risk
(low or intermediate 1 risk) prognostic group of the International Prognostic scoring system.
Therapy of the 5q- syndrome will be discussed in full detail in the special chapter
Lenalidomide Treatment in Lower Risk Myelodysplastic Syndromes. Anemia in these
MDS responds less often to erythroblastic stimulating agents. However, immunomodulatory,
anti-cytokine, and anti-angiogenic agent lenalidomide (CC-5013; REVLIMID, Celgene
Corporation) leads to red blood cells transfusion independence of low risk MDS with del 5q
[174-183]. The del 5q syndrome is now recognized as a distinct pathologic subtype of MDS
with markedly better clinical responses with lenalidomide treatment compared to non del 5q
MDS patients.
Lenalidomide, a structural analogue of thalidomide, is indicated for the treatment of the
5q- syndrome MDS patients, rendering 67% of patients transfusion-independent and inducing
cytogenetic responses in over 40% of them . In contrast, in a large multicenter trial
involving transfusion dependent MDS patients without del(5q) only 26% achieved
transfusion-independence with infrequent cytogenetic improvement [184]. The selective and
specific activity of lenalidomide in MDS remains undefined. Gene expression profiling and
disease- and therapy-associated proteome changes in the sera of MDS patients were used for
monitoring and predicting the response to therapy [185, 186]. Wei et al. [187] provided
evidence that lenalidomide is selectively cytotoxic to del(5q) cells as a result of inhibition of
the haplodeficient dual specificity phosphatases, Cdc25C and PP2A. Phosphorylation of

MDM2 and Fli-1 are important for the level and the activity of both these proteins involved in
the process of erythroid failure in 5q- syndrome and the level of PP2A regulates accumulation
and degradation of p53 and Fli-1 [188, 189]. Biochemical and molecular analyses showed
that inhibition of the haplodeficient PP2A by lenalidomide resulted in hyperphosphorylation
of inhibitory serine residues 166 and 168 on MDM2 and displaced RPS14 to stabilize
MDM2, thereby accelerating p53 degradation [190].
Krnke et al. demonstrated that lenalidomide induces ubiquitination and degradation of
CK1 in del(5q) MDS [191; Figure 3]. Lenalidomide decreased CK1 protein levels without
altering corresponding CSNK1A1 mRNA expression. The lenalidomide-dependent decrease
in CK1 protein level was abrogated by treatment with the proteasome inhibitor MG132 and
the neddylation (NEDD8-activating enzyme) inhibitor MLN4924 (pevonedistat). Therefore,
CRL4cereblon and proteasomes play the important roles in CK1 degradation [191]. CK1
binds cereblon after lenalidomide treatment and is ubiquitinated by CRL4cereblon E3 ubiquitin
ligase. Decreased expression of CSNK1A1 sensitizes cells to lenalidomide. Knockdown of
CSNK1A1 sensitized primary CD34+ cells to lenalidomide, suggesting that haploinsuficiency
of CSNK1A1 might increase lenalidomide sensitivity in del(5q) hematopoietic cells [191].
Other immunomodulatory drugs (IMiDs) as thalidomide, pomalidomide or CC-122, a
non-phthalimide analog of thalidomide, which also bind to cereblon do not induce
ubiquitination and degradation of CK1 in MDS-L cells (cell line of MDS cells with del(5q)
and other abberations) [191].
Figure 3. Schematic diagram of ubiquitination and degradation of casein kinase 1 by CRL4cereblon E3

ubiquitin ligase and proteasomes. Binding of cereblon by lenalidomide induces ubiquitination (marking
CK1 for degradation) and degradation of casein kinase 1. CRBN functions as a substrate recognition
component (substrate receptor) of this E3 ubiquitin ligase enzyme complex. CRL4 cereblon complex
consists of cullin 4A, RING finger protein regulator of cullins (Roc1), and DNA damage binding
protein 1 (DDB1).
68 Ota Fuchs
We showed that the levels of full-length CRBN mRNA are higher in 5q- syndrome bone
marrow and peripheral blood mononuclear cells than in low-risk MDS with normal
chromosome 5 and in healthy controls [192]. These levels correlate with the efficiency of
lenalidomide treatment. The strong decrease of CRBN mRNA levels in the course of
lenalidomide therapy is connected with the loss of sensitivity to lenalidomide and with the
progression of disease to higher-risk form MDS or AML [192].
The mechanism of action of lenalidomide is complex and includes inhibition of a wide
array of proinflammatory cytokines, such as IL-6, and upregulation of T-helper-secreted
cytokines, such as IL-2 and IFN-gamma [193]. It has been also described that IL-2
subsequently activates natural killer (NK) cells. In view of the recently revealed role of IL-6
in the pathogenesis of the 5q- syndrome [65, 194], it is tempting to speculate that the
responses induced by lenalidomide therapy in patients with this disorder are due to down-
regulation of IL-6. Therefore, IL-6 expression and/or upstream regulators, such as TRAF6,
may represent targets, which suggest that agents capable of inhibiting the TRAF6IL-6 axis
may be clinically useful for the management of 5q- syndrome [65, 194, 195].
Lenalidomide inhibits the malignant clone and up-regulates the SPARC and RPS14 genes
expression [196, 197]. Both these genes are localized in CDR on chromosome 5. Induction of
miR-143 and miR-145 in CD34+ cells of MDS patients with the del(5q) abnormality after
exposure to lenalidomide correlates with clinical response [198, 199]. The beneficial effect of
lenalidomide in patients with 5q- syndrome is associated with significant increases in the
proportion of bone marrow erythroid precursor cells and in the frequency of clonogenic
progenitor cells, a substantial improvement in the hematopoiesis-supporting potential of bone
marrow stroma and significant alterations in the adhesion profile of bone marrow CD34+ cells
[198, 199].
Although lenalidomide efficiently reduced a larger fraction of the del(5q) stem cells,
some rare and phenotypically distinct quiescent del(5q) stem cells remained resistant to
lenalidomide [200]. Over time, lenalidomide resistance developed in most of the patients with
recurrence or expansion of the del(5q) clone and clinical and cytogenetic progression. The
resistant clone, insensitive to lenalidomide, overexpresses p53 and sequencing confirmed
TP53 mutation [201, 202]. The presence of easily detectable subclones with inactivated p53,
and thereby a more malignant potential, is important and has prognostic value. Mutation of
TP53 may be one of the molecular events involved in the clonal progression of the 5q-
syndrome to AML [203-206]. The presence of TP53 mutation influences negatively response
to lenalidomide in del(5q) MDS [203, 207]. Immunohistochemical analysis of p53 in bone
marrow biopsies from 85 lower-risk del(5q) MDS patients treated with lenalidomide showed
strong p53 expression in more than 1% of bone marrow progenitor cells in 35% of patients
and was associated with shorter survival and with progression of disease to higher-risk MDS
and AML [208]. The tumor suppressor TP53 gene is located on the short arm of chromosome
17, and its p53 protein product has been directly related to cell cycle regulation and apoptosis
induction [209, 210]. The active form of p53 has a very short half-life (approximately 6 min)
and it is uneasily detectable. However, mutated or inactive forms of the protein accumulate in
the cell nucleus and can be easily detected by immunological methods such as
immunochemistry [208, 211].

Candidate genes were studied using gene deletions or similar approaches to determine
whether they have a role in 5q- syndrome, a subtype of MDS. A major advance occured in
2008 when RNA interference -based approach for all 40 genes located within the 5q- CDR
was used to discover the 5q- disease gene. A deffect in the function of a ribosomal protein
RPS14 that is important for the proper processing of the 40S ribosomal subunit has been
implicated in the pathophysiology of the 5q- syndrome. The RPS14 gene is located in the
deleted region and it seems that this loss affects erythroid differentiation. Haploinsufficiency
of RPS14 gene in 5q- syndrome is associated with deregulation of ribosomal- and translation-
related genes. Dutt et al. [212] have recently found that p53 accumulates selectively in the
erythroid lineage in primary human hematopoietic progenitor cells following expression of
shRNAs targeting RPS14 or RPS19. Thus, 5q- syndrome is similarly as Diamond Blackfan
anemia, where RPS 19 is the most commonly mutated gene, a ribosomopathy [24, 25, 213,
214].
Induction of p53 in both diseases, but not in control samples, led to lineage-specific
accumulation of p21WAF1, CIP1, also known as cyclin-dependent kinase inhibitor 1 or CDK-
interacting protein 1 (a proliferation arrest determinating protein that in humans is encoded by
the CDKN1A gene located on chromosome 6p21.2). The induction of p21WAF1, CIP1 caused cell
cycle arrest in erythroid progenitor cells. Pharmacological inhibition of p53 rescued the
erythroid defect, while a compound that activates p53 through inhibition of HDM2, nutlin-3,
selectively impaired erythropoiesis. The increased apoptosis was observed in the 5q- mouse
bone marrow [41]. In response to diverse stresses, the tumor suppressor p53 diferently
regulates its target genes, inducing cell cycle arrest, apoptosis or senescence. The p53 is
critical regulator of hematopoietic stem cell (HSC) behavior and plays an important role in
regulating HSC quiescence, self-renewal, apoptosis and aging. The p53 promotes HSC
quiescence [215, 216]. The mutant p53 protein can provide escape from apoptosis and
facilitates also the entry of HSC to the cell cycle. Mutations in TP53 are relatively uncommon
in cases of primary (de novo) MDS but have high incidence in patients with therapy related
MDS [217-219]. Garderet et al. [220] found defective erythroid proliferation but not
differentiation capacity related to RPS14 gene haploinsufficiency. They used in vitro model
of erythropoiesis [221, 222] in which mature red blood cells are generated from human
progenitor cells to analyze cell proliferation and differentiation in a homogeneous erythroid
population with RPS14 gene haploinsufficiency in culture. The enucleation capacity of 5q-
clones remained unchanged. Therefore, the decreased erythroid maturation and subsequent
anemia seen in 5q- syndrome is unlikely caused by a specific blockade of late differentiation
and is probably the consequence of the proliferative defect of both pathological and residual
non-deleted clones in the patients bone |marrow [223].
Treatment of zebrafish and murine models of ribosomopathies with L-leucine results in
an improvement of anemia and developmental defects in both diseases (Diamond Blackfan
anemia and 5q- syndrome) [224, 225]. Cmejlova et al. [226] demonstrated that the efficiency
of mRNA translation is significantly depressed in cells derived from Diamond Blackfan
anemia patients, consistent with a pathogenic ribosomal defect. L-leucine is an essential
branched chain amino acid that is known to modulate protein synthesis by enhancing
translation [227-230]. L-leucine has been used to treat Diamond Blackfan anemia patients
70 Ota Fuchs
[231, 232]. Payne et al. [224] confirmed findings from zebrafish model in primary human
CD34 cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, they showed that
loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in
both ribosomopathies models. This effect could be abrogated by rapamycin suggesting that
mTOR signaling may be responsible for the improvement in anemia associated with L-
leucine. Yip et al. [233, 234] reported that L-leucine treatment of cultured erythroblasts
derived from CD34+ cells of healthy controls with RPS14 knockdown and from CD34+ cells
of del(5q) patients resulted in an increase in proliferation, erythroid differentiation and
mRNA translation. These studies support the rationale for ongoing clinical trials of L-leucine
as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS. Leucine and
the other branched-chain amino acids influence protein synthesis by signaling through the
mTOR pathway. The mTOR is central to the control of translation in response to cytokine
signaling and nutrient availability [235, 236]. L-leucine has been used to treat Diamond
Blackfan anemia patients [235, 236]. Three consenting patients withWorld Health
Organizationdefined MDS with isolated del(5q) (a 71-year-old man, a 65-year-old woman,
and a 58-year-old woman; 2 of whom were red cell transfusiondependent and 1 of whom
had no response to erythropoiesis stimulating agent therapy) who were reluctant to initiate
treatment with lenalidomide took L-leucine capsules obtained from a nutritional supplement
store at a dose of 1500 mg orally 3 times daily for 2 to 3 months, approximating the 700
mg/m2 dose chosen for a planned trial of L-leucine in transfusion-dependent patients with
DBA. The DBA trial is registered at www.clinicaltrials.gov as NCT01362595. Although no
adverse events were observed during L-leucine therapy, none of the patients experienced any
improvement in their cytopenias or transfusion needs. Two of the 3 patients subsequently
agreed to take lenalidomide, and both became transfusion-independent during that therapy.
Gene deletion studies indicate that the polyproline domain in the p53 is essential for
effective apoptotic response to stress and inhibit tumorigenesis [237]. Single-nucleotide
polymorphism (SNP), R72P in TP53 exon 4, is linked to cancer and mutagen susceptibility.
There was no association with TP53 mutation and any R72P genotype in either del(5q) or
non-del(5q) MDS [237].
Cenersen, a clinically active 20-mer antisense oligonucleotide complementary to exon 10
of TP53, suppressed p53 expression and restored erythropoiesis in del(5q) MDS patient cells
in culture [238]. Cenersen decreased apoptosis and increased cell proliferation. A Study of
Aezea (Cenersen) in transfusion dependent anemia associated with MDS is in progress.
CK1 and PP2A inhibition in del(5q) MDS are also studied and may be efficient together
with lenalidomide treatment [29, 239].
MDS has long been presumed to be a stem cell disorder, but the final proof has been
lacking [240- 242]. Woll et al. established the existence of rare multipotent MDS stem cells
(MDS-SCs), and their hierarchial relationship to lineage-restricted MDS progenitors [243].
All identified recurrent somatic driver-mutations in low- to intermediate-risk MDS provide
genetic tools to map the identity and fate of MDS-propagating cells in patients in vivo. In
isolated del(5q) MDS, acquisition of del(5q) preceded diverse recurrent driver mutations
[243]. The only low- to intermediate-risk MDS cases in which del(5q) was not the first
identified genomic lesion were four cases of ring sideroblastic anemia in which del(5q) was
preceded by a recurrent SF3B1 mutation. CSNK1A1 is the first gene from the common
deleted region 5q32-q33 in del(5q) where mutation was detected. CSNK1A1 may be the cause
of the initial clonal expansion in patients with 5q- syndrome.
CK1 was shown to colocalize with factors involved in pre-mRNA splicing at nuclear
speckles and to be capable of phosphorylating particular splicing factors within a region rich
in serine/arginine dipeptide repeat motifs [244]. CK1 plays an important role in p53
signaling [245]. Knockdown or downregulation of CK1 in leukemia cells triggers p53
activation [246]. This knockdown of CK1 reduces interaction of MDM2 with p53 thus
increasing p53 activity [247] and inhibits MDMX-p53 binding that leads finally to further
p53 activation [248, 249]. CK1 inhibition increases levels of the inhibitor of the mTOR
kinase (DEPTOR) and inhibits mTOR signaling [250]. Further studies of these signaling
pathways in the mechanism of 5q- syndrome pathogenesis and in lenalidomide treatment
mechanism are necessary.
ACKNOWLEDGMENTS
This work was supported by the research grant NT/13836 from the Ministry of Health of
the Czech Republic.
Patients with MDS-5q- syndrome have macrocytic anemia often with hypoplastic
erythropoiesis and on the contrary thrombocythemia with effective though dysplastic
megakaryopoiesis. Megakaryocytes and erythroid cells are thought to share a common
progenitor MEP (T. P. McDonald et al. Exp. Hematology 1993). There are two key
transcription factors which together with other transcription factors and relevant cytokines
and receptors determine the hemopoietic differentiation of the common stem cell: erythroid
Krppel-like factor (EKLF) for erythroid lineage and Friend leukemia virus integration 1
(FLi-1) for megakaryopoiesis (Pilar Frontelo et al. Blood 2007; G. A. Blobel, Blood 2007; F.
Bouilloux et al. Blood 2008). There is functional cross antagonism between FLi-1 and EKLF
(J. Starck et al. Mol. Cel. Biology 2003). FLi-1 is active only if dephosphorylated (H. Huang
et al. ASH Abstracts 2008). The question is whether both factors play any role in
5q- syndrome.
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Chapter 4
GENETIC MUTATIONS IDENTIFIED

IN MYELODYSPLASTIC SYNDROMES
AND THEIR SIGNIFICANCE
Ota Fuchs*
ABSTRACT
Somatic point mutations are common in MDS and are associated with specific
clinical features but are not included in current prognostic scoring systems. Use of new
molecular biology array-based techniques such as comparative genomic hybridization
and single-nucleotide polymorphism (SNP) analysis led to the identification of genes
involved in epigenetic regulation such as tet (ten-eleven translocation) oncogene family
member 2 (TET2), additional sex combs like 1 (ASXL1), and enhancer of zeste 2 (EZH2).
Combination of new technologies, including next-generation sequencing and mass
spectrometry-based genotyping have been used to identify further somatic mutations in
genes for other epigenetic regulators: UTX (ubiquitously transcribed tetratricopeptide
repeat, X chromosome), DNA methyltransferase 3A (DNMT3A), and isocitrate
dehydrogenase 1/2 (IDH1/IDH2). TET2 may convert 5-methylcytosine (5mC) to 5-
hydroxymethylcytosine (hmC). TET2 is the most frequently mutated gene in MDS known
so far and it may act as tumor-suppressor gene. ASXL1 belongs to the enhancer of
trithorax and Polycomb (ETP) gene group. MDS phenotypes may be caused not only by
loss-of-function of ASXL1 but also by gain-of-function mutations, overexpression of this
gene and so on. EZH2 is a kind of histone methyltransferase. EZH2 is frequently over-
expressed in a wide variety of cancerous tissue types, which reveals it has oncogenic
activity. While defined mutations resulted in dysfunction of histone methyltransferase
activity, suggesting that EZH2 acts as a tumor suppressor for myeloid malignancies, UTX
coding protein is a histone H3 lysine 27 demethylase, and UTX can affect cell
proliferation as well as cell fate decision. Inactivating UTX mutations have been recently
found in multiple cancer types. DNMT3A belongs to the DNA methytransferases
*
Ota.Fuchs@uhkt.cz.
88 Ota Fuchs
(DNMT) gene family. It may be correlated with abnomal methylation status in patients
with MDS. These gene mutations may play key roles in the pathogenesis of MDS. Large
clinical correlative studies are beginning to decipher the clinical importance, prevalence,
and potential prognostic significance of these mutations. Mutations in further genes:
RUNX1/AML1 (Runt-related transcription factor 1, also known as acute myeloid
leukemia 1), TP53 (coding tumor suppressor protein p53) and NRAS (neuroblastoma rat
sarcoma oncogene homolog) were most strongly associated with severe
thrombocytopenia and an increased proportion of bone marrow blasts. Recently,
heterozygous missense mutations in the spliceosomal machinery for pre-mRNA splicing
(spliceosome gene mutations such as U2AF1, SF3B1, SRSF2, or ZRSR2) have been
detected in MDS genomes by massively parallel sequencing technology. The
identification of new genetic lessions could facilitace new diagnostic and therapeutic
strategies in the future. However, investigating the broad spectrum of recurrent somatic
mutations in MDS exceeds capacity of many laboratories when traditional molecular
techniques are used. Next-generation sequencing (high-throughput second generation
sequencing) and mass spectrometry-based genotyping are applicable to increase
diagnostic sensitivity and to improve risk stratication and prognosis of MDS patients in
addition to classical methods (cytomorphology and cytogenetic analysis). Comprehensive
myeloid marker panels have been recently successfully used in diagnostic practice. This
molecular mutation analysis may contribute to risk stratification in possible candidates
for allogeneic stem cell transplantation and in all new clinical research studies in MDS
patients.
Keywords: MDS, TET2, ASXL1, EZH2, DNMT3A, IDH1/2, UTX, RUNX1, TP53, NRAS,
spliceosomal gene aberrations, mutations, high-throughput second generation sequencing,
myeloid deep-sequencing panel
INTRODUCTION
Myelodysplastic syndromes are a heterogeneous group of clonal hematopoietic stem cell
disorders characterized by ineffective hematopoiesis, peripheral cytopenias, frequent
karyotypic abnormalities and risk of transformation to AML [1, 2]. Treatments of MDS
patients are tailored to the predicted prognosis for each each patient. This makes the accurate
prediction of the prognosis very important for the therapy. Cytogenetics and certain clinical
features are the basis for current prognostic scoring systems [3-5]. Patients are stratified into
risk groups. However, more than half of MDS patients have a normal karyotype, and patients
with identical chromosomal abnormalities are often clinically heterogenous [6, 7]. Somatic
mutations are common in MDS (about 50% of all patients had at least one point mutation)
and are associated with specific clinical features [8-21]. Single gene mutations are not
currently used in prognostic scoring systems but have impact on clinical phenotypes and
overall survival. New studies are required to understand the clinical effects of mutations in
various genes and to improve the prediction of prognosis for MDS patients and their therapy.
Untill recently, mutations that are causally linked to the pathogenesis of MDS were largely
unknown. Using novel technologies like high-resolution genome-wide single-nucleotide
polymorphism (SNP)-based arrays (SNP-A) karyotyping and targeted or genome-wide next-
generation sequencing (NGS), various genes have now been identified that are recurrently
mutated [13-18, 20-23]. SNP-A karyotyping can improve the detection of chromosomal
Genetic Mutations Identified in Myelodysplastic Syndromes 89
lesions. Using NGS technologies, the identification of somatic mutations and their
combinations may help to define the clonal architecture of MDS. Technological
developments have enabled the identifivation of many new genetic lesions in MDS patients.
Limited analyses were done but for one or small number of genes and the full impact of
genetics on MDS has not yet been fully realized. However, profound insights into the MDS
pathogenesis have been done.
Three large studies that examined patterns of mutations and their prognostic value in a
large cohort of MDS patients, used samples from 439 [8], 738 [16] and 944 [18] patients with
MDS. 845 of the 944 patients (89.5%) harbored at least one mutation with a median of 3 (0-
12) mutations per sample. 574 of the 845 patients with mutation (67.9%) cases had a normal
karyotype [18]. In 19 cases gene deletions without further mutations were observed. The six
most frequently (>10% of the MDS cases) mutated genes were TET2, SF3B1, ASXL1, SRSF2,
DNMT3A and RUNX1 [18]. Less common mutations (2-10%) involved U2AF1, ZRSR2,
STAG2, TP53, EZH2, CBL, JAK2, BCOR, IDH2, NRAS, MPL, NF1, ATM, IDH1, KRAS,
PHF6, BRCC3, ETV6 and LAMB4. Most of the significantly mutated genes were previously
described in MDS. Newly identified or re-confirmed as recurrently mutated genes include
BRCC3, FANCL, LUC7L2, STAG2 and other cohesin components and binding partners to
chromatin loops such as RAD21, SMC3 and SMC1A or CTCF, GPRC5A, LAMB4 and IRF1.
Mutated genes were grouped into several functonal pathways [18]. These pathways (RNA
splicing, DNA methylation, chromatin modification, transcription, Ras signal transduction,
cohesion and DNA repair pathways) are involved in MDS pathogenesis. Haferlach et al. [18]
devised a novel prognostic model combining mutational status of 14 genes with conventional
risk models including variables used in the International Prognostic Scoring System (IPSS).
This novel model better predicted overal survival compared to a model composed solely of
genetic mutations and the IPSS-R (revised IPSS for MDS) [18]. In a multivariable analysis
that included clinical features and mutations, mutations in five genes (TP53, EZH2, ETV6,
RUNX1, and ASXL1) were independently associated with decreased overal survival [8].
Mutations in one or more of these genes were present in 137 of the 439 patients (31.2%).
Mutations in WT1, IDH2, STAG2 and NRAS correlated strongly with percentage of bone
marrow blasts, whereas SF3B1 mutations predicted a low fraction of blasts. These findings
indicate that mutations in specific genes help explain the clinical heterogeneity of MDS and
that the identification of these mutations would improve the prediction of prognosis in MDS
patients. Gene mutations show significant correlations. The positive correlation of TET2 and
ZRSR2 mutations and negative correlation of ASXL1 and SF3B1 mutations have been
described [16, 18]. Given that many genes are rarely mutated and show the complex patterns
of comutation, much larger sample sizes will be necessary to evaluate genotype-phenotype
correlations [16].
Clinical consequences of all determined mutations are not fully known or are associated
mostly with poor prognosis, more advanced disease and with progression to AML. However,
this comprehensive molecular genetic profiling is promising approach to contribute to
diagnostic accuracy, pathogenesis, biologic subclassification and finally prognostication and
risk stratification in patients with MDS. Considering the decrease of costs for this procedure
in the near future, molecular profiling of multiple target genes will be integrated in
individualized therapeutic decision making for patients with MDS [8, 16, 18].
Uniparental disomy (UPD) can be identified by SNP-A. UPD is caused by chromosomal
non-disjunction or by homologous recombination during mitosis leading to partial duplication
90 Ota Fuchs
of the maternal or the paternal chromosome [24-29]. Some of the regions of UPD included
genes known to be mutated and the potential relationship between UPD and homozygous
gene mutations was therefore investigated [30-35].
Bone marrow genetic abnormalities in MDS have provided important biological and
prognostic information. However, frequent sampling in older patients has been associated
with significant morbidity. The concordance of genetic abnormalities in bone marrow (BM)
and peripheral blood (PB) samples from 201 MDS patients was assessed utilizing SNP-A and
targeted gene sequencing (TGS). 95% (124/130) patients have identical mutations in BM and
PB [36]
All cancers arise as a result of somatically acquired changes in the DNA of cancer cells.
That does not mean, however, that all the somatic abnormalities present in a cancer genome
have been involved in development of the cancer. Indeed, it is likely that some have made no
contribution at all. To embody this concept, the terms driver and passenger mutation have
been used. A driver mutation is causally implicated in oncogenesis. It has conferred growth
advantage on the cancer cell and has been positively selected in the microenvironment of the
tissue in which the cancer arises. A driver mutation need not be required for maintenance of
the final cancer (although it often is) but it must have been selected at some point along the
lineage of cancer development. Driver mutations have equivalent prognostic significance,
whether clonal or subclonal, and leukemia-free survival deteriorated steadily as numbers of
driver mutations increased. Thus, analysis of oncogenic mutations in large cohort of patients
illustrates the interconnection between the cancer genome and disease biology.
Table 1. Frequency and impact of chosen genetic mutations identified in MDS
Gene Prognostic Estimated frequency Additional findings

mutation impact in MDS
SF3B1 Favorable 20-25% highly associated with ring sideroblasts
decreased response to lenalidomide in 5q-
8% (found in 20% of MDS, poor outcome post alloSCT, associated
TP53 Poor
5q- MDS) with complex karyotype, higher BM blasts,
and thrombocytopenia
identifies lower risk MDS with aggressive
EZH2 Poor 5%
course
ETV6 Poor 2-3%
RUNX1 Poor 9-10%
15% (found in 40%
ASXL1 Poor
of CMML)
NRAS Poor 4-8%
DNMT3A Poor 10-15% poor outcome post alloSCT
occurence with TET2 mutationis associated
SRSF2 Poor 12%
with monocytosis
PTPN11 Poor 1%
U2AF1 Poor 7-10%
improved response to azacitidine, poor
TET2 Unclear 20-30% outcome post alloSCT occurence with SRSF2
mutation is associated with monocytosis
Adapted according [37].
A passenger mutation has not been selected, has not conferred clonal growth advantage
and has therefore not contributed to cancer development. Passenger mutations are found
within cancer genomes because somatic mutations without functional consequences often
occur during cell division. Thus, a cell that acquires a driver mutation will already have
biologically inert somatic mutations within its genome. These will be carried along in the
clonal expansion that follows and therefore will be present in all cells of the final cancer.
Frequency and impact of chosen genetic mutations identified in MDS are shown in
Table 1.
MUTATIONS IN EPIGENETIC REGULATORS IN MDS

Mutations in epigenetic regulators can be divided to mutations in regulators of DNA
methylation and to mutations affecting histone function by modification of the DNA-
associated histone proteins [7-12]. Epigenetic regulators influence gene expression (gene
transcription) and might be in line with hypermethylation of CpG islands in the promoters of
key genes involved in cell cycle regulation, apoptosis, tumor suppressor control and in
response to chemotherapy and the consequent silencing of their expression in MDS.
Deregulated DNA methylation patterns in MDS correlate with overall survival of MDS
patients, and with clinical response to hypomethylating agents as I described in special
chapter Epigenetic changes in the pathogenesis and therapy of myelodysplastic syndromes.
However, there is now genetic evidence that DNA methylation is important in MDS,
because several genes that regulate cytosine methylation are somatically mutated in MDS
genomes (DNMT3A, TET2, and IDH1/IDH2).
DNA is methylated almost exclusively at cytosines that are part of a cytosine-phospho -
guanine (CpG) dinucleotide, resulting in 5-methylcytosine (5mC). CpG-dense regios or CpG
islands are located within regulatory elements of approximately half of all human genes and
are generally unmethylated in normal cells with some exceptions of non-transcribed genes on
the inactive X chromosome and imprinted autosomal genes. CpG methylation is catalysed by
a family of DNA methyltransferases (DNMTs) including DNMT1, DNMT3A and DNMT3B.
DNMT1 is required for maintenance methylation during DNA replication. DNMT3A and
DNMT3B function in de novo methylation [38-42].
In myeloid malignancies, methylation changes include simultaneous global
demethylation, increased expression of DNMTs, and de novo methylation of previously
unmethylated CpG islands [43-45]. Profound hypomethylation is a form of genomic
instability and can predispose to aacquisition of mutations, deletions, amplifications,
inversions, and translocations [46]. Hypomethylation can be associated with reactivation of
normally silenced genes or micro RNAs that function in post-transcriptional silencing of
corresponding proteins expression [44, 47]. On the other hand, increased activity of DNMTs
can silence tumor suppressors and other important genes described above [43, 44].
DNA demethylation can occur when DNMT1 is inhibited or absent, by enzymatic
removal of the methyl group, and by oxidative demethylation [45].
Epigenetic silencing is also associated with histone H3 lysine 27 (H3K27) trimethylation.
This modification is associated with closed chromatin and results in transcriptional
92 Ota Fuchs
suppression, suggesting that mutations that decrease H3K27 trimethylation activate

transcription.
MUTATIONS OF THE DNMT3A GENE

Mutations of the DNMT3A gene, located on chromosome 2p23.3 were initially detected
in de novo acute myeloid leukemia (AML) by next generation sequencing technology [48]. In
the same year, Yamashita et al. reported these mutations in AML patients by array-based
genomic resequencing [49]. Following these first reports, mutations of the DNMT3A gene
were detected in about 6% of MDS patients and exhibited equal distribution amongst the
different MDS subtypes [50-53]. Only too small groups of patients were examined and
therefore, there is no multivariate analysis for clinical outcome. Nevertheless, the presence of
DNMT3A mutations correlated with poor clinical outcome in univariate analysis. Patients
harboring DNMT3A mutations tend to progress toward AML and inferior overall survival.
Conditional Mx-Cre-driven excision of the DNMT3A gene to examine the effects of
DNMT3A loss on hematopoietic stem cell (HSC) function has been recently reported [54].
DNA methylation analysis showed specific loci to be hypomethylated and others
paradoxically hypermethylated. Gene expression analysis showed that DNMT3A gene loss
leads to ujp-regulated expression of HSC fingerprint genes and lower expression of genes
with a known role in HSC differentiation. However, the lack of an overt leukemic phenotype
in these studies suggests that DNMT3A gene loss by itself is insufficient to induce disease
[55].
The mechanism by which DNMT3A mutations result in hypermethylation or
hypomethylation needs to be further investigated. DNMT3A mutations are predominantly
heterozygous and constitute mainly monoallelic events. Mutations may result in dominant
negative or gain of function rather than loss of function. The missense mutations (86%) and
truncating mutations (14%) of the DNMT3A gene were described in MDS patients [50-52].
The missense mutations target mainly the methyltransferase domain, which binds to the
methyl donor and catalyzes the methylation of DNA [56, 57]. The most frequently targeted
amino acid is arginine 882 (R882 or Arg882) in 50% of DNMT3A mutations in MDS. This
mutation corresponds to DNMT3B-R823. The protein DNMT3B also catalyzes de novo DNA
methylation and R823 is mutated in ICF (immunodeficiency, centromeric instability, facial
anomalies) syndrome [58]. ICF syndrome without detectable DNMT3B mutation was also
described [59].
MUTATIONS OF THE TET2 GENE

Using an integrated genomic approach including comparative genomic hybridization and
SNP arrays analysis the TET (Ten-Eleven-Translocation) oncogene family member 2 (TET2)
gene was identified as candidate tumor suppressor gene in a variety of myeloid disorders. The
TET family of proteins is able to demethylate DNA by converting 5-methylcytosine (5mC)
into 5- hydroxymethylcytosine (5hmC) [60-63]. The 5hmC can be further oxidized into
formyl- and carboxyl-cytosine (5fc and 5cC), which can subsequently be recognized by
thymine-DNA glycosylase base-excision repair enzymes and replaced by unmethylated

cytosines [64-68]. The TET proteins bind to the transcriptional start site of actively
transcribed genes and keep the chromatin of active genes in an open configuration by the
demethylation of CpG nucleotides. The conversion of 5mC to 5hmC by TET proteins
depends on the cofactors Fe2+ and -ketoglutarate. This dependence shows an link between
the TET proteins and IDH proteins, as IDH1 and IDH2 convert isocitrate to -ketoglutarate.
Therefore, IDH proteins are necessary for supplying the cofactor -ketoglutarate for the TET
proteins [69, 70].
TET2 gene is the so far most frequently mutated gene in MDS and MDS/MPN, and may
help to understand the stem biology of at least some of its subtypes. It is likely that the
mutated form in some way contributes to growth advantage of the malignant clone. Mutations
in the TET2 gene have been identified in 12%-26% of MDS and 46-50% of chronic
myelomonocytic leukemia (CMML) patients [71-76]. TET2 gene mutations appear to be more
prevalent in lower-risk patients, but their presence does not appear to be an independent
prognostic factor. In approximately half of the cases, TET2 gene mutations were bi-allelic,
either by uniparental disomy, deletion or independent mutations in both copies of the gene
[21]. The occurrence of nonsense mutations in the N-terminal part of the coding region in
some cases and deletions that span the entire gene in other patients, strongly suggested that
the mutations result in loss of function. The confirmation was obtained by analyzing mice
with targeted disruption of the TET2 catalytic domain where TET2 was shown as a critical
regulator of self-renewal and differentiation of HSCs [77-80]. The loss of function of the
TET2 protein results in defective 5hmC formation and inhibition of demethylation of DNA
[81]. Therefore, hypomethylating agents might be particularly efficient in patients with TET2
gene mutations. The results of two recent studies are in agreement with this hypothesis [82,
83]. Large prospective clinical trials must be done to confirm this presumption.
MUTATIONS OF THE IDH1 AND IDH2 GENES

Mutations in IDH1 and IDH2 were first described in gliomas [84, 85]. Mutations in the
IDH1gene were then independently found in an AML genome using whole genome massively
parallel sequencing [86]. IDH1gene mutations were present in 15 of 187 AML genomes (8%)
tested and were strongly associated with normal cytogenetic status (13 of 80 cytogenetically
normal samples/16%/). The IDH1 protein is operating in the cytosol and peroxisomes
whereas IDH2 is in mitochondria [87, 88]. IDH1 gene is located at chromosome 2q33.3 and
IDH2 gene at chromosome 15q26.1. As I described in the paragraph about mutations in TET2
gene, IDH1 and IDH2 convert isocitrate to -ketoglutarate, while reducing nicotinamide
adenine dinucleotide phosphate (NADP+) to NADPH. These reactions have important role in
the metabolic pathways and in the protection of cells from oxidative stress-induced
tumorigenesis.
In MDS, the IDH genes are mutated in approximately 3% -10% patients (more frequent
in high risk MDS with aberrant but non-complex cytogenetics where prevalence was 25%) [8,
88-94]. Missense mutations target one single codon in the IDH1 gene (c.394-396 CGT,
encoding amino acid arginin 132) and one or two codons in the IDH2 gene (c.418-420 CGG,
encoding amino acid arginin 140, or c.514-516 AGG, encoding amino acid arginin 172).
94 Ota Fuchs
IDH2 gene mutations in the position c.418-420 CGG, encoding amino acid arginin 140
represent the most frequently occurring mutations in MDS (61%). All these mutations (in
positions IDH1-R132, IDH2-R140 and IDH2-172) reside in the acive site of the proteins [80].
I spite of this cells carrying IDH mutations did not show impaired levels of -ketoglutarate
and NADPH [95, 96]. Mutations are predominantly in one allele leaving the second allele
intact and from this allele is the expression of products normal. The mutant IDH proteins
showed a gain of function as they could convert the -ketoglutarate that is generated by wild-
type IDH proteins into 2-hydroxyglutarate [97]. This oncometabolite 2-hydroxyglutarate
competitively inhibits enzymes that are dependent on -ketoglutarate [97, 98], among which
are the Jumonji family of histone demethylases [99] and the TET proteins. As the function of
the TET2 protein is inbited either by TET2 mutation or by the oncometabolite 2-
hydroxyglutarate formed by mutant IDH proteins, IDH mutations and TET mutations are
mutually exclusive in most MDS patients. However, IDH mutations co-occurred with
approximately 7% of EZH2 mutations and 11% of ASXL1 mutations [8]. More IDH mutations
were found in normal karyotype, isolated del(5q), isolated del(20q), and isolated trisomy 8 [8,
92, 100]. The effect of IDH mutations on clinical outcome of MDS patients is not clear, as
different studies in various groups of patients showed different results [8, 91, 94]. Because of
the low frequency of IDH mutations occurring in MDS the prognostic impact of these
mutations should be confirmed in larger groups of uniformly treated MDS patients and put in
context with other markers. Mutation analysis of IDH1 and IDH2 mutations in MDS patients
may become useful for risk and treatment stratification in the future.
MUTATIONS OF THE EZH2 GENE

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the Polycomb-Repressive
Complex 2 (PRC2), which catalyses the trimethylation of histone H3 on lysine 27 (H3K27)
and involves in genes repression. EZH2 is a member of the Su(var)3,9, enhancer of zest,
Trithorax (SET) domain containing family of histone methyltransferases and is a potential
oncogene and a target for tumor therapy [101-106]. An activating point mutation in the SET
domain of EZH2 (Tyr641) was identified in diverse lymphomas [103]. However, no
activating EZH2 mutations were found in MDS. Instead, EZH2 mutations with a loss of
function of the EZH2 protein were detected in 6% of MDS patients [8, 107-109].
The EZH2 gene is located on chromosome 7q36.1, a region that is frequently affected in
MDS by chromosomal aberrations [6, 107, 109]. Microdeletions of chromosome 7q36.1 co-
occurred with truncating EZH2 mutations that deleted or disrupted the important SET domain
on the remaining allele. Therefore, no functional EZH2 protein is expressed in these cases of
MDS patients [107, 109]. In addition to these EZH2 mutations, biallelic homo- and
heterozygous EZH2 mutations were observed [107, 108]. Truncating and missense EZH2
mutations scattered throughout the all coding sequence were detected and most of these
mutations targeted the SET domain. Analysis of some of these mutants showed loss of
H3K27me3 in affected cells and confirmed the loss of function of mutant EZH2 proteins
[108, 109]. EZH2 mutations occurred associated with normal and non-complex karyotypes
(mainly del(5q), del(20q) and trisomy 8, similarly as IDH mutations) [8, 107, 108] and co-
occurred with 8% of TET2 mutations, 13% of IDH mutations and 22% of ASXL1mutations
[8], indicating that these proteins function in different epigenetic processes and that their
mutations are involved in the pathogenesis of MDS. The co-occurrence of EZH2 and
DNMT3A mutations has not been investigated in large MDS patient cohorts. Most of EZH2
mutations that were found occurred in the absence of a chromosome 7/7q deletion [8, 107-
109].
The presence of EZH2 mutations correlated with adverse prognosis, comparable to the
well-known poor prognosis of patients with a chromosome 7/7q deletion [1, 6, 8, 107, 108].
The correlation of EZH2 mutations with poor prognosis was shown to be independent from
other known risk factors using multivariate analysis [8]. This observation is very important
because most EZH2 mutations were found in lower-risk patients [8, 107]. Since MDS patients
with chromosome 7/7q deletions particularly benefit from treatment with hypomethylating
agents [110], it might be hypothesized that patients harboring EZH2 mutations might benefit
from this treatment as well.
MUTATIONS OF THE UTX GENE

UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome) gene on the X
chromosome codes for a di- and trimethyl H3K27 demethylase, a member of the Jumonji C
family of proteins [111, 112]. UTX occupies the promoter of HOX gene clusters and regulates
their transcriptional output by modulating the recruitment of polycomb repressive complex 1
and the monoubiquitination of histone H2A. Moreover, UTX associates with mixed-lineage
leukemia MLL 2/3 complexes.
The large-scale systematic screening revealed somatic mutations of UTX in several
cancer cell lines including hematological malignancies [113]. This discovery was extended to
patients samples with myeloid malignancies where somatic mutations were also identified
[114]. UTX mutations have been found in 9% (2/7) patients with AML derived from MDS,
none were detected in low-risk MDS (18 patients) [115]. The mutations were present at the
both N- and C- terminus of UTX protein, but mostly in the region adjacent to catalytic
domain required for UTX activity. Functional significance of UTX mutations remains to be
clarified.
MUTATIONS OF THE ASXL1 GENE

ASXL1 gene is similar to the Drosophila additional sex combs gene, which encodes a
chromatinbinding protein required for normal determination of segment identity in the
developing embryo. The protein is a member of the polycomb group of proteins, which are
necessary for the maintenance of stable repression of homeotic and other loci. The protein is
thought to disrupt chromatin in localized areas, enhancing transcription of certain genes while
repressing the transcription of other genes.
ASXL1 gene is located on chromosome 20q11.21 and codes for a nuclear protein of 1084
residues, characterized by an N-terminal helix-turn-helix domain (HARE-HTH) [116] and an
unusual C-terminal plant homeodomain (PHD), which may bind methylated lysines. The
central part of ASXL1 contains an ASH globular domain that may interact with a polycomb-
96 Ota Fuchs
associated deubiquitinase (DUB) [116, 117]. ASXL1 regulates epigenetic marks and
transcription through interaction with polycomb complex proteins and various transcription
activators and repressors [117-119]. However, the role of ASXL1 in leukemogenesis does not
seem to be mediated by the DUB complex [120]. Recent data have shown that ASXL1
interacts with components of the polycomb complex PRC2, namely EZH2 and SUZ 12, two
proteins involved in the deposition of H3K27me3 histone repressive marks. Inhibition of
ASXL1 function leads to loss of H3K27me3 histone marks. ASXL1 also associates with
HP1/CBX5, a component of the heterochromatin repressive complex [121, 122]. HP1
binds to histone H3. JAK2 phosphorylates histone H3 and excludes HP1 from chromatin
[123]. Thus a potential functional link may exist between ASXL1 and JAK2 mutations but
this remains to be demonstrated.
ASXL1 mutations are frameshift and nonsense mutations that are supposed to result in C-
terminal truncation of the protein upstream of the PHD finger. The functional significance of
some reported missense mutations is not clear. Future functional analysis of the truncated
proteins could be extended to the examination of the confirmed missense mutation
Arg402Gln [8]. The most frequent mutation, which accounts for more than 50% of all
ASXL1 mutations, is a duplication of a guanine nucleotide (c.1934dupG). This mutation
leads to frameshift (pGly646TrpfsX12) and has not been found in germ-line DNAs, control
DNAs or other studied types of cancers. This mutation is now generally considered to be a
bona fide mutation [8, 124-126]. ASXL1 mutations are usually heterozygous, suggesting the
haplo-imsufficiency is the key pathological factor. The truncated ASXL1 protein could also
have a dominant negative role in titrating out an interacting protein. Actually, recent data
have demonstrated a loss ASXL1 protein in leukemia samples with ASXL1 mutation,
indicating that these mutations are loss-of-function disease alleles [120, 124-126].
ASXL1 is the second most frequently mutated gene in MDS (in 16% of patients) after
TET2 [8]. In MDS, ASXL1 mutations are more frequent in refractory anemia with excess of
blasts (RAEB) than in other forms such as refractory anemia with ring sideroblasts (RARS)
[8, 89, 119, 120, 124-126]. Therefore, the presence of ASXL1 mutations correlated with poor
overall survival in various MDS patient cohorts. Multivariate analyses indicated that ASXL1
mutations represent an independent poor risk factor for overall survival [8, 126]. Although
chromosome 20q may be affected by deletions in MDS, ASXL1 does not reside in the
commonly deleted region [6, 127]. The adverse effects of ASXL1 mutations on prognosis are
opposite to the correlation of isolated del(20q) with good prognosis [1, 6], suggesting that
del(20q) and ASXL1 mutations represent different pathogenic events in MDS.
MUTATIONS OF THE C-CBL GENE

The casitas B-lineage lymphoma proto-oncogene (c-CBL) is an E3 ubiquitin ligase,
encoded by c-CBL on chromosome 11q23 [32-36, 128-130]. This protein was discovered as
the cellular form of v-Cbl, a retroviral transforming protein of Cas NS-1 retrovirus which
causes pre- and pro-B lymphomas in mice [131, 132]. c-CBL is involved in degradation of
wide variety of activated receptor tyrosine kinases and other tyrosine kinases, including src
kinases. c-CBL seems to have tumor suppressor functions, loss of which promotes
tumorigenesis. On the other hand, once mutated, it is converted to an oncogene protein and
commits to myeloid leukemogenesis through a kind of gain of function causing aberrant

signal transduction [32].
c-CBL mutations were identified in roughly 15% of CMML and in patients with juvenile
myelomonocytic leukemia but were found in fewer than 5% of MDS. c-CBL mutations are
often biallelic, cluster in exons 8 and 9 and usually leave the rest of the gene intact. Mutated
protein inhibits the ubiquitin ligase activity of the wild-type protein [32]. c-CBL mutations are
mutually exclusive of several other commonly mutated genes and are likely associated with a
negative prognosis, although this has yet to be confirmed.
RUNX1 MUTATIONS
RUNX1 (also known as CBFA2 or AML1) is together with TET2 and ASXL1the most
commonly mutated genes in MDS. RUNX1 point mutations are present in 7%-15% of de
novo MDS patients and at a higher frequency in therapy-related disease [133-139].
The RUNX1 protein is a transcription factor which contains a proximal Runt domain
functioning in DNA binding, and distal transactivation domain important for protein-protein
interactions and recruitment of cofactors. Missense mutations of RUNX1are clustered in the
Runt domain and impair DNA binding and function as dominant negatives. Stop codon
mutations and frame shift mutations are found throughout the length of the gene and almost
always disrupt the transactivation domain [137]. In patients with MDS, RUNX1 mutations are
often accompanied by activation of the ras pathway or mutations in these genes [137, 140,
141].
Familial cases of MDS are rare, but are immensely valuable for the investigation of the
molecular pathogenesis of myelodysplasia in general The best-characterized familial MDS is
that of of familial platelet disorder with propensity to myeloid malignancy, caused by
heterozygous germline RUNX1 mutations [142].
RAS MUTATIONS IN MDS

The rat sarcoma (ras) oncogene family encodes 21kD guanosine triphosphate (GTP)-
binding proteins that reside on the inner surface of the cell membrane and that play a role in
signal transduction from membrane receptors [143, 144]. The ras genes acquire transforming
capabilities in association with point mutation of a single nucleotide within the region
encoding the GTP-binding portion of the protein. Roughly 10%-15% of MDS patients have
ras mutation, most commonly codon 12 Nras mutations [145-149]. Ras mutations in MDS
are frequently associated with mutations in other genes, such as RUNX1 [137, 140]. These
additional mutations act together with mutated ras to cause myelodysplasia. The presence of
ras mutations in MDS confers a poor prognosis, with an increased risk of leukemic
transformation [149].
98 Ota Fuchs
TP53 MUTATIONS
The process of carcinogenesis involves the gain of oncogene activity and the loss of
tumor suppressor gene function. A key tumor suppressor gene often lost is TP53, which codes
for p53, is able to induce temporary growth arrest and DNA repair, terminal differentiation, or
apoptosis in response to potentially oncogenic cellular stress such as DNA damage [150,
151]. TP53 is located on the short arm of chromosome 17 at band 13 (17p13) [152]. The
TP53 tumor suppressor gene is mutated in over 50% of human cancers [153]. Alterations of
TP53 are also observed in several classes of hematologic malignancies including MDS [154-
165].
TP53 mutations in MDS patients are associated with progression of disease in some
cases, while being compatible with stable disease or clonal evolution in others. MDS patients
with a TP53 mutation have a significantly poor prognosis regardless of their cytogenetic
findings in most studies. Furthermore, multivariate analysis demonstrated that P53 mutations
were the most significant indicator. The prognoses of the mutated patients were poorer than
those without the mutation in the intermediate subgroups of IPSS [162]. There were no
significant effect of P53 mutations within the high-risk group but the number of the patients
in this group was too small to reach a conclusion [162].
Several reports identified a higher prevalence of TP53 mutations (27%-38%) in therapy-
related MDS/AML than in other hematological malignancies [164].
Patient (18-month-old boy) with der(5;17)(p10;q10) exhibited deletion of the TP53 in
one allele and TP53 mutation (410T>A) in other allele [165]. Since the mutation was not
detected in peripheral blood leukocytes 9 and 13 months before the diagnosis of MDS,
biallelic somatic inactivation of the TP53 gene might play an important role in the occurrence
of MDS. Poor outcome might be associated with resistance to chemotherapy of a minor clone
with TP53 gene alteration. Peripheral blood cell sof his father and mother showed wild-type
TP53 gene [165].
By using sensitive deep-sequencing technology, Martin Jdersten et al. [166, 167]
demonstrated that TP53 mutated populations may occur at an early disease stage in almost a
fifth of low-risk MDS patients with del(5q). Mutations were present years before disease
progression and were associated with an increased risk of leukemic evolution. TP53
mutations could not be predicted by common clinical features but were associated with p53
overexpression. The TP53 mutated clone remained stable during lenalidomide treatment and
expanded at progression. Patients with TP53 mutations had significantly worse outcome. This
information shows clonal heterogeneity in low-risk MDS patients with del(5q), which may be
of importance when assessing the prognosis and selecting the therapy in these MDS patients
[166, 167]. These results were confirmed in next studies [168, 169].
MUTATIONS IN RNA SPLICING MACHINERY

The vast majority of human genes undergo RNA splicing after transcription, which
means that mutations in the RNA splicing machinery could potentially alter the maturation of
messenger RNA (mRNA) for most genes and the subsequent production of protein. Up to
now, genes for four splicing factors were analyzed using next-generation sequencing
approaches for the presence of mutations (SF3B1, SRSF2, U2AF1, ZRSR2) in MDS patients
[170-177]. The results demonstrate that mutations in genes for these splicing factors are
among the most frequent molecular aberrations in MDS, define distinct clinical phenotypes
and show preferential associations with mutations targeting transcriptional regulation. These
mutations were mutually exclusive and less likely occur in patients with complex
cytogenetics or TP53 mutations.
Splicing factor 3b, subunit 1 (SF3B1) is encoded by SF3B1, which is located at
chromosomal band 2q33.1. SF3B1 is a core component of the U2 small nuclear
ribonucleoprotein complex (U2 snRNP). U2 sn RNP is a part of the catalytic site of the
spliceosome that process pre-messenger RNA (pre-mRNA) to mature mRNA and thus
regulates the diversity of splice variants [178]. In addition, RNA splicing is linked to the
epigenetic regulation of gene expression. SF3B1 has also been reported to interact with the
polycomb repressive complex, an important regulator of hematopoiesis [179].
U2 small nuclear RNA auxiliary factor 1 (U2AF1) is encoded by U2AF1, which is
located at chromosomal band 21q22.3. U2AF1 plays a critical role in both constitutive and
enhancer-dependent splicing by mediating protein-protein interactions and protein-RNA
interactions required for accurate 3 splice site selection. U2AF1 recruits U2 snRNP to the
branch point.
Serine/arginine-rich splicing factor 2 (SRSF2) has its gene on chromosome 17q25.1. The
protein encoded by this gene is a member of the serine/arginine (S/R)-rich family of pre-
mRNA-splicing factors, which constitute part of the spliceosome. SRSF2 contains an RNA
recognition motif and S/R-rich domain, which facilitates interaction between different
splicing factors.
Zinc finger (CCCH type), RNA-binding motif and S/R rich 2 (ZRSR2) is an essential
splicing factor, which associates with the U2 auxiliary factor heterodimer and is required for
the recognition of a functional 3 splice site in pre-mRNA splicing, and may play a role in
network interactions during spliceosome assembly. The location of the gene for this splicing
factor is on chromosome Xp22.1.
SF3B1 mutations were prevalent in low risk MDS with ring sideroblasts, while U2AF1
(also known as U2AF35) and SRSF2 mutations were frequent in CMML and advanced forms
of MDS [172]. SF3B1 mutations were associated with a favorable prognosis, while U2AF1
and and SRSF2 mutations are predictive for shorter survival [172].
In another study, SF3B1 mutated MDS patients had lower hemoglobin levels, increased
white blood cells and platelet counts and had more likely DNMT3A mutations [174]. U2AF1
mutated patients had an increased prevalence of chromosome 20 deletions and ASXL1
mutations. SRSF2 mutated patients clustered in RAEB1 and RAEB-2 subtypes and exhibited
pronounced thrombocytopenias. ZRSR2 mutated patients clustered in IPPS int-1 and int-2
risk-groups, had higher percentages of bone marrow blasts and more often displayed isolated
neutropenias. SRSF2 and ZRSR2 mutations were more common in TET2 mutated MDS
patients. Multivariate analysis revealed an inferior overall survival and higher AML
transformation rate for the MDS patients with ZRSR2 mutations and with wild type TET2
[173].
Thol et al., [177] analyzed the cohort of 193 MDS patients and detected 24 (12.4%)
SRSF2, 14 (7.3%) U2AF1, 6 (3.1%) ZRSR2, and 28 (14.5%) SF3B1 mutated patients,
respectively. In univariate analysis, mutated SRSF2 predicted shorter overall survival and
more frequent AML progression compared to wild type SRSF2, whereas mutated U2AF1,
100 Ota Fuchs
ZRSR2, and SF3B1 had no impact on patient outcome. In multivariate analysis, mutated
SRSF2 remained an independent poor risk marker for overall survival and AML progression
[177]. These results show a negative prognostic impact of SRSF2 mutations in MDS.
MUTATIONS IN OTHER GENES IN MDS PATIENTS

Members of the tyrosine kinase signaling pathways are mutated in myeloid malignancies
but infrequently in MDS. The Fms-like tyrosine kinase (FLT3) is rarely mutated in MDS.
Most of the FLT3 mutations found in MDS have been internal tandem duplications (ITD) in
higher-risk MDS. These mutations and N-ras mutations drive progression of MDS and
transformation MDS to AML [180-183]. Mutations of the KIT gene in the extracellular
membrane, juxtamembrane, and tyrosine kinase domains were investigated in 75 patients
with MDS or MDS-derived leukemia [184]. KIT gene mutations were foumd in any of 9
patients with refractory anemia (RA) or 10 patients with refractory anemia with excess blasts
(RARS). Mutations were detected in 2 of 15 (13.3%) patients with RAEB-T or in 1 of 15
(6.6%) patients with CMML and 5 of 26 (19.2%) patients with MDS-AML [184].
Mutations in JAK2 (Janus kinase 2), type II cytokine receptor family member, were also
studied [185]. Mutation V617F, a change of valine to phenylalanine at the position 617, was
found not only in RARS-T but also in RARS MDS patients. Five patients from 101 MDS
patients in original cohort had V617F JAK2 mutation but 2 of those 5 were RARS without
thrombocytosis or any other myeloproliferative features [185].
New mutations in ETV6 (TEL) and GNAS were detected in MDS patients by new
genomic approaches, including next-generation sequencing and mass spectrometry-based
genotyping [8]. ETV6 (ETS variant gene 6), also known as TEL (translocation ets leukemia)
is located on chromosome 12p13 and encodes an ETS family transcription factor. ETS is
derived from human DNA segments homologous to the transforming gene of avian
erythroblastosis virus, E26 [186]. Both translocations and mutations of ETV6 have been
identified in rare cases of MDS and in AML [8, 187]. GNAS (guanine nucleotide binding
protein /G protein/, stimulating activity polypeptide) is the gene on chromosome 20q13.3,
encoding the GS subunit of the heterotrimeric GS protein complex, membrane bound
GTPases containing , , and subunits. Activating GNAS mutation of amino acid R201 was
detected in 3 of 439 MDS patients (0.7%) [8].
Massively parallel sequencing technology identified recurrent somatic mutations in
SETBP1 gene in atypical chronic myeloid leukemia and after this also in MDS [188-190].
This gene encodes a protein which contains a several motifs including a ski homology region
and a SET-binding region in addition to three nuclear localization signals. The encoded
protein has been shown to bind the SET nuclear oncogene which is involved in DNA
replication. SETBP1-mutations were more frequent in ASXL1-mutated CMML. SETBP1-
mutated patients had a significantly inferior overall survival.
Casein kinase 1 was found as target of E3 ubiquitin ligase containing cereblon in its
complex as substrate receptor after del(5q) MDS patients treatment with lenalidomide [191].
CSNK1A1 mutations have been found in 5-7.2% of patients, are associated with older
age, may co-occur with ASXL1 but not TP53 mutations, and are responsive to lenalidomide
[191- 193]. CSNK1A1 mutations have no independent prognostic impact on overall survival
[191-193].
In rare instances, mutations of the RUNX1, CEBPA (CCAAT/enhancer binding protein
alpha), GATA2, SRP72, and TERC, which encodes the telomerase RNA component, or
TERT, coding for the telomerase reverse transcriptase, genes in MDS may be associated with
inherited syndromes that lead to myeloid diseases [142,194- 196].

The clonal architecture of MDS has been studied following the identification of somatic
TET2 mutations [71]. CD34+ cells from MDS patients were fractionated into immature
CD34+ CD38- and mature CD34+ CD38+ progenitors. Although TET2 mutations were
detected in only a small fraction of CD34+ CD38- cells, they were present in a high proportion
of more mature progenitors. Thus, the initial somatic TET2 mutations occured in a CD34+
CD38- cell and was then transmitted to more mature CD34+ CD38+ progenitors. A similar
clonal architecture was observed also in patients with CMML [198]. Although MDS are
defined by cytopenias, dysplastic morphology of blood and marrow cells, and clonal
hematopoiesis, most individuals who acquire clonal hematopoiesis of indeterminate potential
during aging will never develop MDS [197].
Prognostically significant somatic mutations occurred in patients in all risk groups. Most
patients with EZH2 or ASXL1 mutations had low or intermediate-1 risk according to the IPPS.
In contrast, TP53 mutations were observed mainly in patients with intermediate-2 or high risk
according to the IPPS and were strongly associated with thrombocytopenia, an elevated blast
proportion, and a complex karyotype. TP53 mutations were strongly associated with shorter
overall survival. Patients with TP53 mutations and a complex karyotype have very small
number of mutations in other genes. This group is a distinct molecular subclass of MDS with
a unique pathogenic mechanism. The determination of TP53 mutations is necessary in del(5q)
patients for the choose of therapy, because patients with TP53 mutations are often resistant to
lenalidomide therapy. TET2 mutations were the most prevalent genetic abnormality even if
mutations affecting pre-mRNA splicing are also abundantly represented. TET2 mutations
were not strongly associated with clinical features such as cytopenias or blast proportion.
These findings are consistent with the observation that TET2 mutations are present in various
myeloid cancers, including myeloproliferative diseases without defects in hematopoietic
differentiation. Each of the prognosically significant mutations alters the biologic
characteristics and phenotype of MDS in unique ways. The presence or absence of a mutation
in selected genes might be one additional variable added to the IPSS in order to improve this
prognostic system.
Additional studies might resolve some discrepancy in the effect of mutations in splicing
factors. The prognostic signifikance of the splicing factor gene mutations should be
investigated in the context of randomized, prospective clinical trials in MDS in which the
impact of other gene mutations in MDS are also taken into account. Involving these mutations
of the splicing factors to IPSS in future will also improve the prognosis.
Some acquired mutations in MDS are associated with specific clinicopathological
presentations, such as strong association of SF3B1 with ring sideroblasts, ATRX with acquired
102 Ota Fuchs
-thalassemia, RUNX1 with thrombocytopenia, and TP53 with a complex karyotype, therapy-
related disease, and dysgranulopoiesis [197].
Clinicians will be able in near future to detect a broad range of point mutations in
pheripheral blood using new sensitive genotyping methods. These methods will facilitate
diagnosis and prognosis of MDS patients. For example, MDS with a hypocellular bone
marrow (hypo-MDS) is hardly distinguishable from other bone marrow failure syndromes.
Further, approximately 50% of MDS patients with hypo-MDS have normal karyotype by
metaphase cytogenetics. SNP-A karyotyping can improve the detection of chromosomal
lesions that can distinguish hypo-MDS patients from those with aplastic anemia (AA) [199].
NGS technologies showed mutations in SF3B1, ZRSR2, U2AF1, SRSF2, TET2, ASXL1
and BCOR predominantly as driver clones in patients with normal or increased cellularity
(hyper-MDS) compared to hypo-MDS [200]. None of these mutations were exclusively
observed as a driver clone in hypo-MDS.
The levels of mutations were higher in hyper-MDS. Somatic mutations in ASXL1, TET2
and DNMT3A are present also in healthy individuals and in patients with AA and their
presence was associated with an increased risk of these individuals to develop hematologic
malignancies or with increased risk of patients with AA to transformate to MDS and AML
[200].
New technologies will also help in monitoring of response to treatment [7, 8, 82, 83,
201]. The presence of specific mutations (TET2 and perhaps DNMT3A) predicts a higher
likelyhood of response to hypomethylating agent therapy, whereas other mutations (TP53)
predict higher relapse risk and inferior survival after allogeneic stem cell transplantation [202-
204].
ACKNOWLEDGMENTS
Czech Republic and by project for conceptual development of research organisation No
00023736 from the Ministry of Health of the Czech Republic.
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Chapter 5
RISK FACTORS AND PROGNOSTIC SCORING

SYSTEMS IN ARGENTINEAN PATIENTS WITH MDS:
A MULTICENTRIC STUDY
Carolina B. Belli*1,, PhD, Jacqueline Gonzalez2,, MD,

Virna Barcal3,, Bioch, Marcela de Dios Soler4,, MD,
Marcelo Iastrebner5,, MD and Mara Gabriela Flores2,, MD
1
Laboratorio de Gentica Hematolgica, Instituto de Medicina Experimental
(IMEX-CONICET)/ ANM,
Academia Nacional de Medicina, Buenos Aires, Argentina
2
Servicio de Hematologa, Hospital General de Agudos
C Durand Buenos Aires, Argentina
3
CITOMLAB, Buenos Aires, Argentina
4
Servicio de Patologa, Hospital Municipal de Oncologa
M Curie, Buenos Aires, Argentina
5
Servicio de Hematologa, Sanatorio Sagrado Corazn,
OSECAC, Buenos Aires, Argentina
ABSTRACT
One of the most challenging problems in hematology is the heterogeneous group of
clonal disorders that were formally defined as Myelodysplastic Syndromes (MDS) by the
FrenchAmericanBritish Cooperative Group (FAB) in 1982, and subsequently by the
World Health Organization (WHO) in 2001 and in 2008. The bone marrow (BM) is
usually normo/hiper-cellular, displays various morphologic abnormalities and the stem
*
Corresponding Author: Dr. Carolina B. Belli, PhD; Laboratorio de Gentica Hematolgica; Instituto de Medicina
Experimental (IMEX CONICET)/Academia Nacional de Medicina (ANM); Pacheco de Melo 3081, CP1425,
Buenos Aires, Argentina; Phone Number: 54-11-4805-8803; Fax Number: 54-11-4803-9475;
E-mail: cbelli@hematologia.anm.edu.ar.
On behalf of the Argentine MDS Study Group, Argentine Society of Hematology, Buenos Aires, Argentina.
116 Carolina B. Belli, Jacqueline Gonzalez, Virna Barcal et al.
cells show defective capacity for self-renewal and differentiation leading into an
ineffective hematopoiesis and cytopenias.
The clinical course is highly variable, ranging from stable disease over 10 years to
death within a few months due to complications associated with their cytopenias or
leukemic transformation. This great variability in the outcome of MDS complicates
decision-making regarding therapies and prognostic characterization of individual
patients is vital prior initiating treatment. In order to overcome the limitations of the FAB
classification, various scoring systems have been designed during this last 30 years. The
International Prognostic Scoring System (IPSS) has been widely adopted becoming in the
gold standard for risk assessment. In 2012, it has been revised (IPSS-R) defining five
groups of risk based on five cytogenetic groups, new clinical cut-points for relevant
cytopenias and for the percentage of BM blasts. However, the IPSS and IPSS-R exclude
secondary MDS and chronic myelomonocytic leukemia with leukocytosis, which
represent a large proportion of patients. Other disease related factors have been proposed
such us the presence of myelofibrosis, LDH and ferritin level or the transfusion
dependency instead of the hemoglobin level. Some others, related to patients
characteristic, which include age, performance status and the co-existence of
comorbidities, have also been included in different scoring systems.
Argentinean MDS Registry was created in 2008 and is sponsored by the Argentinean
Society of Hematology. The main objective is to collect MDS related data in order to
describe epidemiological characteristics, prognostic factors and scoring risk
stratifications in our population. After 7 years, 14 institutions from Argentine have been
reporting data from 532 patients (89% with de novo MDS) diagnosed mostly since 2007.
The median age was 72 (17-95) years with a gender ratio (M/F) of 1.3. During the
follow-up (median: 18 months, range: 1-129 m), 104 (20%) patients evolved to AML and
211 (40%) died. Gender, percentage of BM blast, hemoglobin level, platelet and
neutrophil counts, cytogenetic groups of risk, presence of myelofibrosis, and red blood
cell transfusion requirement were significant predictive variables for prognosis The IPSS,
IPSS-R, WPSS revised according to hemoglobin levels, MDA score, and Charlsons
Comorbidities Index allowed us to differentiate groups with different outcomes (Kaplan-
Meier and Long-Rank test, p < 0.05).
This chapter thus reviews different prognostic factors and stratifications of risk that
have been published during this past years evaluating them in our population based
registry. Descriptive studies are necessary not only to establish epidemiological features
useful for public health strategies but also to confirm prognosis factors and to validate
scoring systems to properly adapt therapeutic schemes.
INTRODUCTION
One of the most challenging problems in hematology is the heterogeneous group of
disorders that were formally defined as Myelodysplastic Syndromes (MDS) by the French
AmericanBritish (FAB) Cooperative Group in 1982 [1]. MDS are clonal disorders of
hematopoietic stem cells with a propensity to evolve to Acute Myeloid Leukemia (AML).
They are characterized by the presence of cytopenia(s) in combination with a normo-
hypercellular bone marrow (BM) exhibiting dysplasia in, at least, one myeloid cell line. The
stem cells show defective capacity for self-renewal and differentiation leading into an
ineffective hematopoiesis [1-4].
Although MDS are disorders characterized by step-wise genetic progression, the precise
mechanisms responsible for the initiation of idiopathic MDS are currently unknown. And,
Risk Factors and Prognostic Scoring Systems in Argentinean Patients 117
similar to most malignancies, MDS likely arise from a genetically transformed primitive
hematopoietic stem cell, while subsequent genetic and epigenetic changes contribute to their
development and progression. Immune, cytokine, and stromal responses in the host are
important to define the disease phenotype [4-5]. Most patients are initially asymptomatic, and
their condition is incidentally discovered on a routine blood test.
The diverse pathobiology of the disease is manifested by a varied clinical course, with
some patients having more indolent disease and longer life expectancy. Others present with
aggressive variants associated with limited survival of a few months with progression to
AML or death related to the consequences of BM failure including infection, hemorrhage,
and iron overload. Also, clonal evolution is associated with increasingly ineffective
hematopoiesis, progressive impairment of cellular function and worsening peripheral blood
(PB) cytopenia(s) [6-8].
The FAB classification included five categories: refractory anemia (RA), RA with ring
sideroblasts (RARS), RA with excess of blasts (RAEB), RAEB in transformation (RAEB-
t), and chronic myelomonocytic leukemia (CMML) [1]. In 2001, the World Health
Organization (WHO) classification was developed [2] and up-dated in 2008 [3]. It divides
MDS into several categories based on the number of dysplastic cell lines, the proportion of
blasts in the BM, and the presence of a deletion of chromosome 5q. The WHO classification
excluded patients with 20% blasts or more and those with CMML from the category of MDS
[3]. Although their prognostic impact, which is based mainly on BM morphology, is still
impressive [9], both schemes showed limitations to assess prognosis within the respective
morphological groups. Therefore, since the development of the Bournemouth index in 1985,
several useful multi-parameter-based scoring systems have been developed in the past to
define prognostic subgroups [7-8, 10-14].
The assigning of prognosis and the selection of appropriate therapy require careful
application of prognostic scoring systems. Different factors are evaluated in order to offer an
individualized therapy for each patient. These factors can be divided into those related to the
disease and to patients. The first one involves severity of cytopenias, percentage of BM
blasts, and cytogenetic findings, among others, that are included in the majority of scoring
prognostic systems [7-8, 10-14]. On the other hand, the other main group includes patients
age, comorbidities, performance status and other potential complications. Decision making is
complex in this group of patients, with a median age of 70 years old, because ageing is a
multidimensional process involving not only physiological changes but also changes in
functional, social, emotional and cognitive capacities. All these factors can have a significant
impact on the efficacy and tolerability of a potential therapy and have to be exhaustively
assessed before deciding on individual treatment regimens [15].
Argentinean MDS Registry was created in 2008 and is sponsored by the Argentinean
Society of Hematology. The main objective is to collect MDS related data in order to describe
epidemiological characteristics, prognostic factors and scoring risk stratifications in our
population. After 7 years, 14 institutions from Argentine have been reporting data from 532
patients (89% with de novo MDS) diagnosed mostly since 2007. This chapter thus reviews
different prognostic factors and stratifications of risk that have been published during this past
years evaluating them in our population based registry. Descriptive studies are necessary not
only to establish epidemiological features useful for public health strategies but also to
confirm prognosis factors and to validate scoring systems to properly adapt therapeutic
schemes.
PROGNOSTIC FACTORS
BM Blasts and Cellular Morphology
The BM aspirate allows for detailed evaluation of cellular morphology and assessment of
percent of blasts. This visual morphological examination is essential to establish the diagnosis
and the manual count of BM blasts is fundamental for risk assessment. In our population,
63%, 9%, 15% and 13% presented with < 3, 3-4, 5-10 and > 10% BM blast, and these cut-
points proposed by the IPSS-R were useful to predict prognostic differences with median
survivals of 82, 44, 17 and 14 months, respectively (p < 0.001).
Morphological assessment requires skilled morphologists because it can be subjective
particularly in patients with early low risk disease without excess blasts where the diagnosis is
based on dysplasia. Certain laboratory values such as blood cell count and cell volume
measurements are accurate and reproducible. However, certain observations are subtle and
remain observer dependent. Concordance of the diagnosis of MDS in cooperative clinical
trials is generally reported to be nearly 80%, although concordance of sub-classification
among different observers is often considerably less [16]. Inter-observer agreement among
morphologists for recognition of dyserythropoiesis is notoriously poor [17]. Senent et al.
blindly evaluated 50 patients with MDS and after stratifying the degree of agreement
according to the categories required for the assignment of WHO subtypes, this agreement was
not statistically significant for cases with 5-9% blasts in BM, 0.1-1% blasts in PB, or
percentage of erythroid dysplastic cells [18]. Also, Font et al. by reviewing 110 cases
previously diagnosed with MDS, studied whether the threshold of 2% BM blasts was
reproducible. They showed that among MDS WHO 2008 categories, inter-observer
discordance seems to be high in cases with unilineage dysplasia and illustrated that the
threshold of 2% BM blasts as settled by the revised -International Prognostic Scoring
System (IPSS-R; see below) may be not easy to reproduce by morphologists in real practice
[19].
The ability to report myelodysplasia where none is likely to exist was emphasized in
Parmentier et al. blinded study who investigated dyshematopoiesis in BM aspirate squash
preparations of 120 healthy BM donors none of whom would be reasonably expected to have
MDS. More than 10% dysmyelopoiesis could be detected in 46% of BM aspirate squash
preparations with 26% in 2 or more cell lineages and 7% in 3 cell lineages in healthy BM
donors. Donors under the age of 30 years exhibited more dysgranulopoietic and
dysmegakaryopoietic changes compared to the older donors. Female donors showed more
dysgranulopoietic changes than male donors. The concordance rate between the 4
investigators was modest in dysgranulopoiesis but poor in dyserythropoiesis and
dysmegakaryopoiesis [20].
Cytogenetics
Approximately 50% of patients show abnormal karyotype with a heterogeneous pattern

not associated with any particular chromosomal alteration. They may vary from simplex to
complex and are most often characterized by total or partial losses of chromosome material.
The cytogenetic profile shows that total or partial loss of material from chromosomes 5, 7, 17,
20 or a sex chromosome, as well as a relatively high incidence of genetic gains such as
trisomy 8, 19 and 21 are the most frequent alterations while recurrent translocations or other
structural abnormalities are less common [7, 21-23]. The higher prevalence of total and
partial losses suggests the involvement of tumor suppressor genes. However
haploinsufficiency of certain genes located in common deleted regions, as described for
deletion 5q, is being recognized as the relevant mechanism [24].
The presence of recurrently reported cytogenetic abnormality is a co-criterion for a
proper diagnosis and helps in predicting risk [25]. Because the heterogeneity of cytogenetic
alterations in MDS, there is no evidence that a panel of FISH probes could replace routine 20
metaphase cytogenetic analysis. It should be used in specific situations such us metaphases of
bad quality or low level of mitotic index [25]. And, knowing the specific cytogenetic
abnormality can assist in the selection of therapy for certain subgroups of MDS patients (eg,
lenalidomide for patients with 5q- syndrome) [26].
The karyotype was recognized as an independent prognostic factor in 1993 and its
inclusion among different prognostic systems has contributed to the improvement in assessing
prognosis [27]. Morel et al. compared the presence complex karyotypes ( 3 altered
chromosomes) vs. other cytogenetic results [27]. In 1997, the International Prognostic
Scoring System (IPSS) [7] established that some particular cytogenetic alterations were
associated with specific prognosis and clearly differentiated three cytogenetic risk categories:
Good [Normal karyotype, Y chromosome loss, isolated del(5q) or del(20q)], Intermediate
[trisomy 8 and other chromosome defects] and Poor [ 3 cytogenetic alterations and -
7/del(7q)] with median survivals of 46, 29 and 10 months, respectively. This modality was
later adopted by the WHO classification-based Prognostic Scoring System (WPSS) in 2007
[12].
In 2010, based on a coalesced multicentric analysis of 2901 patients with primary MDS
from the German-Austrian group, the International MDS risk analysis workshop (IMRAW),
the Grupo Cooperativo Espaol de Citogentica Hematolgica (GCECGH), and the
International Cytogenetics Working Group of the MDS Foundation (ICWG), five prognostic
subgroups were proposed defining the IPSS-revised (IPSS-R) stratification. After some
modifications, the final categories published were: Very good [del(11q) or Y], Good
[normal, del(5q), del(12p), del(20q), or double incl. del(5q)], Intermediate [7q-, +8, i(17q),
+19, any other single or double, independent clones], Poor [der(3)(q21)/ der(3)(q26), -7,
double incl. -7/7q-, or complex 3 abnormalities] and Very poor [complex >3 abnormalities]
with median overall survivals of 61, 49, 26, 16 and 6 months, respectively [28]. This proposal
was afterward adopted in the IPSS-R version [8] and validated in a series of 630 patients [29].
Figure 1 depicts the survival plots for the proposed cytogenetic stratification applied to our
MDS population.
We have previously described that the presence of an isolated deletion, excluding 7q-,
was a good prognostic finding, while the presence of a monosomal karyotype (MK) was a
high risk marker (IPSS-MK). This alternative but complementary strategy to the original
IPSS system showed an independent prognostic impact and a better discriminating power not
only than the original IPSS categories [23] but also than the initially proposed IPSS-R one in
our series [30]. MK refers to the presence of two or more distinct autosomal monosomies or a
single monosomy in the presence of structural abnormalities [31]. Other published data have
confirmed the bad prognosis associated with MK in MDS [32]. The incorporation of MK into
IPSS-R could further stratify MDS patients with higher-risk IPSS-R (intermediate, high and
very high risk) into four groups, rather than three groups, with different overall survivals [33].
Figure 1. Prognostic relevance of the Cytogenetic Categories of Risk proposed by the IPSS-R in our
Argentinean cohort of 466 evaluable patients. Median overall survivals and number of patients are
shown.
Some MDS patients who show normal karyotype at diagnosis could acquire a cytogenetic
event later in the progression and BM insufficiency will gradually increase. Karyotypic
evolution is relatively frequent in these patients related to a poor clinical prognosis, either
evolving to AML or dying [34-35]. Therefore, both chromosomal and hematologic status
should be accessed repeatedly to evaluate the rate and markers of evolution, especially in
patients diagnosed at very low risk.
Cytopenias
The percentage of patients with more severe cytopenias progressively increases in

parallel with increasing clinical stage of MDS [36]. The major cause of death in up to 30% of
patients is complications relate to cytopenias including bleeding, infections or complications
related to a high transfusion requirement [7-8].
Up to 80% of patients manifest with anemia at diagnosis, and this percentage is increased
in parallel with the evolution of the disease, being one of the hallmarks of MDS. For example,
the percentage of patients with hemoglobin < 8.0 g/dL increased from 11 to 47% as IPSS
progressed from low to high and from 23 to 45% as BM blast count increased from < 5 to 21
30% [36] and also according to FAB classification [37]. Anemia is a major contributor to the
symptomatology of MDS, because it is associated with fatigue, weakness, and shortness of
breath and the presence and degree of the anemia is a well-established negative prognostic
factor [10, 13, 14].
The relationship between severe anemia and poor clinical outcome suggests that anemia
initiates or exacerbates functional decline, particularly, involving cardiovascular disease
which is one of the most common co-morbidity in MDS patients. Also, there is a high
prevalence of patients with transfusion dependence who are prompt to develop secondary iron
overload, associated with clinical organ dysfunction, increased risk of cardiac failure, and
reduced survival.
In our series of 528 patients belonging to the Argentinean MDS, the degree of anemia
shows a good reproducibility and effectiveness to predict clinical outcome. Patients were
distributed according to the IPSS-R cut-points for hemoglobin into: 242 (46%) 10 g/dL, 167
(32%) 8-9.9 g/dL and 119 (22%) < 8 g/dL, with median survival of 82, 37, and 18 months
(Figure 2, p < 0.001).
Figure 2. Prognostic relevance of Hemoglobin cut-points proposed by the IPSS-R in the Argentinean
cohort of 528 MDS patients. Median overall survivals and number of patients for each group are
shown.
We also applied the sex-specific hemoglobin thresholds proposed by Malcovati et al.

instead of transfusion-dependency [38] to our series. These thresholds (males <9, and females
< 8 g/dL) were useful to stratify patients with different life expectancies (p < 0.001) (Figure
3).
Figure 3. Prognostic relevance of sex-specific hemoglobin thresholds in our cohort of 528 MDS
patients. Number of patients and median overall survival for each group are shown.
Thrombocytopenia and platelet dysfunction contribute to hemorrhagic complications in

MDS. The estimated prevalence of thrombocytopenia in MDS ranged from 40% to 65%.
And, the incidence of thrombocytopenia and severe thrombocytopenia, as expected, increased
with FAB and IPSS risk classification [39]. According to IMRAW database and to the
threshold of < 100 x 109/L, 37% were thrombocytopenic and 5% with severe
thrombocytopenia (< 20 x 109/L) [7, 36]. In a MDACC series 43% were thrombocytopenic
(< 50 x 109/L) at referral [13]. In our population, 22%, 19% and 59% presented with platelets
counts of < 50 x 109/L, 50- < 100 x 109/L and 100 x 109/L, respectively, and these cut-
points proposed by the IPSS-R were useful to predict prognostic differences (Figure 4,
p < 0.001).
The degree of neutropenia has been shown to be a risk factor for infection and death in
patients with MDS [40-41]. In an extended analysis of the IMRAW database, 38% of patients
had absolute neutrophil count (ANC) < 1,500/L and 6% with severe neutropenia. Patients
with ANC < 500/L had the highest representation within the RAEB-2 category and the
lowest representation within the RARS category (31% and 2%, respectively) [36]. The ANC
< 800/L (in IPSS-R) has been associated with higher potential infectious risk rather than that
of 1,800/L (in the IPSS) [41]. In our series 17% (92/528) patients presented with ANC
< 800/L associated with shorter overall survival (Figure 5, p < 0.001).
p<0.001
Platelet count 100 x109/L not reached
50- <100 x109/L, 36 months
<50 x109/L, 16 months
Figure 4. Prognostic relevance of platelets counts in the Argentinean cohort of 523 MDS patients.
Median overall survivals for each group are shown.
p<0.001
Neutrophil Count 800/L 77 months
< 800/L 19 month
Figure 5. Prognostic relevance of neutrophil counts in the Argentinean cohort of 528 MDS patients.
Median overall survivals for each group are shown.
Transfusion Dependency
Also related to the degree of anemia, transfusion dependency has been shown to have a
significant effect on survival of patients with MDS. In our series, 47% of patients requires
transfusion support during the follow-up, and transfusion dependency was associated with
shorter survival and prompt evolution to AML (p < 0.001, data not shown).
The decision criteria for transfusion of patients with MDS may vary from clinician to
clinician, center to center, country to country, and from patient to patient depending not only
on their respective hemoglobin level but also on comorbidities and performance status.
Therefore, the inclusion of transfusion dependency into prognostic scoring systems has been
criticized as being subjective a criterion [42].
There is a high prevalence of patients with transfusion requirement that often results in
significant clinical consequences [12]. One of the objectives in MDS patients is to prevent
transfusion related morbidity and mortality, mostly resulting from iron overload, which may
become apparent when the number of red blood cell transfusions exceeds 2040 and serum
ferritin levels exceed 15002000 ng/mL [25]. The severity of transfusion requirement,
calculated as the number of packed red cell units per month, has been significantly related
with cardiac complications [38]. In addition, secondary iron overload determining clinical
organ dysfunction and an increased risk of cardiac failure was associated with reduced
survival in transfusion dependent MDS patients [43].
SCORING SYSTEMS
The evaluation of disease risk and overcome of patients with MDS is one of the most
critical point due to this impressive clinical heterogeneity, and thus the need to develop
prognostic systems that allow risk stratification and help in the timing and choice of therapy
[4, 7-9, 22, 44]. Apart from the intrinsic prognostic value of morphological classifications [1-
3, 9], a number of prognostic scores have been proposed during the last past 30 years and
many of them are currently in use [7-8, 12-14]. The main objective in lower risk patients is
improving the quality of life, and in higher risk patients is to extend the survival trying to
modify the natural history of the disease. Accordingly, therapies vary from supportive care to
intensive chemotherapy and hematopoietic stem cell transplantation.
International Prognostic Scoring System (IPSS)
The International MDS Risk Analysis Workshop (IMRAW) proposed the IPSS based on
816 patients with primary MDS in 1997. The IPSS relies on the primacy of the number of
cytopenias, three cytogenetic groups, and the percentage blasts in the BM to group patients
with MDS into one of four prognostic categories: low risk (score of 0), intermediate 1 risk
(0.5 to 1.0), intermediate 2 risk (1.5 to 2.0), and high risk (> 2.0). Cytopenias were defined for
the IPSS as having hemoglobin level less than 10 g/dL, an ANC less than 1800 /L, and
platelet count less than 100 x 109/L [7]. This system is still the most commonly used score
because it is highly reproducible [22, 45], very simple to calculate and has been very useful in
examining and comparing the outcomes of clinical trials. Therefore, the IPSS has become the
gold standard for risk assessment and is a key element of practice guidelines as those
published by the National Comprehensive Cancer Network (NCCN) or by the Italian Group
[46, 47].
Patients were classified by IPSS as low 157 (42%), Int-1 148 (39%), int-2 48 (13%), and
high 23 (6%) risk, with median survival not reached, 44, 17, and 13 months (p < 0.001,
Figure 6), and time to leukemic evolution (25%) not reached, 31, 10, and 4 months,
respectively (p < 0.001).
We could observe that the proportion of higher risk patient in the present series was
lower than we have previous reported based on retrospective studies from Argentine [22] and
from South-America [45]. This decrease might be related to the current application of the
WHO classification that excludes patients with 20%, or more, of BM blasts, therefore those
patients tend to be less frequently reported in our prospective Registry that includes patients
mostly diagnosed after 2007.
Figure 6. Survival plots according to the International Prognostic Scoring System (IPSS) in our cohort
of 376 evaluable MDS patients. Number of patients and median overall survival for each group are
shown.
WHO CLASSIFICATIONBASED PROGNOSTIC

SCORING SYSTEM (WPSS)
The WHO classification (score 0 to 3) integrated within red cell transfusion dependency
(score 1) and cytogenetic findings, classified according to the original IPSS categories (score
0 to 2), have produced the so-called WPSS. According to WHO categories, patients with: RA,
RARS and MDS with isolated del(5q) score 0; Refractory Citopenia with Multilineage
Dysplasia (RCMD) score 1, RAEB-1 score 2 and RAEB-2 scored 3. In contrast with the four
risk groups identified by the IPSS, the WPSS stratifies MDS patients into five different risk
categories: very low (score = 0), low (= 1), intermediate (= 2), high (= 3-4), or very high
(> 4). The median survival ranges from 103 months in the very-low-risk group to 12 months
in the very-high-risk group. The model was first developed in an initial learning cohort of 426
patients, and was further confirmed in a second cohort of 739 patients. This system is a time-
dependent regression model and, thus, provides prognostic information from initial evaluation
through treatment to follow-up and is even more accurate in identifying patients with a very
low risk [12]. This system has been externally validated in a series of 149 patients and the
authors suggested that WPSS was able to distinguish the truly low risk patients (very low),
who had excellent long-term survival with rare evolution to AML [48]. However, according
to the NCCN Clinical Practice Guidelines, patients with WPSS Very Low, Low, and
Intermediate are considered lower-risk patients [47].
The inclusion of transfusion dependency has been criticized as being a subjective
criterion [42]. Therefore, Malcovati et al. described sex-specific hemoglobin thresholds
(lower than 9 g/dL in males and lower than 8 g/dL in females, Figure 3) that showed a
significant prognostic value comparable to that of transfusion-dependency. They also
recalculated the WPSS risk groups, using sex-specific hemoglobin thresholds (WPSS-Hb)
instead of transfusion-dependency, and obtained highly concordant risk categories. This
modification could provide an objective criterion for prognostic assessment in addition to the
WHO criteria and cytogenetic groups of risk [38]. Another limitation remains in the
subjective WHO classification of the disease, as was discussed above.
WPSS revised according to hemoglobin levels allowed us to stratify 326 patients as very
low 25 (8%), low 142 (44%), intermediate 74 (23%), high 63 (19%), and very high 22 (7%)
risk, with median survival of 105, not reached, 35, 18, and 15 months, and time to leukemic
evolution (25%) of 116, not reached, 32, 12, and 6 months, respectively (p < 0.001).
MD Anderson Prognostic Scoring System (MDACC)
Kantarjian and co-workers developed this system using an initial training cohort of 958
patients, validated in a second test group of 957 patients [13] and, afterward in a series of 775
patients from the Moffitt Cancer Center classified according to the WHO criteria [49]. This
model includes patients with proliferative-type CMML (WBC > 12,000/mL) or secondary
MDS in order to overcome the limitations of previous scores that excluded these patients. It
allows evaluation of all patients that are considered as MDS at any time during their course of
their disease without needed WHO evaluation [44].
The MDACC includes among its variables: performance status (limit of 2); age (limits:
60 and 64); platelet count (limit 3-50-200 x 109/L), hemoglobin (limit 12 g/dL), BM blast
(limit 5-10-19%), white blood cell count (limit 20 x 109/L), transfusion requirement and
cytogenetic findings (chromosome 7 or 3 alterations versus other karyotypes) [13]. Scoring
those variables, this system defined four groups of risk in our population: Low 103 (23%),
Intermediate-1 176 (40%), Intermediate-2 85 (19%), and High 81 (18%), with statistical
differences both in term of overall survival (p < 0.001, Figure 7) and survival free of
evolution to AML (p < 0.001). However, the MDACC ignores the WHO classification and
includes a BM blast range up to 29% (i.e., up to values currently considered as diagnostic of

AML). In addition, it takes into account poor cytogenetic abnormalities exclusively.
p<0.001
(Not reached)
Figure 7. Survival plots according to MD Anderson Prognostic Scoring System in our cohort of 445
MDS patients. Number of patients and median overall survival for each group are shown.
Revised International Prognostic Scoring System (IPSS-R)
The original IPSS system has shown limitations that have become evident over the years.
Among them, it is not a very precise predictor of prognosis in patients with lower risk disease
and that it attributes relatively little weight to cytogenetics. Also, the original IPSS
underweights the clinical importance of severe anemia, neutropenia and thrombocytopenia in
determining the need for therapeutic intervention [36]. Therefore, the IPSS has been revised
(IPSS-R) and published in 2012, trying to address several of the perceived deficiencies of its
predecessor by examining 7012 MDS cases. The new IPSS-R includes a new cytogenetic risk
classification of the disease that divides patients into five cytogenetic categories, new clinical
relevant cut-off for cytopenias (i.e., hemoglobin level, neutrophil and platelet count), and the
new limit of 2% for BM blasts. These variables are used to assign patients to one of the five
risk groups and this score might be adjusted according the age [8]. Using this new IPSS-R a
substantial subset of MDS patients per classical IPSS can be re-classified as higher or lower
risk suggesting that IPSS-R can refine the scoring of an individual MDS patient [8, 50].
As is shown in figure 8, the IPSS-R system showed a good reproducibility [45, 50] also
in treated patients [51-54], includes easy accessible variables, and is expected to be
incorporated by most centers replacing current IPSS until the role of molecular information is
better understood in the prognostication of patients with MDS. Although it is more

complicated to calculate than the original one, online calculators are available to help
(http://www.ipss-r.com).
p<0.001
Figure 8. Revised International Prognostic Scoring System in our cohort of 373 MDS patients. Number
of patients, median overall survivals (A) and time to 25% to AML evolution (B) for each group are
shown.
Recently Della Porta and co-workers refined the WPSS by determining the impact of the
newer clinical and cytogenetic features on a series of 5326 untreated MDS. A strong
correlation was found between the WPSS including the new cytogenetic risk stratification and
WPSS adopting original criteria. Also, a highly significant correlation was found between the
WPSS and IPSS-R risk classifications. Therefore, the clinical choice of the prognostic system
should be on the basis of the differences in the concept of the WPSS and the IPSS-R, and on
specific clinical need [55].
Other Prognostic Variables
Other parameters, such as gender, age morphologic features, and lactate dehydrogenase
(LDH) concentration were used to construct scoring systems in order to define risk groups
differing in terms of survival as well as evolution to AML.
MDS affects more frequently males than females. Our gender ratio, accordingly with
previous reports was 1.3, being 299 (57%) male and 229 females with median survivals of 40
and 77 months, respectively (p = 0.004). Already in the original IPSS publication, gender was
analyzed and its predictive importance for survival and AML evolution was stated, although
weaker than the variables finally included in the IPSS [7]. Women experienced much lower
risk than men in the IPSS low-risk categories but similar risk in the high-risk categories.
Consequently, the IPSS risk categories discriminate much better in women than in men. This
model, if valid, implies that the risk ratios of women and men differ between IPSS risk
categories rendering pairwise gender comparisons within IPSS risk categories substantially
uninformative. Based on main effects and interactions of age (discussed below) and gender,
the Austrian-German collaborative group established an individualized age- and gender-
adapted IPSS to improve prognostication in MDS. This prognostic index (PI) can be
calculated according: PI = [IPSSx 0.59 + sexx -0.31 + age 66x0.51 + (IPSS : sex) x 0.42 +
(IPSS : age 66)x-0.23 + (IPSS : sex : age 66) x -0.58]. To give an example for the proposed
approach, a male patient aged > 66 years with an IPSS level int-1 has an estimated risk of 1.9.
This is almost equal to the average risk of IPSS level int-2, i.e., taking the age and gender of
this patient into account would change his risk estimate from an average int-1 risk to an
average int-2 risk. On the other hand, a female patient aged < 66 years with an IPSS level int-
1 would be assigned -0.1, i.e., a little lower than the average IPSS low-risk category. This
patient would be assigned an average low risk on the IPSS scale [56].
Age is an important indicator of poor prognosis; however, it is not included in the
original or revised IPSS because it could allow underestimation of the severity of MDS in
young patients [57]. However, the effect of age was proposed as an optional additive feature
for overall survival prediction by including age in the formula: (years -70) x [0.05 - (IPSS-R
risk score x 0.005)] and adding the result to the sum of the 5 major variables [8]. A similar
formula was also proposed to adjust the WPSS [55]. We have previously shown that this
IPSS-R adjusted by age helped us to identify 19% of short surviving patients among low risk
IPSS-R patients with a median survival of 34 months [50]. According to our data, age as a
variable showed more prognostic impact in lower- than in higher-risk patients [55].
Idiopathic MDS occurs mainly in older persons. The median age of our series was 72
years old with a range of 17-95 years old, being 397 (75%) patients older than 60 years.
Therefore, most patients with MDS are elderly and typically have co-morbid diseases. The
mortality risk for many of them is not defined by their hematologic disorder alone, but by the
comorbid conditions that coexist with it. About 50% of the MDS patients had one or more
comorbidities and most frequent are: cardiac, hepatic, renal, and liver comorbidities as well as
second tumors. Several groups focused on the description of comorbidities and their potential
use as prognostic markers. Comorbidities influence the life expectancy, independent of
disease related parameters, primarily in the Low and Intermediate risk groups according to the
IPSS, showing a significant impact on both non-leukemic death and overall survival [14, 58].
Age and performance status are imperfect surrogates for this information and models that
explicitly examine extrahematologic conditions can improve on the ability to predict
outcomes in MDS patients [55]. A comprehensive assessment of the severity of comorbidities
at baseline is recommended because not only influences the prognosis but also the choice of
treatment [15, 59]. Older MDS patients, who are more likely to have lower performance
status and major comorbidities than younger patients, may not be able to tolerate intensive
chemotherapy therapy [60].
The importance of measuring comorbidity in consistent and quantifiable ways is being
recognized. The Charlson Comorbidity Index (CCI) was developed in 1987 based on 1-year
mortality data from 559 internal medicine patients and validated within a cohort of 685
patients. The index encompasses 19 medical conditions weighted 16 with total scores
ranging from 037. In the development phase of the index, mortality for each disease was
converted to a relative risk of death within 12 months. A weight was then assigned to each
condition based on the relative risk (RR); for example, RR < 1.2 = weight 0, RR 1.2 < 1.5 =
weight 1, RR 1.5 < 2.5 = weight 2, RR 2.5 < 3.5 = weight 3, and for 2 conditions
(metastatic solid tumor and AIDS) = weight 6 [61]. Hall et al. proposed an electronic
application for rapidly calculating CCI that can increase their use in clinical practice and
research [62]. Our Registry was programmed to calculate automatically the CCI and its
application allowed us to subdivide 528 patients into five categories with prognostic
significance (p < 0.001; Figure 9).
=0
=1
=2
>3 =3
Figure 9. Survival plots according to Charlson Comorbidity Index (CCI) for our cohort of 528 MDS
patients. Number of patients and median overall survival for each group are shown.
The Adult Comorbidity Evaluation-27 (ACE-27) scale, a measure of comorbidity

developed for assessment of patients with solid tumor, has been previously applied to MDS
patients. However, the ACE-27 failed to stratify the prognosis in a series of 840 patients with
MDS [63] or in subjects aged 65 years or older and in the IPSS low-risk subgroup [64].
None of the systems discussed above include impact of comorbidity to the calculation of
the natural history of MDS patients. A specific MDS comorbidity score (MDS-CI) was
developed on a learning cohort of 840 Italian MDS patients, based on definition of
comorbidities according to Sorror et al. [65], and validated on a German cohort of 504
patients. The multivariate analyses showed double weight to cardiac comorbidities, and one
point to moderate-to-severe hepatic, severe pulmonary and renal disease or to solid tumor.
Cardiac disease was the most important comorbid condition from a prognostic point of view,
and the negative interaction between this comorbid condition and severe anemia has a
profound impact on survival of MDS patients, particularly in the lower risk groups according
to disease-related criteria [66]. This MDS-CI provides additional prognostic information on
patients stratified according to WPSS [67] and to the IPSS-R [58].
Myelofibrosis and Other BM Biopsy Determinations
Although BM biopsy may be considered too invasive for elderly patients, it allows for
determination of BM cellularity, architecture, fibrosis, small clusters of immature (CD34+)
progenitor cells or their abnormal distribution/ localization [25]. Therefore, histological
information complements the morphological information obtained from a marrow aspirate in
order to arrive to a proper diagnosis.
The BM is normo-hipercellular in the majority of cases, and is hypocellular according to
age (<30% cellular in patients <60 years old, <20% cellularity in patients 60 years old) in a
minority of them. Hypoplastic MDS needs to be distinguished from both aplastic anemias and
hypocellular AML. Recently, a prognostic model for patients with hypoplastic MDS has been
proposed based on a cohort of 253 patients. Multivariate analysis identified poor performance
status (ECOG 2), low hemoglobin (< 10 g/dL), unfavorable cytogenetics (7/7q or
complex), increased BM blasts (5%) and high serum LDH (>600 IU/L) as adverse
independent factors for survival. This model segregated patients into three distinct risk
categories independent of IPSS score [68].
The immunohistochemistry-based determination of the percentage of CD34+ progenitor
cells is important to avoid underestimation of blast cell count in the smear and when the BM
smear is contaminated with PB cells [25]. In 2005, a group of European experts (European
Myelofibrosis Network, EUMNET) formulated a consensus-based proposal for a semi-
quantitative evaluation of BM fibrosis with the aims of avoiding excessive overlapping and
achieving a high degree of reproducibility in clinical practice [69]. The occurrence of mild
fibrosis is a common feature at diagnosis in these patients and does not correlate with specific
features. However, approximately, 13-20% of patients with primary MDS present with
moderate/severe (grade 2 or 3) BM fibrosis that predicted an inferior overall survival and
leukemia event-free survival [70-71]. The presence of reticulin fibrosis was associated with
multilineage dysplasia, increased cellularity, increased peripheral cytopenias, poorer
cytogenetics, and increased risk of transfusion needs [70]. Similar associations were found in
a series of 43 patients with MDS and CMML-I [72]. Within patients stratified according to
IPSS and WPSS categories, BM fibrosis involved a shift to a one-step more advanced risk
group. CD34+ clusters were associated with an increased percentage of BM blasts, poorer
cytogenetics, and increased risk of transformation to AML [70], and in addition to the IPSS
could lead to an improved prognostic sub-categorization of patients [73]. Although our data
confirm previous results (Figure 10), BM fibrosis showed no incremental prognostic value for
clinical outcome when the IPSS-R was developed [8] and when the WPSS was re-evaluated
[55].
Figure 10. Survival plots according to the presence of BM fibrosis for our cohort of 388 evaluable MDS
patients. Number of patients, grade of BM fibrosis and median overall survival for each group are
shown.
Serum LDH, Ferritine Levels, 2 Microglobulin and Albumin
Ineffective hematopoiesis or the increased turnover and degradation of myeloid cells in

the BM, spleen, and other tissues preceding acceleration of the disease may account for
several different mechanisms underlying an increase in LDH in progressing MDS [53]. A
refinement of the IPSS including the serum LDH has been proposed by the German-Austrian
Cooperative Group. The IPSS + LDH was capable of identifying MDS patients with
unfavorable prognosis within the low and intermediate IPSS risk groups [74]. Also, on
multivariate analysis for survival, LDH was found to be an independent prognostic factor for
survival along with WPSS and age in a reduced series of 149 patients [48], and its serial
determination has been proposed as a suitable follow-up parameter to early recognize disease
progression in low risk patients [75]. High LDH levels along with and higher WPSS risk
groups were significantly associated with a worse survival in a series of 126 patients treated
with azacitidine [76]. Although LDH concentration significantly correlated with IPSS-R risk
groups, it was not an independent predictor of outcome both for survival or evolution to AML
in a series of Italian patients [53].
The negative impact of elevated serum ferritin levels for survival may reflect prior red
blood cell transfusions contributing to iron overload and its complications or may reflect the
severity of the anemia and degree of ineffective erythropoiesis at time of diagnosis. Also,
serum ferritin is an inflammatory marker and this abnormality may reflect the effects of
inflammatory cytokines in MDS. Voso and colleagues showed that ferritin was an
independent prognostic factor in the multivariable analysis for survival, probably reflecting
the negative impact of iron overload itself on the function of vital organs and on the number
of cardiac deaths. However, the prognostic value of transfusion dependence, which
incorporates also features of disease progression, was superior to that of ferritin in predicting
leukemic evolution [53].
Plasma levels of 2 microglobulin (B2M) were prospectively analyzed by Gatto et al. in
553 patients. They found that B2M, with a cut-point of 2 mg/L, was an independent
prognostic variable for survival with weighted significance second only to the karyotype, and
that its incorporation added significantly to the power of the IPSS to stratify MDS patients
[77]. The worse prognosis associated with higher levels of B2M was confirmed by Neumann
et al. [78]. However, when the IPSS-R was developed the authors included this parameter as
provisional. They suggested that renal dysfunction alters these levels and could confound
their results [8].
Komrokji et al. stratified 767 patients into three groups based on albumin concentration
(< 3.5, 3.6-4.0, and > 4.0 g/dL) and hypoalbuminemia was associated with worse outcome.
They found that the albumin level offered prognostic discrimination for outcomes within the
lower and higher IPSS risk groups, as well as with the MD Anderson risk model. In
multivariable analysis, serum albumin was a significant independent co-variate for survival
after adjustment for IPSS, age, serum ferritin, and transfusion dependence [49].
When the larger combined database of 7012 MDS patients was analyzed trying to access
the prognostic impact of other variables not included in the IPSS-R, the results showed that
patient age, performance status, serum ferritin, LDH, and possibly B2M were significant
additive features for survival but not for AML evolution. The authors proposed numerical
values for determining the contribution of each of these features to the patients risk category
that should be added to the raw score of the major variables [8]. Similar results were obtained
when WPSS was re-evaluated [55]. The use of these additional features could be of particular
value for categorization of patients, mostly belonging to the intermediate group of risk
[8, 55].
CONCLUSION AND FUTURE PERSPECTIVES

MDS covers a group of heterogeneous and complex hematologic disorders primarily
found within the older population. The heterogeneity of MDS manifests in the individual
patient as a disease ranging from an indolent condition with a considerable life expectancy to
forms approaching the aggressiveness of AML. Therefore, a risk-adapted treatment strategy is
mandatory.
Prognostic factors may be subdivided into those related to the patients general
characteristics and health condition and those related to the MDS disease itself. Our data
shows that gender, percentage of BM blast, hemoglobin level, platelet and neutrophil counts,
cytogenetic groups of risk, presence of myelofibrosis, and red blood cell transfusion
requirement were significant predictive variables for prognosis (Kaplan-Meier and Long-
Rank test, p < 0.05).
The applied prognostic scoring systems (IPSS, IPSS-R, WPSS revised according to
hemoglobin levels, MDA score, and Charlsons Comorbidities Index) allowed us to
differentiate groups with different outcomes (p < 0.001). During the past 15 years, treatment
has been stratified according to the IPSS risk score, basically into low and high risk. More
recently, IPSS-R and the WPSS have been introduced subdividing patients into five risk
groups with different outcomes in terms of AML evolution and survival. Other efforts such as
the classifications from MDACC have positively contributed to the search for better
prognostic classifications for MDS. Della Porta and co-workers [55] suggested that the
clinical choice of the prognostic system should be on the basis of the differences in the
concept of the IPSS-R and the WPSS, and on specific clinical need.
Cytogenetic alterations are critical in determining the prognosis of patients with MDS.
Recently, developments in molecular technologies have led to major improvements in the
understanding of the molecular pathogenesis of MDS, identifying somatic mutations in 80%-
90% of MDS patients [79-80]. Somatic mutations have a strong effect on survival. The
presence of any of the mutations TP53, RUNX1, TP53, EZH2, or ETV6 [81-82] worsens
prognosis independently of IPSS. ASXL1 and SRSF2 mutations also seem to be associated
with a poor prognosis [83-84]. Including also DNMT3A and TET2, they may thus allow for
better stratification of patients within conventional scoring systems for different types of
treatment [85-86].
In the future it might be necessary to develop predictive scores more than prognostic
scores for different therapeutic approaches, because the number of patients who undergo
specific treatments will increase in the future. There will be pros and cons to every
classification and score as long as our knowledge about the biology of MDS remains limited.
Continuous research into the molecular pathogenesis of this group of disorders and
biomarkers of response to current therapeutic approaches is needed.
ACKNOWLEDGMENTS
The authors thank the investigators of the Argentinean MDSs Study Group organized by
the Argentinean Society of Hematology for the use of MDS Registry database. All authors
gave significant contributions to draft the article, critically revise the content of the
manuscript, approved the final version to be submitted and declare no conflict of interest.
This work was supported by Argentine grants from the Consejo Nacional de
Investigaciones Cientficas y Tcnicas (CONICET), the Agencia Nacional de Promocin
Cientfica y Tecnolgica (ANPCyT) and the Instituto Nacional del Cncer (INC).
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Chapter 6
EPIGENETICS AND EPIGENETIC THERAPY

OF MYELODYSPLASTIC SYNDROMES
Ota Fuchs
ABSTRACT
Myelodysplastic syndromes (MDS) are a goup of clonal hematopoietic stem cell
disorders characterized by ineffective production of blood cells with varying need for
transfusions, risk of infection, and risk of transformation to acute myeloid leukemia
(AML). Epigenetics is the study of changes in gene function that are mitotically and/or
meiotically heritable and that do not entail change in DNA sequence. Epigenetic
modifications refer to the reversible changes, such as DNA methylation, histone
acetylation, and RNA interference that alter gene expression impacting disease biology
and play an important role in the pathogenesis of MDS. Hypermethylation of CpG islands
in the promoters of key genes involved in cell cycle regulation, apoptosis, tumor
suppressor control and in response to chemotherapy and the consequent silencing of their
expression is well documented in MDS. Hypermethylated DNA sequences of key cellular
machinery provide an attractive potential therapeutic target for the treatment of MDS.
Comprehensive analyses of DNA methylation in MDS have recently extended beyond
CpG islands, with the advent of whole genome bisulfite sequencing (WGBS) or reduced
representation bisulfite sequencing (RRBS). DNA methylation and histone modification
not only regulate the expression of protein-encoding genes but also microRNAs (miRs),
such as let-7a, miR-9, miR-34a, miR-124, miR-137, miR- 148 and miR-203. Recurrent
somatic mutations in genes coding proteins involved in DNA methylation (DNTM3A)
and demethylation (TET2) and in covalent histone modifications (EZH2, ASXL1, EED,
JARID2 and SUZ12) have recently been reported in MDS and other myeloid
malignancies. Two forms of MDS (de novo MDS and therapy-related MDS /t-MDS/) are
different in DNA methylation. Hypermethylation in de novo MDS and widespread
hypomethylation in t-MDS have been detected. The only approved way to inhibit DNA
Ota.Fuchs@uhkt.cz.
142 Ota Fuchs
methylation is to use clinically available inhibitors of enzymes - DNA methyltransferases

(DNMTs). 5-Aza-cytidine (Vidaza) and 5-aza-2-deoxycytidine (5-azaCdR; decitabine)
have become the standard in the treatment of patients with higher-risk MDS, in particular
older individuals, where intensive chemotherapy and allogeneic stem cell transplantation
is not possible. These DNMTs inhibitors have both higher response rates and prolonged
survival when compared with traditional cytotoxic therapeutic agents. Several cycles are
necessary to achieve and maintain responses since the effect of azanucleotides is
reversible and malignant clone persists in most responding MDS patients. Somatic
mutational model to predict response to hypomethylating agents in MDS was proposed.
Mutations of TET2, IDH1, IDH2, STAG2, RAD21 and TP53 were more frequent in
responders to hypomethylating agents. However, mutations in ASXL1 and U2AF1 were
more frequent among non-responders to hypomethylating agents. Programmed death 1
(PD-1) signaling may be involved in MDS pathogenesis and in resistance mechanism to
hypomethylating agents. Blockade of this pathway can be a potential therapy in MDS. In
the future, methylation analysis of whole genes, not only of promoters, may identify key
genes in patients with the highest probability of response to azanucleotides and allow a
patient-tailored approach. Another inhibitors of DNMTs (5,6-dihydro-5-azacytidine, 2-
deoxy-5,6-dihydro-5-azacytidine, a second-generation hypo-methylating agent SGI-110
/a dinucleotide of decitabine and deoxyguanosine linked with a natural phosphodiester
linkage/, zebularine, procaine, epigallocatechin-3-gallate, and N-phthalyl-L-tryptophan)
are in preclinical studies or in clinical trials. The second epigenetic target for which drugs
are available is histone deacetylation. There are several histone deacetylases (HDAC)
inhibitors (romidepsin, vorinostat, pracinostat, belinostat, sodium phenylbutyrate,
valproic acid, entinostat, and mocetinostat) in preclinical studies or in clinical trials.
Combination therapies with two types of epigenetic drugs (azanucleotides and HDAC
inhibitors) are also studied.
Keywords: MDS, epigenetics, DNA methylation, histone methylation, histone acetylation,

DNA methyltransferases, azacitidine, decitabine, histone deacetylases, HDAC inhibitors,
histone methyltransferases
INTRODUCTION
MDS are a heterogeneous group of clonal hematopoietic stem cell disorders characterized
by ineffective hematopoiesis, peripheral cytopenias, frequent karyotypic abnormalities and
risk of transformation to AML [1, 2]. MDS are rare in young people and median age of
patients with MDS is approximately 70 years [3, 4]. Current management in MDS includes
supportive care, drug therapy and allogenic stem cell transplantation. MDS patients older than
age 70 years are not good candidates for allogenic stem cell transplantation. Without
intervention, median survival of higher-risk patients (intermediate-2 and high risk patients
according the International Prognostic Scoring System) is about 12 months [5]. The use of
hypomethylating agents (azacitidine and decitabine) improved survival of patients with
higher-risk MDS and their progression to AML was significantly delayed [6-8]. The
improvement of survival is in part attributable to the correction of anemia and transfusions
needs of higher-risk MDS patients. So, the improvement of survival caused by the factual
treatment with hypomethylating agents (DNA methyltransferases inhibitors) is lower [9].
Hypomethylating agents provide also multilineage response in lower-risk MDS patients.
However, the results must be optimized and novel combination strategies are also developed.
Epigenetics and Epigenetic Therapy of Myelodysplastic Syndromes 143
HDAC inhibitors modulate synergistically with DNMTs the epigenetic chromatin structure. It
is very rational to combine DNMTs with HDAC inhibitors and a variety of early phase trials
used this strategy [8, 10-17]. Till now, none of these combination strategies has brought better
results than single agent therapy by DNMTs [18].
HDAC inhibitors impact chromatin conformation by altering the pattern of acetylation of
lysine residues in nucleosomal histones. HDAC inhibitors have also other functions including
induction of reactive oxygen species, inhibition of protein chaperone, alteration of death
receptor pathways, and alterations of NF-B pathway [14, 19, 20].
The relapse rate after termination of the treatment with hypomethylating agents is high.
Prolonged treatment with these agents is necessary in order to delay development of
resistance [21].
THE REGULATION OF GENE EXPRESSION BY DNA METHYLATION

DNA methylation is a covalent modification at position C5 of the cytidine ring in the
context of a CpG dinucleotide. This methylation is catalysed by a family of DNMTs
including DNMT1, DNMT3A and DNMT3B. DNMT1 is required for maintenance
methylation during DNA replication. DNMT3A and DNMT3B function in de novo
methylation [22-25]. CpG rich regions called CpG islands are present in about half of human
gene promoters. Methylation of these CpG islands is associated with transcriptional silencing
from the involved promoters.
When the CpG islands are highly methylated, they bind specific proteins which recruit
transcriptional co-repressors such as histone deacetylases (HDACs). Epigenetic silencing is
also associated with histone H3 lysine 9 (H3K9) methylation.
This modification is associated with closed chromatin and results also in transcriptional
suppression. Alterations in DNA methylation are important in the pathogenesis of MDS [26].
Increasing evidence shows aberrant hypermethylation of genes occurring in and potentially
contributing to pathogenesis of MDS. The tumor suppressor and cell cycle regulatory gene
CDKN2B (cyclin-dependent kinase inhibitor 2B) is an example of hypermethylated gene in
MDS resulting in silenced expression of this cell cycle inhibitor p15INK4B (cyclin-dependent
kinase 4 inhibitor B) and in uncontrolled cell cycle progression and cellular proliferation.
CDKN2B methylation is frequent in refractory anemia with excess blasts in transformation,
therapy-related MDS, and in chronic myelomonocytic leukemia [27-30].
Increased methylation of CDKN2B gene is connected with disease progression.
Methylation level of CDKN2B gene might be used as a marker of leukemic transformation in
MDS [31].
Reversal of aberrant methylation by the treatment with hypomethylating agents leads to
re-expression of silenced tumor suppressor genes and some other genes, often connected with
response to chemotherapy (CDKN2B, cyclin-dependent kinase inhibitor 2A /CDKN2A/
coding for p16INK4A, the cell-adhesion genes /cadherin-1 /CDH-1/, cadherin-13 /CDH-13/,
and immunoglobulin superfamily member 4 /IGSF4/, the pro-apoptotic death-associated
protein serine/threonine kinase gene /DAP-kinase/, the suppressor of cytokine signaling-1
/SOCS1/, the reversion-induced LIM homeodomain containing gene /RIL/, a ligand-
dependent suppressor deleted in colorectal cancer /DCC/, a growth regulatory and tumor
144 Ota Fuchs
suppressor gene hypermethylated in cancer /HIC1/, dinucleosidetriphosphatase-fragile

histidine triad gene /FHIT/ involved in purine metabolism, calcitonin, arachidonate 12-
lipoxygenase /ALOX12/ involved in the production and metabolism of fatty acid
hydroperoxidases, glutathione S-transferase Mu1 /GSTM1/, testes-specific serine protease 50
/TSP50/, O-6-methylguanine-DNA methyltransferase /MGMT/, Krppel like factor 11
/KLF11/, oligodendrocyte lineage transcription factor 2 /OLIG2/, estrogen receptor alpha
/ESR1/, progesterone receptors PGRA and PGRB, RAS association domain family1A
/RASSF1/, functioning in the control of microtubule polymerization and potentially in the
maintenance of genomic stability, and BLU, both tumor suppressors genes located at 3p21.3,
retinoic acid receptor beta /RARB/, a nuclear transcription factor which mediates cellular
signaling, cell growth and differentiation, and neutrophic tyrosine kinase receptor, type 1
/NTRK1/), which is needed to transmit signals for cell growth and survival [32-35].
In the recent years, the discovery of a series of mutations in patients with MDS has
provided insight into the pathogenesis of MDS. Among these alternations have been
mutations in genes, such as IDH1, IDH2, TET2, and DNMT3A, which affect DNA
methylation [36-47]. These mutations are discussed in the special chapter.
Types of Histone Methylation Modification and

Their Regulatory Mechanisms
Histone methylation is carried out by several histone methyltransferases that methylate

lysine (HKMTs) or arginines (PRMTs) in histone tails [48-57]. The four core histones, H2A,
H2B, H3, and H4, make up the nucleosome, the main structural unit of chromatin. Some
specific histone tail modification, such as methylation of histone 3 lysine tail residue 4
(H3K4), are associated with activation of gene expression, while others, such as methylation
of histone 3 lysine 27 (H3K27), are associated with gene repression [58, 59]. These marks are
normally carefully controlled by the interplay of sequence-specific DNA binding transcription
factors and transcriptional cofactors, many of which are histone-modifying enzymes. The end
amino group of lysine can be mono-, di- or tri-methylated. Dependent on this methylation
state, the binding affinity of chromatin-associated proteins varies greatly. Methylation of
histone 3 lysines 4 and 27 is catalyzed by trithorax and polycomb family of proteins. H3K27
is di- and trimethylated by enhancer of zeste homolog 2 (EZH2), a polycomb family protein
[60, 61]. The enzyme that reverses H3K27 methylation was not known until the discovery of
two demethylases, ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX)
and Jumonji domain containing 3 (JMJD3), both of which are members of the Jmje domain-
containing protein family [62]. EZH2 has been reported to be mutated and inactivated in
MDS [63-68], but is also overexpressed in other subsets of MDS [69]. UTX mutations and/or
deletions have also been observed in patients with MDS and chronic myelomonocytic
leukemia [70-73].
Histone Acetylation Status
Acetylation of nucleosomal histones in part regulates gene transcription in most cells.

Differential acetylation of nucleosomal histones results in either transcriptional activation
(hyperacetylation and an open chromatin configuration) or repression (hypoacetylation and

compacted chromatin) [74, 75]. The role of chromatin remodeling in carcinogenesis was
studied with the help of inhibitors of HDACs (HDIs).
HDIs induce the hyperacetylation of nucleosomal histones in cells resulting in the
expression of aberrantly repressed genes (e.g., tumor suppressor genes) that produce growth
arrest, terminal differentiation, and/or apoptosis in carcinoma cells, depending on the HDI
and dose used, and the cell type [76-83]. The inappropriate recruitment of HDACs provides at
least one mechanism by which oncogenes could alter gene expression in favor of excessive
proliferation.
Thus, orally active HDIs with low toxicity towards normal cells and tissues, which would
effectively inhibit tumor growth are needed for epigenetic anticancer therapy. In October
2006, the US Food and Drug Administration (FDA) approved the first drug of this new class,
vorinostat (SAHA, Zolinza) for treatment of cutaneous T-cell lymphoma. Several further
HDIs (romidepsin, belinostat, sodium phenylbutyrate, valproic acid, entinostat, and
mocetinostat) are in clinical trials. HDIs have shown significant activity against a variety of
hematological and solid tumors at doses that are well tolerated by patients, both in
monotherapy as well as in combination therapy with other drugs. Combined DNA
methyltransferase and histone deacetylase inhibition are used in experiments in vitro but also
in clinical trials in MDS patients [10-17].
MicroRNAs and Epigenetic Machinery
MicroRNAs (miRs) belong to a class of small non-coding regulatory RNA that act
through binding to the 3 -UTR of target mRNA and leading to translational repression or
degradation of target mRNA at post-transcriptional level. MiRs can directly target epigenetic
effectors such as DNMTs, HDACs and polycomb repressive complexes. On the other hand,
some miRs (miR-9, 34b/c, 124, 127, 137, 145, 146a, 148, -203, let-7a-3, and others) are
epigenetically regulated [84- 90].
MiR-29b targets DNMT3A mRNA [91-93]. In addition, some isoforms of DNMT3B are
targeted by miR-148 [94]. MiR-26a, 101, 205 and -214 regulates EZH2 [95-100].
Dostalova Merkerova et al. found nine upregulated genes for miRs located at
chromosome 14q32 in CD34+ cells separated from mononuclear cells of bone marrow
obtained from MDS patients [101]. 14q32 region contains 40 miR genes with imprinted
expression controlled by a distant differentially methylated region. For example miR-127, a
member of the 14q32 region, is involved in B-cell differentiation process through
posttranscriptional regulation of BLIMP1, XBP1, and BCL6 genes [101]. BLIMP1 (B
lymphocyte induced maturation protein 1) is a zinc finger transcriptional repressor which
functions as a master regultor of terminal differentiation of B cells into plasma cells. XBP1
(X-box binding protein 1) is transcription factor that regulates MHC class II genes by binding
to a promoter element referred to as an X box. BCL6 (B-cell lymphoma 6 protein) is a
transcriptional represor which regulates Terminal center B cell differentiation and
inflammation.
146 Ota Fuchs
Protein EVI1 and Epigenetic Machinery
EVI1 (the ecotropic viral integration site 1) is encoded by gene on chromosome 3q26
[102-105]. The oncoprotein EVI1 and the DNMT3 co-operate in bindig and de novo
methylation of target DNA [106]. EVI1 forms a bridge between the epigenetic machinery and
signaling pathway [107, 108]. EVI1 represses PTEN (phosphatase and tensin homolog)
expression and activates PI3K/AKT (Protein kinase B)/mTOR via interaction with polycomb
proteins [107, 108]. Overexpression of EVI1 predicts poor survival in MDS and AML [109-
111]. MDS patients with inversion of chromosome 3 and with EVI1 transcriptional activation
achieved morphological and cytogenetic response to azacitidine [112].
Epigenetic Therapy in MDS
Treatment with DNMT inhibitors is a rational strategy with the aim to reinduce the
expression of epigenetically silenced genes for tumor suppressors and other targeted genes,
often connected with response to chemotherapy. Responses to therapy with DNMT inhibitors
are up to now not fully elucidated. We have no clear evidence for DNMT overexpression in
MDS and the decrease in global methylation after treatment with demethylating agents has
not correlated with disease response. Changes in differentiation and/or apoptosis, and
induction of a immune response can be also involved [8, 17, 113-119].
DNA methylation of upstream regulatory element (URE) plays an important role in
downregulation of transcription of PU.1 gene. PU.1 is the transcription factor and tumor
suppressor necessary for myeloid differentiation. Azacitidine treatment demethylated in vitro
URE leading to upregulation of PU.1 followed by derepression of its transcriptonal targets
and onset of myeloid differentiation [114]. DNA demethylation and a shift from a repressive
histone profile to a more active profile that includes the reassociation of RNA polymerase II
(Pol II) with the targeted promoters are necessary for tumor suppressor gene reactivation
[115].
Even if a complete understanding of the mechanism of action of azanucleotides remains
to be elucidated, their pharmacodynamic effects promote enhanced survival independently of
any ability to eliminate the MDS clone. The MDS clone persists in many patients treated by
DNMT inhibitors but this clone is modulated and hematologic function is improved together
with survival of patients.
5-Aza-cytidine (azacitidine, Vidaza) and 5-aza-2-deoxycytidine (5-azaCdR; decitabine,
Dacogen) have become the standard in the treatment of patients with higher-risk MDS, in
particular older individuals, where intensive chemotherapy and allogeneic stem cell
transplantation is not possible.
It has been almost 50 years since the synthesis and antitumor activity of azacitidine (AC)
was described [120, 121]. AC is a pyrimidine nucleoside analog of cytidine and is
characterized by a presence of an extra nitrogen atom at position C5 of pyrimidine ring. This
modification leads to a blockade of cytosine methylation via a covalent trapping of DNMT.
AC is believed to utilize a dual mechanism of action following its phosphorylation: 1)
hypomethylation of DNA at low doses and 2) cytotoxicity due to the incorporation into RNA
and apparent interaction with protein biosynthesis at high doses [122-127]. To overcome
cytotoxicity, a deoxy analog of AC, 5-aza-2-deoxycytidine (decitabine, DAC) was
synthesized, which is incorporated only into the DNA following its phosphorylation [128-
130]. DAC significantly inhibits DNA methylation at lower concentrations and with less
cytotoxicity in comparison with AC [131]. Both, AC and DAC, possess high cytotoxicity at
their maximal tolerated doses and are unstable in aqueous solution [132, 133].
Azacitidine (Vidaza, Celgene Corporation, Summit, NJ, USA or Celgene Europe Ltd.,
Winsdor, UK) was the first drug approved for the treatment of MDS in the United States and
in the European Union [8, 134, 135]. Decitabine (Dacogen, Eisai Inc., Woodcliff Lake, NJ,
USA under license from Astex Pharmaceuticals, Inc., Dublin, CA, USA) received initial
regulatory approval from the US Food and Drug Administration (FDA) in May 2006 for the
treatment of patients with all MDS subtypes. Since then, decitabine has also gained regulatory
approval in Russia, Malaysia, South Korea, the Philippines, Uruguay, Chile, Argentina, Peru,
Colombia, and Brazil, and is considered for approval in other countries [136, 137]. European
Organisation for Research and Treatment of Cancer (EORTC) conducted study, which failed
to reveal a significant improvement in overall survival, time to AML transformation and
death, for low-dose decitabine compared to the best supportive care [138].
Azacitidine Clinical Studies
Azacitidine has been studied in higher-risk MDS in two major randomized multicenter
trials, Cancer and Leukemia Group B (CALGB) 9221 and AZA-001 [139, 140].
Patients with MDS were randomly treated with either azacitidine or best supportive care
in the CALGB 9221 study [8, 134, 135, 139]. A total of 191 patients with a median age of 68
years were used. Azacitidine (AC,75 mg/m2/day) was injected subcutaneously in 7-day cycles
beginning on days 1, 29, 57, and 85 (every 28 days). If a beneficial effect was not
demonstrated by day 57 and no significant toxicity other than nausea or vomiting had
occured, the dose of AC was increased by 33%. Once benefit occured on a particular dosage,
AC was continued unless toxicity developed. Patients were assessed after the fourth cycle.
Those who achieved complete response (CR) continued on AC until either CR or relapse
occured. After 4 months of supportive care, any patients with worsening dinase were
permitted to cross over to treatment with AC. Overall, 59% of patients had either refractory
anemia with excess blasts (RAEB) or RAEB in transformation (RAEB-T) according to
French-American-British (FAB)-defined criteria, and 65% of patients were red blood cell
transfusion dependent. Sixty percent of patients in the azacitidine arm (including 7% of
patients with CR, 16% with a partial response /PR/, and 37% with hematologic improvement
/HI/), compared with 5% of patients in the control arm, responded to treatment (P0.001).
The median time to leukemic transformation or death was 21 months in patients treated with
AC compared with 12 months in the best supportive care (P = 0.007). The median overall
survival was 20 months for AC-treated patients compared with 14 months for patients
assigned to best supportive care. 53% of patients on best supportive care received azacitidine
after crossover. A further benefit of AC over supportive care was a significant improvement
in quality of life (physical functioning, fatigue, dyspnea) in patients treated with AC
compared with patients in the control arm [141]. AC did not increase the rate of infection or
gastrointestinal bleeding above the rate associated with underlying disease [142].
The AZA-001 trial was an international, randomized phase III study designed to test the
hypothesis that AC significantly extends overall survival in patients with MDS compared
148 Ota Fuchs
with standard care regimens including best supportive care, low-dose cytarabine (ara-C, 20
mg/m2 for 14 days every 28 days for at least 4 cycles), or intensive chemotherapy consisting
of induction with higher dose of ara-C (100-200 mg/m2/day for 7 days plus 3 days of
daunorubicin 45-60 mg/m2/day, idarubicin 9-12 mg/m2/day, or mitoxantrone 8-12
mg/m2/day) [8, 134, 135, 140]. A total of 358 patients with higher-risk MDS were randomly
assigned to either azacitidine as in CALGB 9221 or to standard of care. Median age of
patients was 69 years. After a median follow-up of 21.1 months, the median survival time was
significantly better in azacitidine patients compared with standard of care options (24.5 versus
15.0 months, respectively; P = 0.001) irrespective of age, percentage of marrow blasts or
karyotype. In particular, overall survival was prolonged for azacitidine in patients with -7 /
del(7q) cytogenetic abnormality, median overall survival was 13.1 months in the azacitidine
group compared with 4.6 months in the standard of care group (P = 0.00017) [140].
Progression to AML was significantly delayed in patients treated with AC (17.8 months in the
AC group versus 11.5 months in the standard of care group; P0.001). Transfusion
requirements and rate of infections were also significantly improved in azacitidine patients.
Continued azacitidine therapy beyond time of first response improves quality of response
in patients with higher-risk myelodysplastic syndromes in 48% of patients [21, 143]. This
secondary analysis of the AZA-001 phase III study evaluated the time to first response and
the potential benefit of continued AC treatment beyond first response in responders. Overal,
91 of 179 patients achieved a response to azacitidine; responding patients received a median
of 14 treatment cycles (range, 2-30). Median time to first response was 2 cycles (range, 1-16).
Although 91% of first responses occured by 6 cycles, continued azacitidine improved
response in 48% of patients. Best response was achieved by 92% of responders by 12 cycles.
Median time from first response to best response was 3.5 cycles (95% confidence interval
(CI), 3.0-6.0) in 30 patients who ultimately achieved a complete response, and 3.0 cycles
(95% CI, 1.0-3.0) in 21 patients who achieved a partial response.
French group studied a retrospective cohort of 282 higher-risk MDS treated with
azacitidine, including 32 patients who concomitantly received erythropoiesis stimulating
agents (ESA) for a median of 5.8 months after azacitidine onset [144]. Hematologic
improvement was reached in 44% of the ESA and 29% of the no-ESA patients. Transfusion
independence was achieved in 48% of the ESA and 20% of the no-ESA groups. Median
overall survival was 19.6 months in the ESA and 11.9 months in the no-ESA patients.
Platelet doubling after the first azacitidine cycle is a promising independent predictor for
response and overall survival in MDS, chronic myelomonocytic leukemia (CMML) and AML
patients in the Dutch azacitidine patients [145].
Decitabine Clinical Studies
Two studies used 3-day, 9-dose regimens requiring inpatient hospitalization [146, 147]
and two studies used 5-10 day decitabine regimens intended for outpatient administration
[148, 149].
The US D-0007 phase III study compared decitabine (15 mg/m2 continuous 3-hour
intravenous infusion every 8 hours for 3 days) with supportive care in 170 patients with a
confirmed diagnosisof de novo or secondary MDS.
The median age of enrolled patients was 70 years (range, 30-85 years). Most patients
(69%) had intermediate (Int)-2- or high- risk diseaseas defined by the International Prognostic
Scoring System criteria, and were red blood cell transfusion dependent (71%) [146]. No
significant difference was seen in median overall survival (OS) between patients treated with
decitabine and those receiving supportive care (14.0 versus 14.9 months, respectively; P =
0.636). The median duration of response to decitabine treatment was 10.3 months (range, 4.1-
13.9 months). Patients received a median of 3 courses of decitabine treatment
(range, 0-9).
EORTC 06011 phase III study compared decitabine given on a 3-day inpatient regimen
(15 mg/m2 intravenously over 4 hours three times a day for 3 days, every 6 weeks, for a
maximum of 8 cycles) with supportive care. A total of 233 patients with primary or secondary
MDS, or CMML defined by FAB classification (median age, 70 years; range, 60 to 90 years)
were enrolled [147]. 53% had poor-risk cytogenetics, and the median MDS duration at
random assignment was 3 months. The median OS prolongation with decitabine versus best
supportive care was not statistically significant (median OS, 10.1 versus 8.5 months; P =
0.38).
M.D. Anderson Cancer Center ID03-0180 randomized phase II study compared three
outpatient decitabine schedules [148]. In this single-institution study, 95 patients (77 with
MDS, and 18 with CMML) were randomized to receive 20 mg/m2/day intravenously for 5
days, 20 mg/m2/day subcutaneously for 5 days, or 10 mg/m2/day intravenously for 10 days.
Thus, all patients received the same 100 mg/m2 total decitabine dose in each treatment cycle.
Overall, 32 patients (34%) achieved CR and 69 patients (73%) had an objective response.
The 5-day intravenous schedule, which had the highest dose-intensity, was selected as
optimal. The CR rate in that arm was 39% compared with 21% in the 5-day subcutaneous arm
and 24% in the 10-day intravenous arm (P < 0.05). The high dose-intensity arm (the 5-day
intravenous schedule) was also superior at inducing hypomethylation at day 5 and at
activating the expression of the cell cycle inhibitor p15INK4B at days 12 or 28 after therapy.
The 5-day intravenous schedule of decitabine optimizes epigenetic modulation and clinical
responses in MDS.
North American multicenter DACO-020 ADOPT phase II study [149] started on the
results of the study of M.D. Anderson Cancer Center [148] dealing with efficacy and safety of
decitabine in the 5-day intravenous schedule, every 4 weeks in 99 patients with MDS (de
novo or secondary). The primary end point was the overall response rate (ORR) by
International Working Group criteria [150]. Secondary end points included cytogenetic
responses, hematological improvement, response duration, survival and safety. The ORR was
32% (17 complete responses plus 15 marrow complete responses and the overall
improvement rate was 51%, which included 18% of hematologic improvement. Decitabine
can be administered in an outpatient petting with comparable efficiacy and safety to the US
FDA- approved impatient regimen.
Comparison of Azacitidine and Decitabine
Azacitidine and decitabine appear to have similar administration costs. As far as adverse
events azacitidine is well tolerated [8, 134, 135, 140]. Grade 3 and 4 neutropenia was
observed for 91% patients in the azacitidine treated group, and 76% in the best conventional
150 Ota Fuchs
care group. Grade 3 and 4 thrombocytopenia occurred among 85% of patients in the
azacitidine treated group and 80% in the best conventional care group. Higher risk of febrile
neutropenia (23%) was described in the US D-0007 phase III decitabine study. In this study
87% of neutropenia and 85% of thrombocytopenia in response to decitabine were reported. In
AZA-001 study the median OS was 24.5 months for azacitidine compared with 15.0 month
for the best conventional care [140]. Decitabine has not demonstrated a survival advantage
compared with the best conventional care (14.0 versus 14.9 months) [137, 146]. In the
EORTC 06011 study, the median OS was 10.1 months for decitabine and 8.5 months for
supportive care [147]. Comparing results from different studies suggests similar median
number of cycles to first response for azicitidine (2.3 cycles/64 days/ in CALGB 9221 study)
and decitabine (2 cycles/3.3 months/ in the D-0007 study and 2 cycles/2 months/in DACO-
020 ADOPT trial) [139, 147, 149]. For azacitidine treated patients, the median duration of
response was 15 months in CALGB 9221 study [139] and 13.6 months in AZA-001 trial
[140]. For decitabine treated patients, the median duration of response ranged from 8.6
months for EORTC 06011 study [147], 10 months in the DACO-020 ADOPT trial, to 10.3
months in the D-0007 study [146].
Continued exposure to hypomethylating agents is required to maintain response, and
relapse occurs within months of azacitidine or decitabine withdrawal in most cases [151].
Response to hypomethylating agents is difficult to predict with routine clinical variables and
here is the need for biomarkers. Outcome of patients after demethylating agents failure is very
poor [152].
Biomarkers of Sensitivity to Hypomethylating Agents
Only 40% to 50% of patients treated with either azacitidine or decitabine experience
hematologic improvement with these agents, and complete responses occur in as few as10%
to15% of treated patients. A number of research groups have focused on the identification of
methylation patterns that would predict for response in MDS. No such profile exists. Baseline
methylation patterns were not associated with response to hypomethylating agents [153, 154].
The significant correlation was observed between reduced methylation over time and clinical
outcome [153]. Further studies of methylation dynamics both before and after treatment with
hypomethylating agents will be useful to determine the ability of these markers to direct
treatment. DNA methylation of upstream regulatory element (URE) controlling the
transcription of PU.1 gene may be a new biomarker for the prediction which patiens will
benefit from therapy by hypomethylating agents [114].
Several other biomarkers, such as mutations in TET2 gene and levels of miR-29b have
been reported to be associated with responses to azacitidine and decitabine, respectively [155-
158]. TET2 is a protein involved in the conversion of 5-methylcytosine to 5-
hydroxymethylcytosine and could therefore result in passive induction of DNA methylation
[159]. TET2 mutations were recently reported to be associated with clinical response to
azacitidine but not with survival [155, 156]. Contradictory results were obtained by another
research group [157]. TET2 mutations were described in 15% of 86 patients [155]. The
response rate to azacitidine was 82% in the mutated patients and 45% in the nonmutated
patients with wild TET2 gene. Mutated TET2 (P = 0.04) and favorable cytogenetic risk
(intermediate risk: P = 0.04, poor risk: P = 0.048 compared with good risk) independently
predicted a higher response rate. TET2 status may be a genetic predictor of response to
azacitidine, independently of karyotype. Traina et al. [160] showed that higher platelet counts,
lower WBC counts and the presence of TET2 and/or DNMT3A mutations are associated with
better treatment response. Bejar et al. [45] hypothesized that mutations of individual genes
may serve as biomarkers of response for MDS patients treated with hypomethylating agents.
They used massively parallel sequencing to examine 40 recurrently mutated genes in disease
samples from 213 MDS patients treated with azacitidine or decitabine. They examined the
association of mutational patterns with response to treatment and overall survival. They used
competitive murine bone marrow transplant model to test sensitivity of TET2-null and TET2-
wild type hematopoietic cells to treatment with azacitidine. Bejar et al. [45] confirmed the
results of Itzykson et al. [155] who previously reported that 82% MDS patients with TET2
mutations detected by Sanger sequencing respond to treatment with azacitidine. The
mechanism by which TET2 mutations might influence response to hypomethylating agents is
not clear. No pattern of mutation was strongly associated with a lack of response to treatment.
Studies examining samples collected at multiple time points are needed to identify mutations
predictive of acquired resistance or relapsed disease. Stopka et al. [161] observed mutation
status of the 54 myeloid genes. ASXL1 gene became abundantly mutated upon azacitidine
treatment (from 5% to 25%). Negative prognostic significance of association of TP53
mutations with anny of 4 additional genes (RUNX1, ASXL1, EZH2 or ETV6) mutations on
overall survival was demonstrated by Bejar et al. [45] but not in the case of mutations in
individual genes without TP53 mutations (wild type TP53 gene).
Stopka et al. [161] did not confirmed this observation and showed that only ASXL1 gene
mutations associate with significantly shorter overall survival in azacitidine-treated patients.
Expression levels of DNMT1, an enzyme involved in maintenance of methylation patterns,
are regulated by miR-29b [158]. Higher levels of miR-29b were associated with clinical
response to decitabine [158].
Kuendgen et al. [162] analyzed 114 cases (77 patients with MDS, 30 AML and 7
MDS/MPD) treated with azacitidine. Responses were observed in 53 patients (46%).
Azacitidine led to comparable responses in most cytogenetic subgroups, exhibiting broad
efficacy. Only del(20q) led to a higher response rate, requiring confirmatory analyses.
RUNX1 mutations showed an intersting trend to higher response. Lower response rates were
only seen in numerically small subgroups, although for EZH2 mutation the p-value was <0.1.
In patients with TP53 mutations responses are frequent, but the negative impact on survival is
retained, while this is not the case for ASXL1 mutations.
Bally et al. [163, 164] showed that the therapeutic benefit obtained by azacitidine in
patients with TP53 mutations was short lasting suggesting that the precise mechanisms
involved in response to azacitidine are not completly understood. Apoptosis could be a key
determinant of azacitidine mechanism of action [165] and ayacitine fails to eradicate
leukemic stem/progenitor cell populations suggesting that a combined treatment using new
drugs that target this cellular population in vivo are possibly required [166].
Circulating cell-free DNAs from plasma and serum of patients with MDS can be used to
detect genetic and epigenetic abnormalities [167]. The plasma DNA concentration was found
to be relatively high in patients with higher blast cell counts in bone marrow.
Reliable clinical or molecular predictors of benefit from azacitidine therapy in patients
with MDS are not defined. Doubling of platelet count at start of second cycle of azacitidine
therapy compared to baseline was associated with achieving response and survival advantage
152 Ota Fuchs
in a Dutch cohort [168]. To validate this observation, Zeidan et al. [169] analysed a larger
cohort of North American patients, whose data was collected in a prospective clinical trial
with a longer median follow-up. We found a significant association between platelet count
doubling after first cycle of azacitidine therapy and probability of achieving objective
response. Among patients with MDS or oligoblastic acute myeloid leukaemia (< 30% bone
marrow blasts, n = 102), there was a statistically significant reduction in risk of death for
patients who achieved platelet count doubling (n = 23, median OS, 21.0 months) compared to
those who did not (n = 79, median OS, 16.7 months, adjusted hazard ratio (no/yes) = 1.88,
95% confidence interval, 1.03-3.40, P = 0.04). Nonetheless, the addition of this platelet count
doubling variable did not improve the survival prediction provided by the revised
International Prognostic Scoring System or the French Prognostic Scoring System.
Identification of reliable and consistent predictors for clinical benefit for azacitidine therapy
remains an unmet medical need and a top research priority.
Resistance to Hypomethylating Agents
There is a subgroup of patients with MDS who do not respond to therapy with
hypomethylating agents and a large, growing cohort of patients that lose progress while on
azacidine or decitabine therapy [170]. Since the mechanism of resistance to hypomethylating
agents are not known, selection of therapy is largely empiric but must take into account the
age, comorbidities, and performance status of the patient, as well as the characteristics of the
disease at the time of treatment failure. Higher intensity approaches and allogeneic stem cell
transplantation can yield improved response rates and long-term disease control but should be
limited to a selected cohort of patients who can tolerate the treatment-related morbidities. For
the majority of patients who likely will be better candidates for lower intensity therapy,
several novel, investigational approaches are becoming available. Among these are newer
nucleoside analogues, inhibitors of protein tyrosine kinases, molecules that interact with
redox signaling within the cell, immunotherapy approaches, and others.
In clinical trials, some patients do not respond to hypomethylating agents initially
(primary resistance) and most patients who initially respond to treatment, eventually relapse
(secondary resistance) despite continued therapy with hypomethylating agents. Most primary
mechanisms of resistance are based on metabolic pathways. The primary resistance is caused
by the insufficient intracellular concentration of nucleoside triphosphates resulting from
deoxycytidine kinase deficiency (DCK mutations or aberrant gene expression), increased
deamination by cytidine deaminase (CDA), or high dNTP pools. Higher ratio of CDA/DCK
in a subset of patients means that decitabine is less activated through mono-phosphorylation
by DCK and more inactivated through deamination by CDA in non-responders. Secondary
resistance is likely due alternate progression pathways as a less aberrant DNA methylation
was found during the treatment with hypomethylating agent than at diagnosis, and there were
no significant changes in decitabine metabolism gene expression [171].
Wu et al. [172] measured the mRNA expression of metabolism (humaman equilibrative
nucleoside transporter 1/hENT1/, hENT2, DCK and CDA) and apoptosis (BCL2L10) genes
and found that the hENT1 mRNA level was significantly higher in response compared with
non-response patients (P = 0.004). Furthermore, the DCK mRNA level was significantly
reduced for relapse (P = 0.012) compared with those with continued marrow complete
remission. These findings indicate that the decitabine metabolic pathway affects its
therapeutic effects, lower hENT1 expression may induce primary resistence and down-
regulated DCK expression may be related to secondary resistence.
Epigenetic Regulation of Mesenchymal Stromal Cells
Hematopoietic stem cells (HSC) reside in the bone marrow in a specific niche where
interaction with mesenchymal stromal cells (MSC) governs quiescence, proliferation and fate
decisions of HSC [173]. It has recently been shown that diseased HSC in low-risk MDS
reprogram MSC to remodel the niche [174]. Increasing evidence also suggests that an altered
stromal microenvironment in MDS contributes to ineffective hematopoiesis. Azacitidine is
currently the standard treatment for higher-risk MDS. Azacitidin induces direct cytotoxicity
to cancer cells and inhibits DNA methyltransferase, allowing reversal of epigenetically
silenced genes, thus restoring impaired hematopoiesis.
Huberle et al. [175] asked whether treatment with azacitidine also exerts a direct effect on
the stromal microenvironment in MDS. They examined the effect of azacitidine on primary
MSC in vitro as wellas the functional interaction between MSC and HSC after azacitidine
treatment. Primary MSC were isolated from bone marrow, cultured under defined conditions
until confluency and used up to 4 passages. Identity of MSC was confirmed by presence of
characteristic cell surface markers by flow cytometry. MSC from MDS patients showed
reduced proliferation potential and altered morphology compared to healthy MSC. Treatment
with azacitidine had no effect on cell surface marker expression of MSC. Annexin/propidium
iodide staining determined that azacitidin did not affect viability or healthy of MDS MSC up
to a concentration of 100 M. Analysis of growth kinetics revealed decreased proliferative
capacity of healthy MSC atb 100 M azacitidine while MDS-MSC were more sensitive, with
decreased growth at 10 M azacitidine. Azacitidine significantly altered the ability of MDS-
MSC to differentiate towards the adipogenic lineage while osteogenic differentiation was not
affected. Gene expression and secretion of epigenetically regulated cytokines osteopontin
(OPN) [176, 177] and stromal cell-derived factor 1 (SDF1) were significantly decreased upon
azacitidine treatment of MDS-MSC. Co-culture experiments showed that azacitidine
pretreatment of MSC led to increased apoptosis of MDS CD34+ hematopoietic cells and
inhibited short-term and long-term hematopoietic colony formation. This effect on CD34+
progenitors was recapitulated by addition of neutralizing OPN antibody to untreated MSC.
Notch gene expression was upregulated in MDS CD34+ cells but not in healthy progenitors
after contact with azacitidine pretreated MSC. Therefore, azacitidine directlys modulates
MSC function and alters nichecomposition leading to suppression of hematopoietic
progenitors in MDS. These data provide evidence that epigenetic regulation of MSC plays a
role in MDS and suggest an additional mode of action by which azacitidine exerts its
therapeutic activity.
Immunomodulatory Effect of Hypomethylating Agents
Several authors studied the consequences of hypomethylating agents on the immune

system of patients with MDS or AML. Treatment with azacitidine alleviates auto-immune
154 Ota Fuchs
MDS-related manifestations [178] and patients treated with a combination of azacitidine and
donor lymphocyte infusions (DLI) after hematopoietic stem cell transplantation (HSCT) seem
to be less frequently subject to aggressive graft-versus-host disease (GVHD) [179, 180].
These findings, as well as data from murine models [181, 182] showed that azacitidine has
immunomodulatory properties against GVHD. Enhanced generation of regulatory T cells
(Tregs) through demethylation of the FOXP3 receptor could be involved [181, 182]. A
similar induction of Tregs has been noted in patients receiving HMA after HSCT [183, 184],
though the functionality of these expanded Tregs is debated [185, 186].
Hypomethylating agents also induce anti-tumoral immune responses by restoring the
expression of tumor antigens at the surface of MDS cells. They induce for instance CD8+ T
cell response to cancer testis antigens in MDS/AML patients [187, 188].
MDS and AML patients with multilineage dysplasia show several immunological
abnormalities. In this clinical setting, by combining flow cytometry and CDR3 spectra typing
Fozza et al. [189] monitored the kinetic of the T-cell repertoire during azacitidine treatment,
in order to explore its potential ability to reverse the immune derangement typical of these
disorders. They firstly demonstrated by flow cytometry an increase in both CD4+ and CD8+
T-cell frequencies after starting treatment. Moreover, when monitored by spectra typing their
patients showed significant changes in their T-cell receptor (TCR) CDR3 profiles, which
were much more evident in helper T-cells. In fact, the frequency of BV (beta variable)
subfamilies showing a skewed CDR3 profile significantly decreased from baseline to the
following evaluations in CD4+ T-cells (81% vs. 70%). This pattern was even more
pronounced in patients responding to azacitidine (90% vs. 61%). Our data show that the
overall derangement of the T-cell repertoire detectable in patients with MDS and AML with
multilineage dysplasia gradually improves during azacitidine treatment. These findings
therefore suggest that azacitidine could be potentially able, not only to restore the
hematopoietic function, but also to reverse the immune derangement typical of these
hematologic disorders.
Hypomethylating agents can be double-edged swords in anti-tumoral immune responses
[190]. Azacitidine demethylates the promoter of PD-1 on T cells, restoring expression of this
auto-inhibitory eceptor, potentially leading to exhaustion of anti-tumoral T-cell mediated
responses [191, 192].
New Hypomethylating Agents
Current hypomethylating agents are limited due to route of administration and potency as
inducers of DNA hypomethylation. An oral compound or an agent with a better
pharmacodynamic profile could improve hypomethylating therapy of MDS patients. Initial
results with an oral formulation of 5-azacytidine have been reported [193].
2, 3, 5-triacetyl-5-azacytidine demonstrates significant pharmacokinetic improvements
in bioavailability, solubility, and stability over the parent compound 5-azacytidine [159]. In
vivo analyses indicated a lack of general toxicity coupled with significantly improved
survival. Pharmacodynamic analyses confirmed its ability to suppress global methylation in
vivo. Esterified nucleoside derivatives may be effective prodrugs for azacitidine and
encourages further investigation and possible clinical evaluation [194].
A new salt derivative, oral decitabine mesylate, is used in ongoing trials. A barrier to
efficacious and accessible DNMT1-targeted therapy is cytidine deaminase, an enzyme highly
expressed in the intestine and liver that rapidly metabolizes decitabine into inactive uridine
counterparts, severely limiting exposure time and oral bioavailability. Oral administration of
3,4,5,6-tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase, before oral
decitabine extended decitabine absorption time in mice and nonhuman primates and widened
the concentration-time profile [195]. Therefore, the exposure time for S-phase-specific
depletion of DNMT1 is increased without the high peak of decitabine levels that can cause
DNA damage and cytotoxicity [195].
5,6-dihydro-5-azacytidine (DHAC) and 2-deoxy-5,6-dihydro-5-azacytidine (DHDAC)
are hydrolytically stable. There is no evidence of significant genotoxicity and/or
mitochondrial toxicity on mammalian cells [196]. Both compounds are a less toxic alternative
of azacitidine and decitabine and may also be of therapeutic interest.
Another compounds actively being studied in clinical trials are SGI-110 and CP-4200, a
second generation hypomethylating agents [197]. One of the limitations of the nucleoside
analogues in the clinical trials has been the side effects, such as thrombocytopenia and
neutropenia, which are probably caused by cytotoxic effects associated with the drugs
incorporation into the DNA or RNA independently of their DNA hypomethylation value.
This has encouraged the search for inhibitors of DNA methylation that are not incorporated
into DNA or RNA.
Zebularine is a cytidine deaminase inhibitor [198-201] that also displays antitumor and
DNA demethylating properties [198]. Zebularine is a cytidine analog that contains a 2-(1H)-
pyrimidinone ring [199].
The drug procainamide, approved by the FDA for the treatment of cardiac arrythmias,
and procaine, a drug approved by the FDA for use as a local anesthetic, were proposed as
non-nucleoside inhibitors of DNA methylation [202]. This action is thought to be mediated
by their binding to GC-rich DNA sequences. Both, procaine and procainamide, are
derivatives of 4-amino-benzoic acid.
Dietary phytochemicals, tea catechins, polyphenols, particularly (-)-epigallocatechin-3-
gallate decreased the levels of 5-methylcytosine, DNMTs activity, mRNA and protein levels
of DNMT1, DNMT3A and DNMT3B and also decreased histone deacetylase aktivity and
stimulated re-expression of the mRNA and proteins of silenced tumor suppressor genes [203].
N-phthalyl-L-tryptophan (RG108) and its dicyclo-hexyl-amine salt effectively blocked
DNA methyltransferases in vitro and did not cause covalent enzyme tramping in human cell
line [204, 205]. Incubation of cells with RG108 resulted in signifiant demethylation of
genomic DNA without any detectable toxicity. RG108 caused demethylation and reactivation
of tumor suppressor genes.
Combinations of Hypomethylating Agents with Histone Deacetylase

Inhibitors or Other Drugs
In vitro, most of histone deacetylase inhibitors (HDACI) have been shown to have
synergistic activity when combined with either azacitidine or decitabine. Therefore, phase I
and II clinical trials were performed. Combination of decitabine and valproic acid was safe
and active and time to response was accelerated [12]. A randomized phase II trial has been
156 Ota Fuchs
conducted at M.D. Anderson Cancer Center comparing decitabine versus decitabine in

combination with valproic acid [136]. Results did not show any significant benefit with the
combination of decitabine and valproic acid. Sometimes, ATRA (all-trans retinoic acid) was
added in these combinations [206]. Adding valproic acid to decitabine was not associated
with improved outcome in the treatment of patients with MDS or elderly patients with AML.
Future therapies may consider combining hypomethylating agents with better HDAC
inhibitors and using different schedules [207]. Similar results were obtained with combination
of azacitidine and further potent HDAC inhibitors, MS-275 (Entinostat), suberoylanilide
hydroxamic acid (SAHA, Vorinostat), a benzamide HDACI (Mocetinostat, MGCD0103,
clinical trial NCT02018926) [136, 206].
Phase I trial for combination of lenalidomide with azacitidine has shown this combination
to be very safe and clinically active in MDS [208]. In this study 18 patients (2 intermediate
1-, 10 intermediate 2- and 6 high- risk) were enrolled with median age 68 years (range, 52 to
78 years). Interval from diagnosis was 5 weeks (range, 2 to 106 weeks) and follow-up was 7
months (range, 1 to 26 months). Azacitidine (75 mg/m2/day) on days 1 through 5 and
lenalidomide (10 mg) on days 1 through 21 were used. The combination of lenalidomide and
azacitidine is well tolerated with encouraging clinical activity. Therefore, phase II multicenter
study of the azacitidine (75 mg/m2/d for 5 days) and lenalidomide (10 mg/d for 21 days; 28-
day cycle) combination in patients with 36 higher-risk MDS was performed [209]. The overal
response rate was 72%; 16 patients (44%) achieved a complete response (CR), and 10 (28%)
had hematologic improvement. Median CR duration was 17 + months; median overal survival
was 37 + months. TET2/ DNMT3A/ IDH1/2 mutational status was associated with response.
This clinical trial was registered as NCT00352001. Currently, combination therapies of
azacitidine with lenalidomide appear to be promising thus making them an appealing option
for treatment in these patients [210-213].

The aim of current clinical research of hypomethylating agents for MDS patients is to
achieve more continuous hypomethylation without undue toxicity, and combining
hypomethylating drugs with other drug classes [12, 136, 190, 206-213]. Azacitidine prolongs
survival by a median of only 9.5 months. Therefore, novel therapeutic approaches are clearly
needed. Despite the encouraging results with azacitidine and decitabine, it is obvious that it
will be important to have access to second generation agents with the capacity to increase
faster early response rates with acceptable toxicity profiles. Preliminary results with oral
formulation of both azacitine and decitabine are promising and these forms could improve
hypomethylating therapy of MDS patients in future. Another approach is to develop
combination strategies using either azacitidine or decitabine. Several such approaches are
currently studied and many are promising but not yet fully understood. Including of cytidine
deaminase inhibitor in these combinations appears to be important for better results but it
needs new clinical studies. A strictly targeted approach is the use of an orally available,
selective, potent inhibitor of the mutated IDH2 protein, AG-221 [214]. However, IDH1/IDH2
mutations are relatively rare in MDS, with a frequency of 4-12%, and confer gain of function
to mutated cells. Oncometabolite, 2-hydroxyglutarate, is accumulated and inhibits histone and

DNA demethylases and determines block of differentiation.
Currently, no standard care regimens have been established for patients after failure of
hypomethylating agents. Santini et al. [215] in their review discussed treatment options after
loss of response or progression on azacitidine. In addition, they described optimization of
first-line treatment along with potential biomarkers for identifying and monitoring response
during treatment with azacitidine.
ACKNOWLEDGMENTS
Czech Republic and by the project (Ministry of Health, Czech Republic) for conceptual
development of research organization (Institute of Hematology and Blood Transfusion,
Prague).
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Chapter 7
INTRODUCTION OF MILD ORAL CHEMOTHERAPY

FOR ELDERLY PATIENTS WITH HIGHER-RISK
Akira Horikoshi
Department of General Internal Medicine,
Nerima-Hikarigaoka Hospital, Tokyo, Japan
ABSTRACT
Myelodysplastic syndromes (MDS) are primarily a group of acquired bone marrow
disorders that occur in the elderly in whom many factors indicate a poor prognosis. In
particular, MDS patients with intermediate-2 or high-risk scores on the international
prognosis scoring system, corresponding to higher-risk MDS, show a very poor
prognosis.
Recent studies have indicated that azacitidine, a hypomethylating agent, as compared
to conventional care regimens for the treatment of higher-risk MDS patients, who are
ineligible for intensive treatment, remarkably improves overall survival. However, the
duration of the median treatment was long (13 months, 9 cycles) and the cost was high.
Because the number of elderly patients with higher-risk MDS is increasing in Japan,
we developed a novel mild therapeutic approach that is convenient and useful for such
patients. In this study, this oral chemotherapy regimen consisted of cytarabine ocfosfate
(1--D-arabinofuranocylcytosine-5-stearylphosphate) and etoposide. The data presented
here were obtained from several of our previous studies. In this trial for elderly patients
with higher-risk MDS, the therapy response rate was 60%, the median survival time in
the responsive group was 857 days, and adverse effects were not observed. Furthermore,
this regimen could be used for the treatment of elderly patients with acute myeloid
leukemia (AML; de novo AML and AML following MDS). The efficacy of this regimen
for additional 3 cases treated after completion of the previous studies was described. Our
results suggest that this therapy is well tolerated, cost-effective, and useful for the
treatment of elderly patients with higher-risk MDS.
176 Akira Horikoshi
INTRODUCTION
The incidence and prevalence of cancer increase with age. Myelodysplastic syndromes
(MDS) are a group of acquired bone marrow disorders that primarily occur in the elderly in
whom many factors indicate a poor prognosis. These include a poor performance status, being
immunocompromised, having a higher proportion of unfavorable karyotypes, and decreased
functional reserve of normal organ systems. In particular, MDS patients with intermediate-2
or high-risk scores on the International Prognostic Scoring System (known as higher-risk
MDS) show a very poor prognosis [1].
Recent studies have indicated that azacitidine, a hypomethylating agent, remarkably
improves overall survival as compared to conventional care regimens for the treatment of
higher-risk MDS patients [2-4]. However, the median duration of treatment was long and the
cost was high. Because the number of elderly patients with higher-risk MDS is increasing in
Japan, we developed a novel oral chemotherapeutic approach that is convenient and useful for
such patients. Here, we describe the treatment and the results based on several of our previous
studies.
TREATMENT DESIGN
Patient eligibility and study design have been reported previously [5]. In brief, patients
were eligible if they were over 65 years of age with higher-risk MDS, had refractory anemia
with excess of blasts (RAEB), or RAEB in transformation (RAEB-T) as defined by the
French-American-British (FAB) criteria [6], or had acute myeloid leukemia (AML)
diagnosed according to the revised FAB criteria [7]. Patients with acute promyelocytic
leukemia were excluded.
Between 1997 and 2008, we enrolled 4 patients with RAEB and 6 patients with RAEB-T
(5 men and 5 women), as well as 12 patients with AML (8 men and 4 women). After
completion of the study, we encountered additional eligible patients: 2 patients with RAEB-
T and 1 patient with AML evolving from MDS. All these patients were men.
Figure 1. Structural formula of SPAC (cytarabine ocfosfate). MW = Molecular weight.

Introduction of Mild Oral Chemotherapy for Elderly Patients 177
Table 1. Clinical and laboratory characteristics of MDS patients
*1: congestive heart failure *2: diabetes mellitus *3: hypertension.
A cytogenetic study was performed as described previously [5]. To assess response to

treatment, we applied standardized response criteria outlined previously [8] for MDS patients.
In AML patients, complete remission (CR), and partial remission (PR) were defined as
described in a previous report [9].
Subjects received oral induction therapy with cytarabine ocfosfate (SPAC: 1--D-
arabinofuranocylcytosine-5-stearylphosphate) (Figure 1) at 300 mg/day and etoposide (EP)
at 50 mg/day after meals 2 times daily for 14 days. The therapy period was shortened, if a
patients performance status (PS) worsened and severe cytopenia occurred. The response was
evaluated following 1 cycle of this regimen (induction therapy). Some patients received
additional SPAC and EP treatment after induction therapy depending on their clinical
condition.
Clinical features and therapy outcomes were evaluated for their prognostic value for
overall survival (OS) using Kaplan-Meier curves. Survival curves were compared using the
log-rank test and the generalized Wilcoxon test.
RESULTS
Clinical and biological characteristics of a group of 10 higher-risk MDS patients and 12
AML patients are shown in Tables 1 and 2, respectively which are reproduced from our
previous report [5]. The median ages of the MDS group and the AML group were 76 years
and 70 years, respectively. Three 90 year old men with AML were included. The patients had
178 Akira Horikoshi
a variety of complications, and some patients had poor PS scores. Abnormal karyotypes
including complex abnormalities were frequently detected in both groups.
Table 2. Clinical and laboratory characteristics of AML patients
*1: hypoplastic leukemia *2: relapse *3: ubenimex *4: hydroxyurea *5: daunorubicin, one injection *6:
L-asparginase+vincristine+prednisolone *7:
cyclophosphamide+doxorubicin+vincristine+prednisolone *8: ara-C+aclarubicine *9: ara-
C+aclarubicine+G-CSF *10: hypertension *11: hyperlipidemia *12: diabetes mellitus *13: non-
Hodgkin lymphoma *14: atrial fibrillation*15: disseminated intravascular coagulation *16: old
myocardial infarction *17: congestive heart failure
Response and toxicity to this regimen for both groups is shown in Tables 3 and 4, which
are also reproduced from the same report [5]. Six of the 10 (60%) MDS patients went into
remission and 5 AML patients achieved a CR (CR rate 41.7%), including 2 patients, both
aged 90 years. Occurrence of reduction of SPAC dosage and the shortening of therapy
duration were mainly due to PS and, severe cytopenia rather than to toxicity. Although bone
marrow suppression occurred after treatment, no serious adverse events were observed. The
typical induction procedures of 2 MDS patients (Patients 7 and 9) are shown in Figures 2 and
3, respectively.
The range and median of the OS time was 1.5 to 41 months and 602.5 days, respectively,
for MDS and 2 to 41 months and 312 days, respectively, for AML patients. Additional SPAC
+ EP therapy in MDS patients produced a prolonged survival time (Table 3). Patient 7 was
administered 7 days of cyclic SPAC + EP (at 1-6 month intervals) as post-remission therapy.
This patient survived for 41 months with good quality of life as an outpatient.
The efficacy and the low toxicity observed in the outpatient setting makes this treatment
an attractive option for pos-tremission therapy in elderly individuals.
Figure 2. Treatment procedure of patient 7.
Figure 3. Treatment procedure of patient 9.

180 Akira Horikoshi
As reported previously [5], the difference in OS between the AML and MDS groups was
not statistically significant (Figure 4a, reproduced from the report). No statistical differences
were observed in comparisons of higher age versus lower age, high white blood cell (WBC)
counts versus low WBC counts, complex abnormal karyotypes versus others, and whole
abnormal karyotypes versus normal karyotypes. However, there was a clear survival
advantage for MDS and AML patients achieving a CR or a PR, as shown in Figure 4b
(reproduced from the report). The difference in the median survival time was significant: 790
days in the CR + PR group compared with 174 days in the nonresponsive (NR) group. We
calculated that the median survival time in the CR + PR group of MDS was 857 days.
Table 3. Result of chemotherapy with SPAC and EP for MDS patients
*1:5,31,2011 *2:SPAC 200mg/day *3:partical remission *4:complete remission *5:ubenimex *6:short duration of
daunorubicin +are-C +6MP + orednisolone.
The additional 3 cases treated after completion of this study were as follows: Case 1 was
referred to us because of pancytopenia due to MDS (RAEB). He had been treated with
cyclosporine and blood transfusions. One year later at 75 years old, his peripheral blood
examination showed hemoglobin (Hb) 5.7g/dl, WBC 680/l, and platelet count (PLT)
17,000/l. Bone marrow aspiration revealed hyperplasia with 55% blasts. Abnormal
karyotypes were not observed. Although the conversion from MDS to overt AML had
occurred, a CR was achieved 2 months later, after 7 days of SPAC + EP therapy. Severe
complications were not observed. However, he died after the first relapse. His survival after
the start of this therapy was 14 months.
Case 2, 84 years old, was diagnosed with RAEB-T with 23.4% blasts in the bone
marrow. Hematological data showed Hb 8.1g/dl, WBC 1600/l, and PLT 38,000/l. His
karyotype showed 46, XY, del(7)(q22). After induction therapy for 14 days, a CR was
achieved. No severe complications were observed. He is being treated with 7 days of cyclic
SPAC + EP post-remission therapy. His CR has been maintained for 8 months.
Case 3, 77 years old, was diagnosed with RAEB. Bone marrow aspiration showed
hyperplasia with 6.4% blasts. An abnormal karyotype of 47, XY, +8 was observed. He had
been treated with cyclosporine and blood transfusion for 5 months. However, his
pancytopenia was growing worse. His peripheral blood examination showed Hb 4.9g/dl,
WBC 465/l, and PLT 11,000/l. Bone marrow aspiration showed hyperplasia with 20.4%
blasts. After induction therapy for 10 days, a CR was achieved. He has been observed as an
outpatient without blood transfusions for 3 months.
Figure 4. Kaplan Meier curves for overall survival analysis of the patients. A. AL vs. MDS (dashed
line). B. CR+PR vs. NR (dashed line).
182 Akira Horikoshi
DISCUSSION
SPAC is rapidly transformed into cytarabine (ara-C) when orally administered [10]. The
pharmacokinetics of SPAC after oral administration of 150-300 mg/m2/day is comparable to
that of continuous infusion of ara-C at 20 mg/m2/day [11]. Two phase II studies [12, 13] and
one multi-institutional study of SPAC combined with G-CSF [14] for acute leukemia and
MDS showed the effectiveness of this regimen at a dose of 100-400 mg/day for more than 14
days.
As previously reported [15, 16], low-dose EP is effective against acute lymphoblastic
leukemia and MDS, and the synergistic effect of ara-C and EP has been demonstrated in
clinical [17] as well as in vitro studies [18]. In addition, the sequence-dependent anti-tumor
effect of this combination has been observed for L1210 ascites tumors in mice [19, 20].
Administration of EP 6 hours before ara-C injection generated the best therapeutic results,
and the amount of ara-C incorporation into the DNA of L1210 cells increased to more than
200% of the control by a 3 and 6 hours pre- administration of EP. The authors of this study
surmised that the period of repair of DNA damage due to EP might be the critical time in
which ara-C incorporation into DNA increase. As SPAC is slowly converted to ara-C in the
liver, and ara-C is released into the blood over a prolonged period of time [10], a synergistic
anti-tumor effect would be produced with concurrent administration of EP.
As reported previously [9], the plasma concentration of ara-C was comparatively higher
in AML patients treated with SPAC + EP combination therapy. The accumulation of ara-C
during the days of SPAC administration may be the reason for the higher concentrations. It is
possible that the higher ara-C concentrations cause more marked myelosuppression and
induce a reduction of abnormal clones.
Nadirs, especially in WBC counts, were observed in all cases of AML and MDS,
although to varying degrees (Tables 3 and 4). Three AML patients (No. 17, 19, and 20) and 2
MDS patients (No. 1, and 10) with longer-lasting nadirs responded well to this treatment.
As described in the results, only the achievement of a CR or a PR was predictive of
longer survival, and the difference between the CR + PR and NR groups was significant
(Figure 4b). Patients in poor clinical condition (PS scores of 3-4) were able to achieve a CR
or PR, indicating low toxicity and an absence of treatment-induced complications (Tables 1-
4).
The therapeutic efficacy of SPAC and EP among higher-risk MDS patients in this study
was satisfactory. Remission was achieved in 60.0% of the patient. In addition, 2 higher-risk
MDS patients and 1 patient with AML evolving from MDS achieved a CR. Although the CR
rate was good for higher-risk MDS with SPAC + EP treatment, post-remission therapy and
maintenance therapy with SPAC + EP were needed for a long CR duration as described in the
results. However, some patients received only weak or no post-remission therapy owing to
their debilitated physical and psychological conditions.
In term of cost-effectiveness, it should be noted that older individuals are at risk of more
frequent and more prolonged hospitalization for neutropenic infections compared to younger
individuals, and of more prolonged deconditioning after hospitalization, which may result in
the need for costly rehabilitation. Therapy cost itself affects patients budget for a family and
the patients mental health. We compared the costs of 1 cycle of azacitidine (75 mg/m2/day
for 7days) (> 1.33 m2) and SPAC + EP for 14 days and found that azacitidine treatment cost
USD 8034.32, whereas SPAC + EP cost only USD 496.5 (calculated as USD 1 = JPY 120).
Although there is a system of health insurance, called a major medical expense supply in
Japan, the huge difference in cost between the two therapies is clear. Medical expenses may
affect not only an individual but also public finances. As previously reported [4], the median
treatment period and the median of treatment cycles are 13 months and 9 cycles, respectively
for azacitidine. The total therapeutic costs calculated from the cost of 1 cycle of azacitidine
rise rapidly over time.
Table 4. Result of chemotherapy with SPAC + EP for AML patients
*1:number of days from the start of therapy till the day showing the lowest counts of WBC and PL *2:
5,31,2011 *3:SPAC:200mg/day *4 no remission *5: complete remission *6:ubenimex
*7:daunorubicin+ara+prednisolone *8:aularubicin+ara-C+6MP+prednisolone *9: high-dose ara-C
As the number of elderly higher-risk MDS patients is increasing in most industrialized

countries, novel therapeutics are needed. We have introduced an approach to the oral
treatment for such refractory patients. We hope this information will be helpful to clinicians
involved in the management of older patients with blood disorders.
CONCLUSION
Although age is a risk factor for myelotoxicity, mucositis, neurotoxicity, and
cardiotoxicity from cytotoxic chemotherapy, our oral chemotherapy regimen with SPAC and
EP for higher-risk MDS in elderly patients is well tolerated and shows few adverse effects.
Oral agents are particularly suitable for the management of elderly patients, as they may
be administered at home, and the dose may be adjusted on a daily basis. We believe that
184 Akira Horikoshi
SPAC + EP therapy can be cost-effective and potentially useful for elderly patients with
higher-risk MDS in poor condition, although our studied patient population was small.
ACKNOWLEDGMENTS
I would like to thank my colleagues from the Division of Hematology and Rheumatology
Department of Internal Medicine at the Nihon University School of Medicine, the Department
of General Internal Medicine at Nerima-Hikarigaoka Hospital, Seihuusou Hospital, and my
wife, Kyoko Horikoshi, for her warm support of my medical research.
APPENDIX
If readers want to receive more information about cytarabine ocfosfate (SPAC), trade
name Starasid, please contact NIPPON KAYAKU CO. LTD. http://www.nipponkayaku.
co.jp/english/.
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dose etoposide: a potential therapy for myelodysplastic syndromes. Br J Haematol,
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186 Akira Horikoshi
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392.
Chapter 8
LENALIDOMIDE TREATMENT IN LOWER RISK

Ota Fuchs
ABSTRACT
MDS with chromosome 5q deletion [del(5q)] is now recognized as a distinct
pathologic subtype of MDS with markedly better clinical responses with the
immunomodulatory and cereblon binding drug lenalidomide (CC5013, Revlimid)
treatment compared to non-del(5q) MDS patients.
Treatment with lenalidomide results in transfusion independence in vast majority of
patients. Several mechanisms of action are believed to contribute to the therapeutic effect
of lenalidomide. They include the effect on the immune system associated with cytokine
production, T- and natural killer cells co-stimulation. Lenalidomide stimulates
erythropoiesis with substantional improvement in the hematopoiesis-supporting potential
of bone marrow stroma and significant decrease in the adhesion of bone marrow CD34+
cells.
Lenalidomide has anti-inflammatory effects and inhibits angiogenesis. The exact
mechanism of action of lenalidomide on del(5q) clones is not fully known, but there
appears to be several candidate (tumor suppressor) genes whose expression may be
modulated by lenalidomide treatment. The upregulation of secreted protein acidic and
rich in cysteine (SPARC) after treatment with lenalidomide is particularly interesting
given its location at 5q 3132 and its role as a tumor suppressor with its anti-proliferative,
anti-adhesion, anti-angiogenic properties.
The levels of activin-A increased 4 fold and significant deregulation of genes
involved in extracellular matrix interactions and erythropoiesis relative to healthy
controls were found. Dual specificity phosphatases [the cell division cycle 25 C
(Cdc25C), and the protein phosphatase 2Ac (PP2Ac)] are important cell cycle
regulators of G2-M checkpoint and their inhibition by lenalidomide leads to G2 arrest
Ota.Fuchs@uhkt.cz.
188 Ota Fuchs
and apoptosis. Genes for both these phosphatases are located in the 5q deleted region.
Hyperphosphorylation of the murine double minute 2 protein (MDM2) and human
analogue (HDM2), as a result of inhibition of PP2Ac phosphatase activity, stabilizes
MDM2 or HDM2, permitting p53 degradation and transition to G2 arrest and clonal
suppression. Lenalidomide resistance in del(5q) MDS is associated with PP2Ac over-
expression.
Patients with mutated TP53 were shown to have poorer erythroid and cytogenetic
responses to lenalidomide and a higher potential for AML evolution. Cereblon seems to
have an important role in lenalidomide action in MDS. Lenalidomide binds to cereblon
which acts as the substrate receptor of E3 ubiquitin ligase CRL4 cereblon. This E3 ubiquitin
ligase in the absence of lenalidomide ubiquitinates cereblon itself and the other
component of CRL4cereblon complex, DNA damage binding protein 1 (DDB1) but in
presence of lenalidomide it changes its specificity and ubiquitinates casein kinase 1
(CK1) and degrades it in proteasomes. CK1 is a serine/threonine kinase and the CK1
gene (CSNK1A1) is located on 5q32 in commonly deleted region (CDR). Inhibition of
CK1 sensitizes 5q- syndrome cells to lenalidomide. High level of full length cereblon
mRNA in mononuclear cells of bone marrow and of peripheral blood seems to be
necessary for successful lenalidomide treatment of 5q- syndrome.
Bone marrow aspirates of patients who responded to lenalidomide showed before
treatment decreased expression of the set of genes needed for erythroid differentiation.
Lenalidomide seems to overcome differentiation block in del(5q) patients with decreased
expression of these genes compared to the non-responders.
Keywords: del5q MDS, immunomodulatory agents, lenalidomide, erythropoiesis, cell cycle,

apoptosis, SPARC, dual specificity phosphatases, MDM2, p53, E3 ubiquitin ligase
CRL4cereblon, cereblon, casein kinase 1
INTRODUCTION
Interstitial deletions involving long (q) arm of chromosome 5 are one of the common
cytogenetic abnormalities in MDS patients [1-17]. The presence of del(5q), either as the sole
karyotype abnormality or as part of a more complex karyotype, is present in 10-15% of
patients with de novo MDS and has distinct clinical implications for MDS [1]. Outcomes
among MDS patients with deletion 5q vary greatly, both in terms of overall survival (OS) and
risk of transformation to AML. The presence of additional chromosomal abnormalities or an
excess of blasts shortens OS and increases risk of transformation to AML. Del(5q) MDS
patients frequently have symptomatic anemia, and its treatment has traditionally consisted of
red blood cell (RBC) transfusions and, for some, iron chelation therapy. The 5q- syndrome, a
subtype of low risk MDS, is characterized by an isolated 5q deletion. Although the length of
the deleted area varies from case to case, deletion in the band 5q32-33 is common. The 5q-
syndrome is characterized by a female predominance, severe refractory macrocytic anemia,
normal or elevated platelet counts, abnormal hypolobulated megakaryocytes, stable clinical
course with relatively rare progression to acute myeloid leukemia (AML). MDS with isolated
del(5q) in which the sole cytogenetic abnormality is del(5q) is a distinct entity with the risk of
evolution into AML of approximately 10% [11, 18]. It is characterized by macrocytic anemia
with or without other cytopenias and/or thrombocytosis. Myeloblasts comprise less than 5%
of bone marrow and less than 1% of peripheral blood.
Lenalidomide Treatment in Lower Risk Myelodysplastic Syndromes 189
Lenalidomide [3-(4-amino-1-oxo1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione;
Revlimid; Celgene Corporation, Summit, NJ, USA] is 4-amino-glutarimide analog of
thalidomide (Figure 1) with potent immunomodulatory, antiangiogenic and direct neoplastic
cell inhibitory activity [19-26]. Thalidomide was synthesized in Germany, in 1954, from -
phtaloylisoglutamine, to be used as a sedative and antimetic drug. In 1957, after a short period
of preclinical studies, thalidomide was approved for first trimester gestational sickness in
humans. The appearance of malformations, such as phocomelia in the newborn, resulted in its
ban three years later [27-30]. The US Food and Drug Administration (FDA) approved
thalidomide in 1998 for the treatment of erythema nodosum leprosum [27]. A small but
consistent fraction of transfusion-dependent MDS patients achieved transfusion independence
by treatment with thalidomide [28-30].
Lenalidomide was developed in order to avoid thalidomide side effects (sedation and
neuropathy), and to increase efficacy [19-24]. Lenalidomide shares a number of structural and
biological properties with thalidomide (Figure 1) but it is safer and more potent than
thalidomide. Oral lenalidomide was first studied in a single-center trial [31].
Figure 1. Chemical structures of immunomodulatory drugs (IMiDs), today also known as cereblon-
binding drugs. Lenalidomide and pomalidomide are synthetic compounds derived by modifying the
chemical structure of thalidomide.
Erythroid and cytogenetic responses were achieved in a study of 43 patients with MDS,
particularly in patients with isolated del(5q31-33) [31]. Lenalidomide was administered in
three different dosing schedules: 25 mg daily, 10 mg daily, and 10 mg daily for 21 days of
each 28-day cycle [31]. The erythroid response rates were highest in patients with the
International Prognostic Scoring System (IPSS) low or intermediate-1 risk MDS. Transfusion
independence was achieved in 20 of 32 patients (63%), and three additional patients had
reduced red blood cells transfusion needs [31]. Ten of 12 patients (83%) with del(5q31)
experienced major erythroid responses, defined as sustained transfusion independence,
compared with a 57% response rate in patients with a normal karyotype and a 12% response
rate in patients with other cytogenetic abnormalities. Complete cytogenetic remissions were
achieved in 75% of the del(5q31) patients (9 of 12 of these patients), with one additional
patient achieving at least a 50% decreases in abnormal metaphases [31]. Myelosuppression
190 Ota Fuchs
(neutropenia and/or thrombocytopenia) was the most common adverse event, but it was dose-
dependent, favoring the 10 mg daily dose for 21 days of each 28-day cycle.
MULTICENTER PHASE II TRIALS OF LENALIDOMIDE

After encouraging results of a single-center trial (MDS-001) [31, Table 1], the effect of
lenalidomide on red blood cell (RBC) transfusion-dependent del(5q) MDS cases of low and
intermediate-1 IPSS risk assessment was investigated in a large multicenter phase II study
(MDS-003). This trial led to FDA approval of lenalidomide in the USA as well as to approval
in several other countries for treatment of RBC transfusion-dependent anemia due to low or
intermediate-1 risk MDS associated with a chromosome 5q deletion with or without
additional cytogenetic abnormalities [32]. The initial schedule was 10 mg of lenalidomide for
21 days every 4 weeks, but the treatment schedule was subsequently amended so that the 10
mg dose was given every day because of the shorter interval between initiation of treatment
and a response in the pilot study. Of the 148 transfusion-dependent patients who were
included in the study, 46 were treated on the 21-day schedule and 102 received continuous
daily dosing. Overall, 112 (76%) patients responded to treatment with a median time to
response 4.6 weeks. Among these, 99 no longer needed transfusions by week 24, while the
remaining 13 patients had a reduction of 50% or greater in the number of transfusions
required. There was no significant difference in response rate between the two treatment
schedules. Response rate was independent of additional chromosomal aberrations. Patients
with pretreatment thrombocytopenia had an inferior outcome. Almost half of the patients,
including some with complex karyotypes, had a complete cytogenetic response. Neutropenia
and thrombocytopenia were the most common treatment-associated adverse events. Most
other adverse events were of low or moderate severity and included pruritus, rash, diarrhea,
and fatigue. Adjustment of the lenalidomide dose due to intolerance was required in 124
patients, including 93 of those receiving continuous daily dosing and 31 of those receiving
21-day dosing. Thirty patients discontinued lenalidomide treatment because of adverse events
including thrombocytopenia or neutropenia, rash AML, anemia, facial edema, congestive
heart failure, urticaria, diarrhea, weight loss, renal insufficiency, cerebrovascular accident,
dementia, dyspnea, pyrexia, and pneumonia. However, the European Medicine Agency
(EMA) did not approve lenalidomide for this indication.
Their concern, based on results of the MDS-003 trial, was that lenalidomide may trigger
progression to AML in some patients with del(5q). Data comparing long-term outcomes in
lenalidomide-treated and untreated patients with MDS with del(5q) are limited but it is now
clear that the concern of the EMA has not been confirmed by a recent study [33].
New results led to the approvement by EMA of lenalidomide for use in low- or
intermediate-1 risk MDS patients who have the deletion 5q chromosomal abnormality and no
other chromosomal abnormalities, are dependent on RBC transfusions, and for whom other
treatment options have been found to be insufficient or inadequate.
Table 1. Trials in lenalidomide approved for use in MDS by the United States Food and Drug Administration (FDA)
Trial Lenalidomide dose Number of patients Results Comments

MDS-001 phase I/II Varying doses of oral lenalidomide, 43 patients with MDS and transfusion- This study showed the potential of lenalidomide in Responses were definied according to the
study ranging from 25 mg daily down to dependency or symptomatic anemia. All erythropoietin-resistant del(5q) MDS but also a role modified International Working Group
10 mg for 21 days of every 28-day patients were refractory to erythropoietin of lenalidomide in erythropoietin-refractory non- (IWG) criteria [66]. Neutropenia and
cycles [31]. or had high endogenous erythropoietin del(5q) MDS patients. 83% of patients with thrombocytopenia were common side
levels. 88% of patients had low- or del(5q31) MDS responded compared to 53% of effects.
intermediate-1 risk IPSS risk assessment. patients with a normal karyotype and 12% of
60% of patients had del(5q) chromosomal patients with other karyotype abnormalities [31].
aberration.
MDS-002 phase II 10 mg of lenalidomide orally either 215 patients at a median age of 71 years 26 patients were identified as transfusion- Ebert et al. [64] identified a set of
study for 21 of 28 days, or with with low or intermediate-1 risk MDS independent with a median hemoglobin rise of 3.2 erythroid-specific genes with decreased
continuous dosing [46]. with or without non-del(5q) g/dL. A further 17% achieved a reduction of expression in non-del(5q) MDS responders
chromosomal abnormalities. pretreatment transfusion requirements. The duration on patient samples from this trial.
of response was at least 24 weeks.
MDS-003 phase II 10 mg of lenalidomide orally for 21 148 transfusion-dependent, lower-risk, 64% of patients became transfusion-free (at least 56 MDS-003 was a single-arm study. Factors
study of 28 days with possible dose del(5q) patients. 111 had a single del(5q) days of transfusion independence) and at least 1 predicting response to lenalidomide in
reductions in case of adverse events chromosomal abnormality, and 37 g/dL increase in hemoglobin. 44% of patients del(5q) MDS were a platelet count
to 5 mg daily and 5 mg daily every patients had additional chromosomal achieved a complete cytogenetic remission (absence decrease by at least 50% in non-
other day [32]. abnormalities. of the del(5q) cytogenetic abnormality). thrombocytopenic patients at base-line
(before starting with lenalidomide
treatment).
MDS-004 phase III 10 mg of lenalidomide orally for 21 205 del(5q) MDS patients were 56.1% of patients treated with 10 mg of 52 patients (25.4%) progressed in this
study of 28 days, 5 mg of lenalidomide on randomized 1:1:1 to lenalidomide 10 lenalidomide, 42.6% of patients treated with 5 mg phase III study to AML with almost 3
days 1 to 28, or placebo on days 1 mg/day, lenalidomide 5 mg/day, or lenalidomide and 5.9% of patients with placebo years of follow-up.
to 28 of 28-day cycles [69]. placebo in the double-blind treatment achieved red blood cell transfusion independence for
phase. at least 26 weeks. Median time to response was
about 4 weeks, median maximum of hemoglobin
increase was 6.3 g/dL. Complete cytogenetic
response rates were 29.4%, 15.6%, and 0% for
lenalidomide 10 mg, lenalidomide 5 mg, or placebo.
MDS-005 phase III 10 mg of lenalidomide orally once 375 low risk (low- or intermediate 1-risk) Study start date: November 2009; estimated study Clinical trials.gov Identifier:
study daily (administered as one 10 mg MDS patients without del(5q) who do not completion date: December 2018; estimated primary NCT01029262 (A servise of the U.S.
lenalidomide capsule and 2 placebo respond to erythropoiesis-stimulating completion date: April 2016 (final data collection National Institutes of Health). This
capsules) or placebo (3 placebo agent therapy and require regular red date for primary outcome measure). international a phase III trial, double-blind
capsules) once daily for up to 4 blood cell transfusions. is being conducted at more than 70 medical
years (see Steensma DP-The centers in 14 countries, including 9 centers
Hematologist March 1, 2011 and in the United States and Canada.
Celgene MDS 005).
192 Ota Fuchs
Kuendgen et al. [33] have evaluated clinical outcomes of 295 lenalidomide-eated patients
from two clinical trials (MDS-003 and MDS-004, Table 1) and 125 untreated red blood cell
transfusion-dependent patients with del(5q) low- or intermediate-1-risk MDS from a large
multicenter registry. Median follow-up was 4.3 years from first dose for lenalidomide-treated
patients and 4.6 years from diagnosis for untreated patients. Risk factors for AML
progression and mortality were assessed. In conclusion, lenalidomide treatment did not
increase AML progression risk, but instead confered a survival benefit in red blood cell
transfusion-dependent patients with del(5q) low- or intermediate-1-risk MDS.
The long term effect of lenalidomide on the 5q- syndrome was investigated
retrospectively in 168 patients treated in four MDS trials [34]. For those patients who
achieved transfusion-independency, the median duration of response was 2.2 years. In
multivariate analysis cytogenetic response, lower bone marrow blast percentage, and lower
baseline transfusion burden were independent predictors of prolonged overall survival.
List et al. [35] reported the long-term outcomes (median follow-up 3.2 years) in lower
risk del(5q) MDS patients treated with lenalidomide in the MDS-003 trial. RBC transfusion
independence was achieved in 97 of 148 treated patients (65.5%), with a median response
duration of 2.2 years. Partial or complete cytogenetic response was achieved by 63 of 88
evaluable patients (71.6%). Median OS was longer in patients achieving RBC transfusion
independence than in non-responders. Similarly, cytogenetic response was higher and with a
trend toward reduced relative risk of AML progression in patients achieving RBC transfusion
independence than in non-responders.
Current recommendation state is that treatment with lenalidomide in del(5q) MDS should
be continued until disease progression [35-37]. The question whether interrruption of
lenalidomide treatment for patients in remission would be beneficial has been also addressed
[36]. It is important for several reasons: 1) it could reduce costs and side effects; 2) it could
facilitate disease progression to AML. Different mechanisms have been discussed to explain
AML progression. Evidence that pre-therapeutic telomere length was significantly shorter in
those patients who ultimately transformed to AML than in those who did not, was presented
[38]. Transformation to AML is occasionally observed, paticularly in patients without a
cytogenetic response to lenalidomide. Jdersten et al. [39] performed molecular studies in a
patient with classical 5q- syndrome with complete erythroid and partial cytogenetic response
to lenalidomide, who evolved to high-risk MDS with complex karyotype. Immunohisto-
chemistry of pretreatment marrow biopsies revealed a small fraction of progenitors with
overexpression of p53 and sequencing confirmed a TP53 mutation. TP53 mutated subclones
have not previously been detected in 5q- syndrome and indicates heterogeneity of this
disease. Subsequently, TP53 mutations with a median clone size of 11% (range, 1% to 54%)
were detected in 10 from 55 (18%) low-risk MDS or intermediate-1 risk patients with del(5q)
by next-generation sequencing [40]. TP53 mutations are associated with strong nuclear p53
protein expression. Patients with mutation had significantly worse outcome. TP53 mutations
may lead to genetic instability and disease progression. This clonal heterogeneity in low-risk
MDS patients with del(5q) may be of importance when assessing the prognosis and selecting
the therapy in these patients. It has been speculated that continuous administration of
lenalidomide may lead to selective pressure on stem cells that induces genomic instability,
resulting in acute leukemia transformation [41].
Longest transfusion-free intervals are achieved in patients low-risk MDS patients with
del(5q) who are exposed to lenalidomide 6 months beyond complete cytogenetic remission
[40, 42-45]. Lenalidomide should not be withdrawn prematurely in patients who achieve
transfusion independence as partial cytogenetic remission patients seem to have a higher
relapse rate than complete cytogenetic remission patients.
Response rate to lenalidomide in low- or intermediate-1-risk MDS without a 5q deletion
who became transfusion dependent after erythropoiesis-stimulating agents (ESA) treatment
was investigated also in a multicenter, phase II study (MDS-002) [46, Table 1]. 214 patients
received 10 mg oral lenalidomide daily or 10 mg on days 1 to 21 of a 28-day cycle. The most
common grade 3/4 adverse events were neutropenia (30%) and thrombocytopenia (25%). 56
(26%) patients achieved transfusion independence after a median of 4.8 weeks of treatment
with a median duration of transfusion independence of 41.0 weeks. A 50% or greater
reduction in transfusion requirements occured in 37 additional patients, yielding a 43%
overall rate of hematologic improvement (in comparison with 76% in the case of low- or
intermediate-1-risk MDS patients with del(5q)). Responding karyotypes included trisomy 8
(n = 3), -Y (n = 3), deletion 11q (n = 2), and deletion 17p (n = 1). While clonal suppression
represents the main mechanism of lenalidomide action in MDS patients with del(5q), the
restoration and promotion of effective erythropoiesis is the main mechanism in non-del(5q)
MDS patients [47]. When endogenous EPO levels were considered, together with previous
ESA treatment, one could identify patients whose response rate in terms of transfusion
independence was clearly higher, reaching 42.5%, for serum EPO 100 U/L [48].
Sibon et al. [49] have recently reported 31 lower-risk non-del(5q) MDS patients with
anemia refractory to ESA and treated with lenalidomide. Twenty patients from this group also
received an ESA. An erythroid response was obtained in 15 patients (48%), including 10 of
the 27 (37%) previously transfusion-dependent patients, who became transfusion-
independent. Nine of responders relapsed, whereas 6 (40%) were still responding and
transfusion free after 11 months. Median response duration was 24 months.
These studies were based on scientific knowledge because small deletions in several
ribosomal genes, including RPS14, were found in CD34+ cells not only in patients with
del(5q) but also in patients with non-del(5q) MDS. This observation suggested that
deregulated ribosomal biogenesis may not be limited to del(5q) MDS [50-52]. Czibere et al.
[53] showed that lower risk non-del(5q) MDS patients with RPS14 haploinsufficiency tend to
have prolonged survival. Defective ribosomal biogenesis has a lead role in disrupting
erythropoiesis in a variety of anemias. Disruption of ribosomal biogenesis has been clearly
demonstrated in multiple ribosomopathies to greatly perturb p53 signaling [54-63].
Bone marrow aspirates of patients who responded to lenalidomide showed before
treatment decreased expression of the set of the genes needed for erythroid differentiation.
Lenalidomide seems to overcome differentiation block in del(5q) patients with decreased
expression of these genes compared to the non-responders [63]. Thus, lenalidomide restored
erythroid differentiation potential by upregulation of the suppressed erythroid gene signature
(genes for - and -globin, ankyrin 1, band 3, band 4.2, carbonic anhydrase, ferrochelatase
and glycophorin B) [64].
The Groupe Francophone des Mylodysplasies conducted a multicenter phase 2 trial
with lenalidomide in intermediate-2 (19 patients) and high-risk MDS (28 patients) with
del(5q) [65]. Forty seven patients (24 males and 23 females, with a median age of 69 years,
range, 36-84 years) were treated. Forty three patients of 47 patients had transfusion-dependent
anemia. Patients received 10 mg lenalidomide once daily orally during 21 days every 4
weeks. In patients without response after 8 weeks, the lenalidomide dose was increased to 15
194 Ota Fuchs
mg/day in the same time schedule during an additional 8 weeks. If no response was found in
this additional time of treatment, lenalidomide was discontinued. Thirteen of the 47 patients
(27%) achieved response according to International Working Group (IWG) 2006 criteria [66].
Median duration of overall response was 6.5 months, 11.5 months in patients who achieved
the complete remission. Grade 3 and 4 neutropenia and thrombocytopenia were seen in most
patients.
RANDOMIZED PHASE III PLACEBO-CONTROLLED STUDY

OF LENALIDOMIDE IN DEL(5q) PATIENTS
This study [67-70, Table 1] examined the safety of lenalidomide in a randomized phase
III trial (MDS-004) in low-/int-1-risk myelodysplastic syndromes (MDS) with a del(5q)
abnormality.
Similar criteria to those used in the MDS-003 study were chosen. Two hundred five
patients were randomized to receive treatment with either lenalidomide 10 mg orally daily for
21 days of each 28-day cycle, lenalidomide 5 mg orally daily for 28 days of each 28-day
cycle, or placebo. Erythroid responses were assessed at 16 weeks. Nonresponders were then
in open-label treatment and they were excluded from the efficacy analysis. Red blood cell
transfusion independence was achieved in 53.6% of patients treated on 10 mg arm, 33.3% on
5 mg arm and 6% on the placebo arm. Cytogenetic response rates were also highest in the 10
mg arm (41.5% of patients), while in 5 mg arm (17.4%) and in the placebo arm (0%). The
median rise in hemoglobin at the time of the best response was also higher in patients treated
with the 10 mg lenalidomide. No difference in the rate of AML transformation among three
arms was found. This study confirmed that the preferred starting dose of lenalidomide in
patients with del(5q) low-/int-1-risk MDS remains 10 mg.
Health-related quality of life (HRQL) outcomes were assessed using the Functional
Assessment of Cancer Therapy-Anemia in 167 RBC transfusion-dependent patients with
IPSS low- or intermediate-1-risk del5q31 MDS treated with lenalidomide versus placebo in a
randomized phase III clinical trial, MDS-004 [71]. Clinically important changes in HRQL
from baseline were observed at weeks 12, 14, 36, and 48 among responders in both treatment
groups (5mg and 10 mg lenalidomide). Lenalidomide treatment may be effective in
improving HRQL outcomes [71].
FURTHER CLINICAL STUDIES OF LOWER RISK MDS PATIENTS

WITH DEL(5q) TREATED WITH LENALIDOMIDE
Many of the initial clinical and laboratory observations obtained in the MDS-003 trial
were confirmed in the study of Le Bras et al. [72]. Ninety five lower risk MDS patients (low
and intermediate 1 risk in IPSS, 25 males and 70 females with a median age of 70.4 years)
with del(5q) were treated with 10 mg of lenalidomide daily, 21 days every 28 days for at least
16 weeks. Patients with at least a minor erythroid response after 16 weeks were treated in the
same way until disease progression, treatment failure or treatment-limiting toxicity.
Erythroid response was evaluated according to IWG 2000 criteria [73]. Sixty two of the
95 patients (65%) achieved erythroid response according to IWG 2006 criteria [66]. In these
62 patients, 60 patients (63% from 95 patients) achieved red blood cell transfusion
independence. Median time to transfusion independence was 16 weeks (range 8-33 weeks).
Fifteen patients who achieved transfusion independence were analyzed for cytogenetic
response (20% of complete and 40% of partial cytogenetic response). The rest of these 15
patients (40%) had no cytogenetic response. Six (6.3%) patients progressed to AML and 15
patients died, including 6 patients who had achieved transfusion independence. In the MDS-
003 trial, the primary endpoint was hematological response, while in the study of Le Bras
[72] et al. transfusion independence. The cytogenetic remission rate was higher in the MDS-
003 trial (73% versus 60% in the study of Le Bras et al. [72]). Neutropenia and
thrombocytopenia were the most common adverse events in both studies [74].
A Japanese multiinstitutional study MDS-007 in MDS patients with del(5q) treated with
lenalidomide has been recently performed [75, 76]. This study was targeted on morphologic
analysis and evaluation of the relationship among erythroid response, change of morphologic
findings and cytogenetic response. MDS-007 trial was a single-arm, open-label study. Eleven
patients were enrolled in this study, including 5 patients with transfusion-dependent anemia
and 6 patients with transfusion-independent symptomatic anemia. Nine patients showed less
than 25% of bone marrow erythroblasts before therapy with lenalidomide and no patient had
more than 40% of bone marrow erythroblasts at that time.
Eight patients showed a rapid increase of bone marrow erythroblasts to more than 40%
on day 85. All patients except one achieved a major erythroid response as defined by either
transfusion independence or by rapid increase of hemoglobin level in most patients on day
169 of lenalidomide therapy. One patient without any hematologic response by day 169,
achieved a major erythroid response on day 218.
Erythroid response could be achieved even without a cytogenetic response. No patient in
this analysis showed a hematological relapse prior to cytogenetic one. These findings
suggested that lenalidomide can improve anemia by more than one mechanism of action and
also through mechanism different from del(5q) elimination.
Oliva et al. [77] studied 45 patients with anemia and lower risk del(5q) MDS who
received lenalidomide 10 mg/day. Lenalidomide was well tolerated with 82% obtained
erythroid response within 24 weeks. Durable 69% response was described at 52 weeks.
Cytogenetic response occurred in 29 patients (64%). 10 patients achieved a complete
cytogenetic response.
Snchez-Garcia et al. [78] used time-dependent multivariate methodology to analyse the
influence of lenalidomide therapy on OS and AML progression in 215 patients with IPSS low
or intermediate-1 risk and del(5q). The 5-year time-dependent probabilities of OS and
progression to AML were 62% and 31% for patients receiving lenalidomide and 42% and
25% for patients not receiving lenalidomide. Achievement of RBC transfusion independency
or cytogenetic response after lenalidomide was associated with longer OS in multivariate
analysis. Response to lenalidomide resulted in a substantial clinical benefit and did not appear
to increase AML risk [78].
Cerqui et al. [79] presented results of lenalidomide treatment of 21 RBC transfusion-
dependent elderly patients with multiple comorbidities in a single-centre real-world study.
Of 18 evaluable patients (median follow-up: 22 months), 17 achieved an erythroid
hematologic response and 16 RBC transfusion independence. Cytogenetic response rate was
196 Ota Fuchs
80%, median OS was 48 months (range 3-164), and 5-year leukemia-free survival was 84%.
Three patients progressed to AML.
One patient with baseline TP53 mutation, achieved an erythroid hematologic response
and partial cytogenetic response and did not progress to AML. Lenalidomide was very
effective and well tolerated even in these unselected elderly patients with multiple
comorbidities and did not appear to increase the risk of inducing AML [79].
Polish group presented a retrospective analysis of low-risk MDS with isolated del(5q)
treated with lenalidomide outside the clinical trials [80]. 36 RBC transfusion-dependent
patients have been included in the study. Patients received lenalidomide 10 mg/day on days 1-
21 of 28-day cycles. 91.7% of patients responded to lenalidomide treatment.
Erythroid response was achieved in 72.2% of patients, 19.4% achieved minor erythroid
response and 8.4% of patients did not respond to treatment. Median duration of response was
16 months (range 6-60 months). Treatment was well tolerated.
Hematological toxicity (grade 3 and 4): neutopenia in 16 (44%) patients and
thrombocytopenia in 9 (25%) patients was described [80]. Two patients (5.5%) progressed to
high-risk MDS and two subsequent proggressed to AML. A Kaplan-Meier estimate for
overall survival at 5 years was 79.0 8.8% [80].
Austrian group showed the results of a retrospective, multicentre, observational analysis
of 50 MDS patients, who received lenalidomide and their data support the use lenalidomide
therapy for lower-risk MDS poatients in clinical practice [81]. Fourty-six percent of the
patients (23 patients) suffered from isolated del(5q) MDS (5q- syndrome) while 6 patients
(12%) showed del(5q) plus additional aberrations or isolated del(5q) but >5% blasts in the
bone marrow (5 patients).
The remaining 16 patients (32%) had MDS with other WHO classifications. Seventy
percent belonged to lower IPSS risk classes. Lenalidomide doses were 10 mg a nd 5 mg on
days 1-21 of a 28-day cycle. Patients received 11 months of treatment, with a median therapy
period of 3.5 months, median follow-up was 3.9 months.
Transfusion independence during two months following lenalidomide therapy was 64%.
The two patients that had already progressed to AML at study entry did not show any
response to lenalidomide therapy. Of the remaining 48 MDS patients, eleven developed
AML. Six of these 11 patients had del(5q) with >5% bone marrow blasts and subsequent 5
patients had additional aberrations or other MDS. Median OS was not reached in the
observation period.
The main reason for discontinuation of lenalidomide therapy was disease progression (16
patients), while the most limiting toxicity of lenaslidomide (cytopenia) caused discontinuation
in only 2 patients [81].
TREATMENT OF DEL(5q) PATIENTS WITH RELAPSE DURING

LENALIDOMIDE EXPOSURE
At the time of relapse of transfusion dependence, bone marrow aspiration would be
performed in order to evaluate the patient for morphological or cytogenetic progression. If
there is no progression, the patient is without drug next 3 to 4 monthes. Thereafter, the patient
is re-exposed to lenalidomide. Second responses are regularly seen, probably by epigenetic
mechanism [82]. In the case of progressive disease, salvage therapies including demethylating
agents or allogeneic bone marrow transplantation are necessary [83].
THERAPY WITH LENALIDOMIDE IN COMBINATION

WITH ANOTHER DRUG IN MDS
In order to maximize the potential benefit from lenalidomide therapy combination

strategies were developed. Lenalidomide in attempt to improve outcome of patients can be
combined with erythropoiesis-stimulating agents (ESA), such as erythropoietin or
darbepoietin alpha. This therapy is based on preclinical observations shoving that
lenalidomide significantly potentiated erythropoietin receptor signaling. The addition of
erythropoietin (40, 000 U/week) for an additional 8-week course had the beneficial effect in
low and intermediate-1 risk MDS patients who had failed prior treatment with lenalidomide
monotherapy for 16 weeks [84]. To evaluate the potential benefit of the combination of
lenalidomide and ESA, Park et al. [85] tried the association in three del5q MDS patients, who
were resistant or partially responding to lenalidomide alone. Lenalidomide had two different
actions, one on the disapperance of the 5q- clone and the other one on the stimulation of the
erythroid production in combination with ESA.
In low to intermediate-1 risk non-del(5q) MDS, lenalidomide treatment is less effective
with a lower response rate (25%) and shorter response duration than in the same risk MDS
with del(5q) [45]. Combination of lenalidomide with another drug could improve outcome of
patients with low to intermediate-1 risk non-del(5q) MDS. Ezatiostat hydrochloride (Telintra,
TLK199), a tripeptide glutathione analog is a reversible inhibitor of the enzyme glutathione
S-transferase P1-1 (GSTP1-1) inhibitor [86-92]. This inhibitor was developed for the
treatment of cytopenias associated with lower risk MDS. Ezatiostat activates jun-N-terminal
kinase (JNK), promoting the growth and maturation of hematopoietic progenitors, while
inducing apoptosis in human leukemia blasts [86]. The ability of ezatiostat to activate the
caspase-dependent pathway may help eliminate or inhibit the emergence of malignant clones.
Alternatively, ezatiostat increases reactive oxygen species in dysplastic cells and contibutes
by this effect also to apoptotic death [86]. Based on these mechanisms of action, response
rates, non-overlapping toxicities, and tolerability observed in a single agent ezatiostat phase 1
and 2 studies in MDS [88-90, 93], a study of the combination of ezatiostat and lenalidomide
was conducted to determine the safety and efficacy of ezatiostat with lenalidomide in non-
del(5q) low to intermediate-1 risk MDS. Eighteen patients (median age 73 years; range 57-82;
72% male) were enrolled in the study [90]. Thirteen patients (72%) were intermediate-1 risk
and 5 patients (28%) were low risk. Four patients had abnormal cytogenetics. Twelve patients
(67%) were red blood cell transfusion-dependent and 2 patients (11%) were were platelet
transfusion-dependent. Three of 8 (38%) patients achieved transfusion independence
including 1 responder who did not respond to prior lenalidomide. Ezatiostat caused clinically
significant reduction in red blood cell and platelet transfusions. Since ezatiostat is non-
myelosuppressive, it is a good candidate for combination with lenalidomide. The
recommended doses of this combination regimen for future studies is the ezatiostat.
Lenalidomide and azacitidine are active in patients with lower- and higher-risk MDS.
These agents may complement each other by targeting both the bone marrow
198 Ota Fuchs
microenvironment and hypomethylating action on the malignant clone. Phase I combination

trial of lenalidomide and azacitidine in patients with higher-risk MDS was a multicenter,
single-arm, open-label study [94]. Twenty five patients were screened and enrolled in this
therapy and their response was assessed after four and seven cycles of the tretment.
Azacitidine was administered at 75 mg/m2 daily for five consecutive days, and lenalidomide
10 mg daily for 21 days, of a 28-day cycle. Of 18 evaluable patients, 12 (67%) responded to
therapy; 8 (44%) achieved complex response, 3 (17%) had hematologic improvement and one
(6%) had bone marrow complex response. Of those who responded, eight experienced relapse
or disease progression at a median 7.5 months from initial response (range, 3 to 17 months).
Two patients transformed to AML, one patient at 7 and one patient at 11 months from initial
response. Another report from the same research group on three MDS patients with normal
cytogenetics who relapsed on monotherapy and achieved a complete response with
combination of lenalidomide and azacitidine has been recently published [95]. This
combination was also studied by the other American, French, German and Australian groups
[96-99]. The combination of lenalidomide and azacitidine is feasible and seems to be
effective in lower risk MDS with del(5q) [97] and even in a very high risk patient groups with
advanced MDS or AML and a del(5q) [94-96, 98, 99]. The hypomethylating agents
azacitidine and decitabine are most commonly used to treat patients with higher-risk MDS.
Romiplostim (AMG 531, Nplate) is an Fc-peptide fusion protein (peptibody) that acts as
a thrombopoietin receptor agonist. It has no amino acid sequence homology with endogenous
thrombopoietin. Romiplostim stimulates megakaryopoiesis and thrombopoiesis by binding to
and activating the thrombopoietin receptor and downstream signaling [100-103].
Romiplostim appeared well tolerated in patients with lower risk MDS and thrombocytopenia
[104]. Low platelet counts in patients with MDS may be due to the underlying disease or due
to treatment with disease-modifying agents, and platelet transfusions are often the only
treatment for clinically significant thrombocytopenia or bleeding. Randomized phase II study
evaluating the efficacy and safety of romiplostim treatment of patients with low or
intermediate-1 risk MDS receiving lenalidomide was performed [105]. This was double-
blind, placebo controlled, dose finding study that evaluated the effect of romiplostim on the
incidence of clinically significant thrombocytopenia events (grade 3 or 4 thrombocytopenia
and/or receipt of platelet transfusions) and the safety of romiplostim in patients with low or
intermediate-1 risk MDS receiving lenalidomide. Thirty nine patients (median age 74 years;
range, 39 to 90) were randomized into treatment groups receiving placebo, 500 g
romiplostim, or 750 g romiplostim by weekly subcutaneous injections in combination with
lenalidomide (one 10 mg capsule by mouth daily for each 28-day cycle). Fifteen patients
(39%) had platelet counts 50x109/L and 7 (18%) had del(5q). Treatment continued for a
total of four cycles. Twelve patients (31%) discontinued the study. Disease progression to
AML was reported in 1 patient in the romiplostim 500 g group. Response was 8% for the
placebo, 36% for 500 g romiplostim, and 15% for 750 g romiplostim groups. Romiplostim
appeared to be well tollerated in low or intermediate-1 risk MDS patients receiving
lenalidomide.
It is possible that the effect of lenalidomide could be augmented by addition of another
immunomodulation agent, cyclosporine A. A single-arm, open-label study of the efficacy and
safety of lenalidomide in combination with cyclosporine A in red blood cell transfusion-
dependent both 5q- and non 5q- MDS patients started at Weill Cornell Medical College in
New York.
Other drugs are tried and will be probably used in combinations with lenalidomide in the
treatment MDS patients with del(5q) in the future. Dexamethasone and lenalidomide rescue
erythropoiesis, alone and in combination, in RPS14- and RPS19- (ribosomal proteins of small
ribosomal subunit) deficient cells [106]. L-leucine was also studied in RPS14- and RPS19-
deficient cells [107-110]. The combined use of L-leucine and lenalidomide might be
considered for therapy in MDS patients with the del(5q) since there is evidence to suggest
that these two drugs act through different mechanism and their effect may be synergistic.
MECHANISMS OF ACTION OF LENALIDOMIDE

Lenalidomide shares a number of structural and biological properties with thalidomide,
but it is safer and more potent than thalidomide. Both drugs appear to function through four
mechanisms: immunomodulatory, anti-inflammatory, anti-angiogenic and direct neoplastic
cells inhibitory [111, 112]. Lenalidomide has a direct erythropoiesis stimulating effect.
Shortly, Wei et al. [113] demonstrated that the haplodeficient enzymatic targets of
lenalidomide within the commonly deleted region are two dual-specificity phosphatases, the
cell division cycle 25C (Cdc25C) and the protein phosphatase 2A (PP2A). These
phosphatases are coregulators of G2-M checkpoint in the cell cycle and thus, their inhibition
by lenalidomide leads to G2 arrest and apoptosis of del(5q) specimens. The mechanism of
action is different in non-del(5q), where lenalidomide restores and promotes effective
erythropoiesis with no direct cytotoxic effect [113]. Lenalidomide promotes erythropoiesis
and fetal hemoglobin production in human CD34+ cells [114]. The increased fetal
hemoglobin expression was associated with epigenetic effect on chromatin (an increase in
histone 3 acetylation on the -globin gene promoter).
In MDS patients with del(5q), allelic deletion of the RPS 14 gene is a key effector of the
hypoplastic anemia. Impairment of ribosomal biogenesis liberates free ribosomal proteins to
bind to and trigger degradation of HDM2 (human homologue of the mouse double minute 2
protein /MDM2/) with consequent p53 transactivation in response to nucleolar stress
independently of DNA damage [115, 116]. Overexpression of p53 is typical for erythroid
precursors of primary bone marrow of MDS patients with with del(5q). Lenalidomide inhibits
the haplodeficient PP2A resulting in hyperphosphorylation of inhibitory serine 166 and serine
186 residues on MDM2, and displaces binding of RPS14 to suppress MDM2 autoubiquitation
whereas PP2A overexpression promotes drug resistance. Lenalidomide promotes p53
degradation by inhibiting HDM2 autoubiquitination in erythroid precursors of MDS patients
with with del(5q) bone marrow [117].
The similar epigenetic modulation of gene for p21(CIP1/WAF1) by lenalidomide was
described in both lymphoma and multiple myeloma [118]. A potent cyclin-dependent kinase
inhibitor p21(CIP1/WAF1) decreases activity of cyclinE-CDK2 or cyclinD-CDK4/6
complexes, and thus functions as a regulator of cell cycle progression. The p21 protein can
mediate cellular senescence and also interact with proliferating cell nuclear antigen (PCNA),
a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication
and DNA damage repair.
Most MDS patients including those with del(5q) become refractory to erythropoietin
(EPO). EPO is an essential glycoprotein that facilitates red blood cell maturation from
200 Ota Fuchs
erythroid progenitors and mediates erythropoiesis [119]. EPO acts through EPO-receptor
(EPO-R) and the signal transducer and activator of transcription 5 (STAT5) [47, 119].
Disruption of STAT5 results in a variety of cell-specific effects, one of which is the impaired
erythropoiesis [120]. Lenalidomide relieves repression of ligand-dependent activation of the
EPO-R/STAT5 pathway. Ebert et al. [64] showed that target genes of this pathway are
underexpressed in lenalidomide-responsive MDS patients wihout del (5q). Lenalidomide
promotes erythropoiesis in MDS by CD45 protein tyrosine phosphatase inhibition [121].
CD45 phosphatase is overactivated in MDS and may inhibit phosphorylation of STAT5
stimulated by EPO-R. Lenalidomide is able to restore EPO-R/STAT5 signaling that is
essential for hematopoiesis. Lenalidomide restores and promotes effective erythropoiesis in
non-del(5q) without direct cytotoxic effect.
A deregulated immune system plays the important role in pathogenesis of MDS.
Deregulation is caused by the alteration of cytokines in the bone marrow microenvironment,
deffective T-cell regulation and diminished natural killer (NK) cell activity. Deficiences in T
cells, NK cells and interferon- (IFN-) production were described in the bone marrow and
peripheral blood of MDS patients [122, 123]. Lenalidomide exhibits potent T-cell
costimulatory properties and augmented production of IL-2 and IFN- [19, 124]. Akt
(proteinase B) signaling pathway and transcription factor AP1 (activator protein 1) are
involved in T-cell activation [124]. Increased numbers and activation of NK and NK T-cell
populations were also observed in peripheral blood cells cultured with lenalidomide [125,
126]. Anti-inflammatory effects of lenalidomide is based on inhibition of proinflammatory
cytokines and chemokines, such as TNF-, IL-1, IL-6, IL-12, monocyte chemotactic
protein-1 and macrophage inflammatory protein-1. On the other hand, lenalidomide elevates
anti-inflammatory cytokine IL-10. Interestingly, haploinsuficiency of miR-145 and miR-146a
in 5q - syndrome increases IL-6 levels by elevation of interleukin-1 receptor-associated
kinase 1 (IRAK1), Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP),
tumor necrosis factor receptor-associated factor-6 (TRAF6), and NF-B [127-135]. RPS14,
miR-145, and miR-146 were significantly increased and TNF-, IL-1 and IL-10
significantly downregulated during the treatment with lenalidomide [136-138].
Messingerova et al. [139] found elevated activities of lactate dehydrogenase (LDH) and
matrix metalloproteinase 9 (MMP-9) in the blood plasma of MDS del(5q) patients as
compared with healthy controls. This was stabilized to control values after lenalidomide
treatment. Similar behavior we registered also for the thioredoxin and calnexin contents in
blood plasma. Peripheral blood mononuclear cells (PBMC) from patients with MDS del(5q)
prior to and after treatment with lenalidomide did not exhibit any detectable amount of P-
glycoprotein (P-gp) gene transcript. However, we detected a measurable amount of multidrug
resistance associated protein 1 (MRP1) mRNA in PBMCs from three patients prior to
lenalidomide treatment and in one patient during lenalidomide treatment but it was not
present prior to treatment. These data indicated on usefulness of applied protein markers
estimation for monitoring of MDS del(5q) patient treatment effectiveness by lenalidomide.
Expression of MRP1 seems to be independent on lenalidomide treatment and reflects
probably the molecular variability in the ethiopathogenesis of MDS del(5q).
Angiogenesis, the formation of new blood vessels, plays an important role in the growth
and progression of MDS. The vascular endothelial growth factor (VEGF) and to a lesser
extent IL-6 are cytokines that stimulate the formation of blood vessels. Increased levels of
these cytokines have been shown in MDS [140]. Anti-angiogenic effects of lenalidomide are
independent of immunomodulatory effects and are mediated through endothelial cell

migration inhibition [141-144]. The mechanism by which lenalidomide inhibited VEGF-
induced endothelial cell migration may be related to VEGF-induced inhibition of Akt
phosphorylation. Furthermore, loss of anti-angiogenic effect of lenalidomide predicted
disease progressionand an increased risk of transformation to AML [145].
Lenalidomide does not affect DNA synthesis but inhibits cytokinesis of MDS cells.
Cytokinesis occurs as the final stage of cell division after mitosis. A contractile ring, made of
non-muscle myosin and actin filaments assembles in the middle of the cell adjacent to the cell
membrane. Formins are Rho-GTPase effector proteins that are involved in the polymerization
of actin and effects microtubule during meiosis, mitosis, the maintenance of cell polarity,
vesicular trafficking and signaling to the nucleus [146, 147]. Diaphanous (mDia)-related
formin mDia1 is encoded by DIAPH1 located on the long arm of chromosome 5 (5q31.3) and
lies between the two commonly deleted regions in MDS patients with 5q- syndrome. It is not
clear whether mDia1 plays a role in lenalidomide effect on cytokinesis. Knock-out of
DIAPH1 in mice has T cell responses and myelodysplastic phenotype [148-150].
The clinical effect of lenalidomide is associated with significant increases in the numbers
of erythroid, myeloid and megakaryocytic colony-forming cells and a substantial
improvement in the hematopoiesis-supporting capacity of bone marrow stroma. Lenalidomide
induces significant alterations in the adhesion profile of hematopoietic progenitor cells,
including over-expression of membrane ligands (CXCR4/CD184, CD54/ICAM1, CD11a and
CD49d where CD is cluster of differentiation) and overproduction of soluble stromal cell-
derived factor-1 (SDF-1) and of ICAM1 in the bone marrow microenvironment. CXC4 is C-
X-C chemokine receptor type 4 also known as fusin or CD184. ICAM1 (intracellular
adhesion molecule 1 also known as CD54) is a cell surface glycoprotein. All these effects
favor the maintenance of CD34 + cells in the bone marrow [151]. Lenalidomide-mediated
induction of the SLAM antigen CD48 on patients CD34+ cells may be associated with the
drugs apoptosis-inducing effect through co-stimulatory interactions between CD34+ cells
and cytotoxic lymphocytes in the bone marrow microenvironment.
ROLE OF CEREBLON IN THE EFFICACY OF

LENALIDOMIDE IN DEL(5q) MDS
Down-regulation of the cereblon (CRBN) gene expression is associated with the
immunomodulatory drug lenalidomide resistance and poor survival outcomes in multiple
myeloma patients. However, the importance of the CRBN gene expression in patients with
myelodysplastic syndrome (MDS) and its impact on lenalidomide treatment of these patients
are not clear. We evaluated the cereblon expression in mononuclear cells isolated from bone
marrow [23 lower-risk MDS patients with isolated 5q deletion (5q-), 37 lower-risk MDS
patients with chromosome 5 without the deletion of long arms (non 5q-) and 24 healthy
controls] and from peripheral blood [38 patients with 5q-, 52 non 5q- patients and 25 healthy
controls] in order to gain insight into the role of cereblon in lower-risk MDS with or without
5q deletion and into the mechanisms of lenalidomide action. 5q- lower-risk MDS patients
have the highest levels of CRBN mRNA in comparison to both lower-risk myelodysplastic
syndromes with chromosome 5 without the deletion of long arms and the healthy controls
202 Ota Fuchs
[152]. CRBN gene expression was measured using the quantitative TaqMan real-time PCR.
High levels of CRBN mRNA were detected in all lenalidomide responders during the course
of the therapy. Significant decrease of the CRBN mRNA level during treatment by
lenalidomide is associated with loss of response to treatment and disease progression. These
results suggest that, similarly to the treatment of multiple myeloma, high levels of full length
CRBN mRNA are necessary for the efficacy of lenalidomide in lower-risk 5q- patients [152].
French group (Sardnal et al. [153]) described a higher distribution of G alleles than in
healthy controls at the site of A/G polymorphism located at the site 29 nucleotides before the
transcriptional start site of the CRBN gene as a biomarker of lenalidomide responders in
low/int-1-risk MDS without del(5q). However in our study [152], allele A was predominant
(81.5% of A and 18.5% of G alleles in the whole group of 5q- low-risk MDS patients (Table
2). Moreover in lenalidomide responders we found even bigger diference (86.7% of A and
13.3% of G alleles). A/G polymorphism in healthy controls was 75% of A and 25% of G
alleles.
In addition, the nonresponder to lenalidomide therapy was homozygote for G at the
position of this polymorphism. However in 6 non-del(5q) patients from MDS-005 study
analysed in our laboratory (all non-responders to lenalidomide therapy), the majority was AA
homozygotes that is in conclusion with Sardnal et al. [153, Table 2].
Krnke et al. [154] demonstrated that lenalidomide induces ubiquitination and
degradation of casein kinase 1 (CK1) in del(5q) MDS. Lenalidomide binds to cereblon
which acts as the substrate receptor of E3 ubiquitin ligase CRL4cereblon. This cullin-4 really
interesting new gene (RING) E3 ubiquitin ligase complex in the absence of lenalidomide
ubiquitinates cereblon itself and the other component of CRL4cereblon complex, DNA damage
binding protein 1 (DDB1) but in presence of lenalidomide it changes its specificity and
ubiquitinates CK1 in del(5q) MDS (Figure 2).
Two CDRs of chromosome 5 have been identified in del(5q) MDS: a distal locus (5q32-
33) is deleted in typical 5q- syndrome [isolated del(5q)] whereas deletions in a proximal locus
(5q31) are proper of forms of MDS with higher rate of progression.
Moreover, patients with deletions involving the centomeric and telomeric extremes of 5q
have a more aggressive disease phenotype and telomeric extremes of 5q have a more
aggressive disease phenotype and additional chromosomal lesions [155-159]. CK1 gene
(CSNK1A1) is located on chromosome 5q32 in a distal CDR. Inhibition of the serine-
threonine kinase CK1 sensitizes 5q- syndrome cells to lenalidomide [160]. Recently,
missense mutations in CSNK1A1 have been described [160-163] in patients with MDS.
Mutant CK1 enhanced expression of the -catenin and -catenin-related genes in the Wnt/
-catenin signaling pathway in the CD34+ cells and expansion of hematopoietic stem cells but
did not activate p53 signaling pathway. CSNK1A1 mutations were described only in 5-7.2%
of del(5q) MDS patients, all in exons 3 and 4 of CSNK1A1. As no CSNK1A1 mutation was
found in non-del(5q) MDS patients, CSNK1A1 mutation is highly specific for del(5q) MDS
patients. All del(5q) MDS patients with CSNK1A1 mutation responded to lenalidomide.
Preferential inhibition of cell growth in del(5q) MDS patients with CSNK1A1 mutation over
wild-type cells was observed after pharmacological inhibition of CK1 using a CK1
inhibitor. Thus, development of CK1 inhibitors may provide a new therapeutic opportunity
in del(5q) MDS patients with CSNK1A1 mutations.
Table 2. Analysis of the A/G polymorphism located at -29 nucleotides upstream of the transcription start site of the CRBN gene
(located on chromosome 3 at nucleotide 3179746; NC_000003.12; Homo sapiens, GRCh38)
/according to Sardnal et al. Leukemia 2013; 27: 1610-3/
Homozygot Heterozygot Homozygot Number of

Number of patients Number of G alleles
A AG G A alleles
MDS with isolated del(5q) 31 13 2 75 (81.5%) 17 (18.5%)
MDS with isolated del(5q) responders
12 4 0 28 (87.5%) 4 (12.5%)
to lenalidomide therapy (LT)
MDS with chromosome 5 without the
deletion of long arms (non5q-) and non- 5 2 0 12 (85.7%) 2 (14.3%)
responders to LT
Healthy controls 6 3 1 15 (75.0%) 5 (25.0%)
204 Ota Fuchs
FURTHER PROGNOSTIC FACTORS FOR THE EFFICACY OF

LENALIDOMIDE IN DEL(5q) MDS AND NON-DEL(5q) MDS
Lenalidomide is an effective drug in low-risk MDS with isolated del(5q) but not all
patients respond. Role of TP53 mutations and karyotype complexity in disease progression
and outcome was studied [39, 40, 157, 164, 165]. The negative prognostic impact of TP53
mutations has been reported. Recently, it was reported that strong p53 expression in >1% of
bone marrow progenitor cells correlated with TP53 mutations. Pyrosequencing analysis of
laser-microdissected cells with stong p53 expression confirmed TP53 mutations, whereas
cells with moderate expression were predominantly of wild-type p53 [164]. This study
validates p53 immunohistochemistry as strong and clinically useful predictive method in
patients with lower risk del(5q) MDS [164]. TP53 status was also assessed on RNA by the
functional analysis of separated alleles in yeast (FASAY) method. This assay evaluates the
transcriptional activity of p53 on p53-responsive promoter stably integrated in the yeast
genome [165]. This method together with next-generation sequencing are a reliable and cost-
effective methods to detect all TP53 aberrations including insertions and deletions. TP53 gene
status was not related to thrombocytopenia.
Figure 2. Schematic diagram of ubiquitination and degradation of casein kinase 1 by CRL4cereblon E3

ubiquitin ligase and proteasomes. Binding of cereblon (CRBN) by lenalidomide induces ubiquitination
(marking CK1 for degradation) and degradation of casein kinase 1. CRBN functions as a substrate
recognition component (substrate receptor) of this E3 ubiquitin ligase enzyme complex. CRL4 cereblon
complex consists of cullin 4A, RING finger protein regulator of cullins (Roc1), and DNA damage
binding protein 1 (DDB1).
TP53 polymorphism in exon 4 has been implicated in susceptibility and predidposition to

solid tumors. This polymorphism lies proximal to the DNA-binding domain and adjacent to
the proline-rich, proapoptotic domain of the p53. Substitution of a cytosine (C-allele) for the
guanine (G allele) results in a proline (P) rather than arginine (R) residue at position 72 of the
p53. Although McGraw et al. [166] found no significant difference in response rates by
genotype, the 2-year estimate for lenalidomide response duration was more than doubled for
CC compared with both CG and GG allele dosage.
Mallo et al. [14, 157] and Jonasova et al. [167] described platelet count as a prognostic
factor for response low risk del(5q) MDS patients to lenalidomide treatment. By multivariate
analysis, the most important predictor for lenalidomide treatment failure was a platelet
count <280 x 109/l.
New cytogenetic tools such as fluorescence in situ, hybridization (FISH) and single
nucleotide polymorphism array (SNP-A)-based karyotyping, increased the diagnostic yield
over metaphase cytogenetics. Sugimoto et al. [168] have recently found with help of these
new cytogenetic tools that normal karyotype and gain of chromosome 8 were predictive of
response to lenalidomide in non-del(5q) patients with myeloid malignancies. Mutational
analysis of TET2, UTX, CBL, EZH2, ASXL1, TP53, RAS, IDH1/2, and DMT3A was
performed and revealed 13 mutations in 11 patients from 21 non-del(5q) patients, but did not
show any molekular markers of responsivenes [168].
The responses of non-del(5q) MDS patients to lenalidomide seem to be inversely
correlated to the pre-treatment EPO levels [169].
A gene signature and correlation with presence of somatic mutations in lenalidomide
responsive cases is under investigation in non-del(5q) MDS patients. Cereblon levels may be
also important biomarker for lenalidomide efficacy [152].

Lenalidomide is currently the treatment of choice for lower risk transfusion-dependent
del(5q) MDS patients, and remains a treatment alternative for the management of anemia in
lower risk MDS without 5q deletion MDS patients with adequate neutrophil and platelet
counts [31-37, 45, 49, 83, 111, 168-180]. Lenalidomide has also activity in higher risk MDS
and AML with del(5q) and even in non(del5q) MDS [168, 169].
Though the mechanism of lenalidomide action has not been definitively determined, it is
clear that there is a difference between mechanisms in MDS with del(5q) and in
non-del(5q) MDS.
In MDS with del(5q), lenalidomide acts through inhibition of phosphatase activity in the
commonly deleted region of the long arm of chromosome 5. This phosphatases play a key
role in in cell cycle regulation. The inhibition of these phosphatases by lenalidomide leads to
G2 arrest, followed by apoptosis of del(5q) specimens. The direct cytotoxic effects of
lenalidomide on the del(5q) clone are also very important. Lenalidomide inhibits the
malignant clone and up-regulates the SPARC (secreted protein acidic and rich in cysteine)
gene mapping to the commonly deleted region in 5q- syndrome patients [181]. However,
SPARC is dispensable for murine hematopoiesis [182]. While haploinsufficiency of the
RPS14 gene appears to be a key contributor to erythropoietic failure associated with del(5q)
MDS, the critical genes responsible for clonal dominance in del(5q) high-risk MDS and AML
are less well-defined. It is known that this deleted region is different in del(5q) high-risk MDS
and AML [183]. The effect of lenalidomide in these cases needs to identify further biologic
206 Ota Fuchs
features accounting for the response, thereby allowing rational use of this drug, both alone
and in combination with another agents.
In MDS with non-del(5q), an increased expression of adhesion molecules caused by
lenalidomide treatment leads to recovery and maintenance of the CD34+ cells through
interactions between the hematopoietic and stromal cells. This effect of lenalidomide on the
bone marrow microenvironment causes abrogation of the function of pro-apoptotic and pro-
inflammatory cytokines. Lenalidomide is capable to increase red blood cell production
independently of ribosome dysfunction. Lenalidomide restores and promotes effective
erythropoiesis without direct cytotoxic effect. Lenalidomide activates the EPO-R/STAT5
pathway.
Lenalidomide is nowadays an accepted standard treatment for del(5q) MDS. In non-
del(5q) disease, its role is more difficult ot define. Studies have shown that about 18% of
patients treated with a standard dose of 10 mg/day on 21 out of 28 days might achieve
erythroid transfusion independence rates that last 6 months or longer. The responses to
lenalidomide seem to be inversely correlated to the pre-treatment EPO level. The higher the
EPO level, the lower the responses. In the absence of other cytogenetic or molecular
predictive factors that allow to discern which patient benefit most from treatment, its
incorporation into the treatment algorithm is dependent on the available alternatives,
including erythropoietic agents, immunosupressive treatments and experimental strategies
like thrombopoietin receptor agonists or the antagonists of transforming growth factor beta.
Given that 90% of responses to lenalidomide occur within four months of treatment, patients
not responding within this time frame should discontinue therapy [169]. Future studies are
needed to find prognostic biomarkers for lenalidomide response in non-del(5q) MDS.
The presence of multiple cellular and genetic abnormalities in MDS is common and
suggests that combination therapy targeting different mechanisms of action may be beneficial,
particularly in higher-risk disease, for which both microenvironment and cell regulatory
mechanisms play a role. The optimal dose, schedule and duration of treatment is still an area
of active investigation, especially in the use of lenalidomide combinations with other drugs.
ACKNOWLEDGMENTS
This work was supported by the research grant NT/13836-4/2012 from the Ministry of
Health of the Czech Republic.
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Chapter 9
THE OLD AND THE NEW-INTEGRATING PROGNOSTIC

MODELS AND MUTATIONAL ADVANCES WITH
EPIGENETIC AND CELLULAR THERAPIES
FOR MYELODYSPLASTIC SYNDROMES
Uma Borate and Antonio Di Stasi

The University of Alabama at Birmingham,
Birmingham, AL, US
ABSTRACT
Myelodysplastic syndromes (MDS) is a heterogeneous group of diseases
characterized by an indolent or aggressive course based on age, blood and blast counts
and aberrant cytogenetics, within a background of characteristic molecular alterations.
While supportive treatment is the mainstay approach for patients with low-risk disease,
hypomethylating agents are now the first-line treatment for patients with higher-risk
MDS, often used as a bridge to allogeneic hematopoietic stem cell transplantation
(HSCT), which remains the only curative option only for about 40-50% of patients.
However, considering HSCT-related morbidity and mortality risks it is a suitable
therapeutic option only for younger patients (generally up to 70years of age) without
significant comorbidities, and since the prognosis of patients who lose response or
progress while on hypomethylating agents is extremely poor alternative strategies are
needed. We discuss how to improve current prognostic models like IPSS and IPSS-R by
integrating newer somatic mutations and discuss new epigenetic modulators being
evaluated as promising new treatment of patients with MDS.
Additionally, in order to reduce relapse rate and treatment related morbidity and
mortality, we will discuss a newer interesting approach under pre-clinical investigation
consisting of chimeric antigen receptor (CAR) modified T-cells redirected against
myeloid antigens, as preparatory conditioning to an allogeneic HSCT for MDS and/or
acute myeloid leukemia (AML). Newer gene modification strategies are available
potentially enabling the specific targeting of myeloid blasts whilst sparing normal
E-mail:uborate@uabmc.edu, Phone: 205-934-7841, Fax: 205-975-8394.
E-mail:adistasi@uabmc.edu, Phone: 205-934-0688. Fax: 205-975-8394.

220 Uma Borate and Antonio Di Stasi
hematopoietic cells, with the goal of extending CAR T-cell therapies also to HSCT-
ineligible patients, and they warrant investigations in MDS.
INTRODUCTION
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic
disorders, defined by peripheral blood cytopenias due to ineffective hematopoiesis and
dysplastic morphologic changes with or without clonal chromosomal abnormalities.
MDS has an increased risk of progression to acute myeloid leukemia (AML) [1, 16].
MDS RISK FACTORS

Demographics
The most significant risk factor for developing MDS is advanced age, with yearly
incidence rates increasing 10-times for people in their 8th decade of life compared with the
general population [1] Men also have an increased incidence rate compared to women (4.4 vs.
2.2 per 100,000), as do whites compared with blacks (3.3 vs. 2.4 per 100,000) [1].
Pre-Existing Cancers and Therapy for Previous Cancers
Secondary MDS accounts for approximately 10% of all MDS diagnoses. Most cases
result from chemotherapy for other solid tumors like breast, colon or lung cancers, with
alkylating agents, topoisomerase inhibitors and radiation therapy [2]. The typical latency
period for secondary MDS after exposure to alkylating agents or radiation therapy is 5 to 7
years. The risk appears to be dose-dependent and is associated with unbalanced translocations
involving chromosome 5 or 7, or complex cytogenetics [3-5]. MDS after exposure to
topoisomerase inhibitors is less common, with a latency period of approximately 2 years and
is associated with a balanced translocation involving 11q23 (the MLL gene) [6].
Secondary MDS and AML is also seen in patients with Hodgkins and non- Hodgkins
lymphoma who have undergone autologous bone marrow transplantation. In these patients,
rates of secondary MDS/AML were approximately 7% at 10 years and 20% at 20 years of
follow-up. All of these patients were heavily pretreated with alkylating agents and
topoisomerase inhibitors, and received cyclophosphamide/total body irradiation or busulfan/
cyclophosphamide preparative regimens for their transplantation [7-11]. Other studies have
shown an association between MDS and other independent hematological premalignancies
like MGUS [12] and after use of purine analogs for lymphoid malignancies [13]. MDS may
also evolve from other antecedent hematologic disorders, particularly polycythemia vera,
after treatment with alkylating agents [14].
The Old and the New-Integrating Prognostic Models 221
Environmental Factors
MDS can arise after environmental and occupational exposure to organic solvents, such
as benzene and its derivatives has been seen in patients working in the rubber and oil
industries [15, 16] Case-control studies confirm an increased risk of MDS from exposure to
agricultural chemicals and radiation [17, 18].
A casecontrol study consisting of 403 newly diagnosed MDS patients and 806
individually gender and age-matched patient controls from 27 major hospitals in Shanghai,
China, examined the relationship of lifestyle, environmental, and occupational factors to risk
of MDS. The study showed that for all MDS subtypes combined anti tuberculosis drugs were
an independent risk factor, while benzene, hair dye new building and renovations and
pesticides were relative risk factors. Risk factors of MDS subtype refractory cytopenias with
multiple dysplasia (RCMD) were benzene as an independent risk factor and traditional
Chinese medicines, pesticides and herbicides as relative risk factors [19]. Smoking tobacco
was significantly associated with refractory anemia with excess of blasts (RAEB). Education
was shown as an independent protective factor against all subtypes of MDS.
A meta-analysis of 10 studies, examined the association between smoking and the
development of MDS showed the odds ratio for MDS developing in smokers was 1.45 (95%
CI, 1.21, 1.74), indicating a 45% increase in risk. Alcohol increased the risk, albeit not
significantly [20].
CYTOGENETICS AND PROGNOSIS

The prognosis of patients with this disease varies significantly based on both patient and
disease characteristics. While cytogenetics play a central role in the different prognostic
systems, our knowledge of somatic mutations and epigenetic abnormalities in this disease is
increasing rapidly. These findings are likely to be incorporated into future prognostic models
and may help guide therapeutic options.
The clinical behavior of MDS in terms of rate of progression of marrow failure,
transfusion dependence, risk of progression to AML and overall survival varies greatly
among patients with this disease. Various classifications have been proposed over the years in
an attempt to better prognosticate patient outcomes. The French-American-British (FAB)
classification is the oldest scheme for the classification of MDS. (Table 1) It divides MDS
into five subtypes based on the bone marrow and peripheral blood morphology, namely
percentage of blasts in the peripheral blood and bone marrow, peripheral blood absolute
monocytosis, and the presence or absence of ring sideroblasts.21 However, other features of
the disease including cytogenetic abnormalities were not included, due to our very limited
knowledge of these changes when the FAB classification system was developed.
The World Health Organization (WHO) re-classified MDS in 2000 and 2008 (Table 2)
based on clinical data, but the system remained predominantly a morphologic classification.
The WHO 2008 classification system sub-classifies the low-grade MDS (i.e., less than 5%
marrow blasts) based on the number of lineages demonstrating dysplastic changes. Patients
with refractory cytopenia with unilineage dysplasia (RCUD), including RA, refractory
neutropenia, refractory thrombocytopenia, and RA with ring sideroblasts (RARS), have a
more favorable outcome compared with those low-grade MDS patients with multilineage
dysplasia, including refractory cytopenias with multilineage dysplasia (RCMD) and RCMD
with ring sideroblasts.
Table 1. FAB classification of Myelodysplastic Syndromes
Peripheral blood Bone marrow

Refractory anaemia (RA) Blasts 1% Blasts < 5%
Monocytes 1 x 109/l Ringed sideroblasts 15%
Refractory anaemia with ringed Blasts 1% Blasts < 5%
sideroblasts (RARS) Monocytes 1 x 109/l Ringed sideroblasts > 15% of
erythroid precursors
Refractory anaemia with excess Blasts < 5% Blasts 5% - 20%
blasts (RAEB) Monocytes 1 x 109/l
Chronic myelomonocytic leukaemia Monocytes > 1 x 109/l Blasts 20%
(CMML) Blasts < 5%
Refractory anaemia with excess Blasts 5% Or blasts > 20% - 30%
blasts in transformation (RAEB-t) Or Auer rods Or Auer rods
It lowers the blast threshold for the diagnosis of AML from 30 to 20%, eliminating the
diagnosis of refractory anemia with excess blasts in transformation (RAEB-t) by FAB
criteria.
Refractory anemia with excess blasts has been subdivided into RAEB-1 (5-9% marrow
blasts) and RAEB-2 (10-19% marrow blasts) again based on differences in prognosis. The
2008 WHO classification also adds a category for unclassifiable cases that do not fit into
other categories and for atypical presentations of MDS, such as those with extensive fibrosis
[22]. The WHO classification does include one genetically-defined subtype, MDS with
isolated del(5q).
However, aside from marrow morphology, both the FAB and WHO systems do not
include factors that have been shown to affect the course of MDS.
Factors known to influence MDS outcomes include patient age, comorbid conditions
[23], performance status, transfusion dependence, LDH, cytogenetics, and marrow fibrosis.
Multivariate analyses have demonstrated the significant impact of marrow cytogenetic
abnormalities on the prognosis of patients with MDS [24]. A clonal abnormality is defined by
the International System for Human Cytogenetic Nomenclature as two or more cells with the
same chromosome gain or structural rearrangement, or three cells with the same chromosome
loss [25].
The most widely accepted MDS risk assessment model is the International Prognostic
Scoring System (IPSS), which uses marrow blasts, cytogenetic changes, and the number of
peripheral blood cytopenias to classify MDS cases into 4 prognostic subgroups: low risk
(score 0), intermediate-1 risk (score 0.5-1.0), intermediate-2 risk (score 1.5-2.0) and high risk
(score > 2.5) [26](Table 3).
Table 2. WHO classification of myelodysplastic syndromes
Disease Blood findings Bone marrow findings

Refractory anaemia (RA) Anaemia Erythroid dysplasia only
No or rare blasts < 5% blasts
< 15% ringed sideroblasts
Refractory anaemia with ringed Anaemia 15% ringed sideroblasts
sideroblasts (RARS) No blasts Erythroid dysplasia only
< 5% blasts
Refractory cytopenia with multilineage Cytopenias (bicytopenia or pancytopenia) Dysplasia in 10% of the cells of two or more myeloid cell lines
dysplasia (RCMD) No or rare blasts < 5% blasts in marrow
No Auer rods No Auer rods
< 1x109/L monocytes < 15% ringed sideroblasts
Refractory cytopenia with multilineage Cytopenias (bicytopenia or pancytopenia) Dysplasia in 10% of the cells in two or more myeloid cell lines
dysplasia and ringed sideroblasts No or rare blasts 15% ringed sideroblasts
(RCMD-RS) No Auer rods < 5% blasts
< 1x109/L monocytes No Auer rods
Refractory anaemia with excess blasts -1 Cytopenias Unilineage or multilineage dysplasia
(RAEB-1) < 5% blasts 5-9% blasts
No Auer rods No Auer rods
< 1x109/L monocytes
Refractory anaemia with excess blasts -2 Cytopenias Unilineage or multilineage dysplasia
(RAEB-2) 5-19% blasts 10%-19% blasts
Auer rods Auer rods
< 1x109/L monocytes
Myelodysplastic syndrome - unclassified Cytopenias Unilineage dysplasia: one myeloid cell line
(MDS-U) No or rare blasts < 5% blasts
No Auer rods No Auer Rods
MDS associated with isolated del(5q) Anaemia Normal to increased megakaryocytes with hypolobated nuclei
Usually normal or increased platelet count < 5% blasts
< 5% blasts Isolated del(5q) cytogenetic abnormality
No Auer rods
The system was developed prior to the WHO classification, and therefore, incorporated
21-30% marrow blasts. Importantly, this model only applies to patients at the time of
diagnosis with de novo MDS who received supportive care alone. Therapy-related MDS
generally has a much worse prognosis than de novo disease.
Table 3. IPSS Prognostic Variables and Weights
Score Value
Prognostic Variable 0 0.5 1.0 1.5 2.0
BM blasts (%) <5 5-10 11-20 21-30

Karyotype* Good Intermediate Poor
Cytopenias 0/1 2/3
Scores for risk groups are as follows: Low, 0; INT-1, 0.5-1.0; INT-2, 1.5-2.0; and High, 2.5.
*
Good, normal, -Y, del(5q), del(20q); Poor, complex ( 3 abnormalities) or chromosome 7 anomalies;
Intermediate, other abnormalities.
These risk groups showed significantly different overall survival and risk of AML
transformation. Median survival for low-risk patients was 5.7 years, 3.5 years for
intermediate-1 risk, 1.2 years for intermediate-2 risk, and 0.4 years for the high risk disease
group.
The IPSS has several limitations, the most important of which is it does not characterize
patients with lower-risk disease (IPSS low or intermediate-1 risk) very well in terms of
prognosis. This group accounts for two-thirds of patients with MDS, some of whom may
possibly benefit from early intervention. As this system was developed at initial diagnosis in
patients with MDS, it cannot be used reliably during the course of the disease and its
evolution. Furthermore, the model was based on cytogenetic information from 816 patients,
two thirds of whom had a normal diploid karyotype [26]. The prognostic significance of less
common single or double cytogenetic changes (i.e., other than del(5q), chromosome 7
abnormalities, trisomy 8, del(20q) and Y) could not be accurately assessed; these were
arbitrarily included in the intermediate risk karyotype group. However, less common
cytogenetic changes could impact prognosis [27].
Schanz et al. evaluated a greater number of MDS cases (2902 patients) with cytogenetic
data from several different groups, and were able to propose a new comprehensive
cytogenetic scoring system [28]. This data set allowed the investigators to estimate the
prognostic impact of less common single and double cytogenetic changes [29]. Nineteen
cytogenetic abnormalities were identified.
Cytogenetic categories have a more significant effect on survival compared with marrow
blast percentage [13]. They divided these cytogenetic abnormalities into five prognostic
subgroups: very good, good, intermediate, poor and very poor [28] (Table 4).
The Old and The New-Integrating Prognostic Models and Mutational Advances 225
Table 4. Cytogenetic classes according to Schanz et al.
This new cytogenetic scoring system provided the foundation for the revised IPSS (IPSS-
R) [30](Table 5). The major differences between the IPSS-R and its predecessor include five
cytogenetic subgroups rather than three, classification of the less common cytogenetic
abnormalities, division of the lowest marrow blast subgroup into < 2% blasts and 2 to < 5%
blasts, and weights for the degree of individual cytopenias. A significant proportion of
patients within the IPSS lower-risk group (27%) would be upstaged in the IPSS-R
classification, while 18% in the higher risk group would be downstaged by IPSS-R.
The score by IPSS-R correlated with the risk of dying and risk of leukemic
transformation (Table 6).
AML 25%: Time to evolution to AML in 25% of risk population.
Table 5. IPSS-R Prognostic Factors and Weights
0 0.5 1 1.5 2 3 4
Cyto Very Good Int Poor Very
Good Poor
Blasts <2% 2 - < 5% 5 - < 10% >10%
HGB 10 g/dL 8-10 g/dL <8 g/dL
Platelet 100,000 50,000 - <50,000
100,000
ANC 0.8 <0.8
Table 6. IPSS-R and Prognosis
Very Low Low Intermediate High Very High

(< 1.5) (> 1.5-3) (> 3 4.5) (> 4.5 6) (> 6)
OS (years) 8.8 5.3 3.0 1.6 0.8
AML, 25% (years) NR 10.8 3.2 1.4 0.7
Table 7. WPSS classification Prognostic Variables and Weights
Variable 0 1 2 3
WHO class RA, RARS, 5q RCMD RS RAEB-1 RAEB-2
Karyotype Good Intermediate Poor
Transfusions No Regular
Risk groups: Very low (score = 0), Low (score = 1), Intermediate (score = 2), High (score = 3-4), Very
high (score = 56).
However, there are features that clearly have a negative impact on but are not included in
either the IPSS or IPSS-R, including advanced age, performance status, comorbid illnesses,
transfusion dependence, abnormal marrow blast percentage and marrow fibrosis. As
transfusion requirements were found to be an independent prognostic factor in the survival of
MDS patients, another scoring system that specifically takes into account the transfusion
needs of the patient was developed [31].
This scoring system uses the 2008 WHO MDS classification and is called the WHO
classification-based Prognostic Scoring System (WPSS) [32]. (Table 7) The WPSS can also
be applied at any time during the course of the disease. However, one major limitation of the
WPSS scoring system is the requirement of accurate information about prior transfusion
requirements of the diagnosis of MDS which is not always available. The WPSS
classification has now been modified to include hemoglobin levels instead of transfusion
requirements [33].
The global MD Anderson Cancer Center (MDACC) model can be used to evaluate
patients with chronic myelomonocytic leukemia (CMML) and treatment-related
myelodysplastic syndrome, at any time during the course of the disease [34]. These patients
had been excluded from both the IPSS and IPSS-R. This scoring system incorporates age,
performance status, and transfusion requirement, which adversely affect the prognosis of
MDS patients, but are not included in the IPSS or IPSS-R. The degree of anemia,
thrombocytopenia and leukocytosis (for CMML) are also included in the model. The global
MDACC model has not been validated but is intended to evaluate all MDS patients anytime
during their course of their disease without WHO classification of their disease pathology.
Patients with 0 to 4 points had a median survival of 54 months and a 3 year survival of 63%.
Patients with 5 and 6 points had a median survival of 23 to 30 months and 3-year survival of
30 to 40%. Patients with 7 to 8 points had a median survival of 13 months and a 3-year
survival rate of 13 to 19%. Patients with 9 or more points had a median survival of 5 to 10
months and a 2% 3-year survival. She has a combined score of 8 due to performance status 2
(2), age over 65 years (2), platelet count 50,000 to 199,000/microliter (1), hemoglobin less
than 12 g/dL (2), and transfusion dependence (1).
Table 8. MDACC MDS Lower Risk Prognostic Model
Characteristics Points
Unfavorable cytogenetics 1
Age 60 years 2
Hemoglobin < 10 g/dL 1
Platelets
<50 x 109/L 2
50-200 x 109/L 1
Bone Marrow blasts 4% 1
Score Median Survival 4-year OS (%)

_________________________________________________________
0 NR 78
1 83 82
2 51 51
3 36 40
4 22 27
5 14 9
6 16 7
7 9 NA
Table 9. Somatic mutations in MDS [21-40]
Mutation Chromosome Frequency Function Clinical significance in

Location (%) mutated cases
TET2 4q 20-26 Control of cytosine Inconsistent impact on
hydroxymethylation survival: improved
response to azacitidine
RUNX1 21q Up to 20 Transcription Factor Decreased survival
ASXL1 20q 10-15 Epigenetic regulator Decreased survival
EZH2 7q 2-6 Polycomb group protein Decreased survival
DNMT3A 2p Up to 8 Transcription Factor Decreased survival and
increased risk of
sAML
CBL 11q 1 Signal Transduction Unknown
IDH1/IDH2 2 q/15q 5-10 As IDH1/Cell metabolism, Decreased survival
epigenetic regulation (unknown for IDH2)
Patients with lower risk MDS remain quite heterogeneous in terms of prognosis. These
patients have a wide range of outcomes. To better prognosticate in this category, Garcia-
Manero et al. evaluated outcomes in a series of 856 patients with low or int-1 disease by IPSS
seen at the M. D. Anderson Cancer Center over a 30 year period (1976-2005) [20] Using
multivariate analysis they developed a risk assessment model for patients with lower risk
MDS. Unfavorable karyotype included all karyotypes except for del(5q) alone and normal
diploid. Patient age, degree of cytopenias and elevation of the bone marrow blast above 4%
were included in the model [35]. (Table 8) The model has been validated by Bejar et al. [36].
Characteristics were selected from multivariate analysis model in patients with lower risk
MDS. Each characteristic is associated with a number of points. Score is calculated by adding
all points. Each score allows calculation of median survival in months and probability of
survival at four years.
Over the last 2-3 years, several new and clinically significant somatic mutations have
been identified in varying frequencies in MDS (Table 9).
Some of these appear to affect the prognosis of the disease. TET2 is one of the most
frequently mutated genes in MDS; TET2 mutations appear to be prognostically favorable in
MDS [37]. The 5-year overall survival is 76.9% in mutated versus 18.3% in unmutated
patients (p = 0.005). The 3-year leukemia-free survival is 89.3% in TET2 mutated versus
63.7% in unmutated patients (p = 0.035). In multivariate analysis, the presence of TET2
mutation was an independent favorable prognostic factor irrespective of the MDS subtype.38
RUNX1 point mutations have been identified in MDS and MDS-related AML [39, 40]. About
20% of MDS patients may have these mutations, with a higher incidence in secondary MDS
compared to de novo disease. This mutation is associated with a worse prognosis compared
with those without the mutation [41].
The ASXL1 gene is an epigenetic regulator and its mutations are a poor prognostic factor
for overall survival independent of other established risk factors in multivariate analyses [42,
43]. Mutations of the EZH2 gene located on chromosome 7 have been discovered in several
myeloid malignancies including MDS [44-46]. Over-expression of EZH2 gene is generally
associated with poor prognosis in MDS, especially in lower-risk cases [46, 47]. DNMT3A
mutations are found in approximately 8% of de novo MDS cases. DNMT3A mutations may
occur early in the course of MDS and patients with DNMT3A mutations have worse overall
survival and an increased risk of progression to acute leukemia [48, 49]. IDH1/2 genes are
mutated in approximately 10% of MDS patients [50]. In MDS with sole del(5q) cytogenetic
abnormality, mutant IDH has been associated with poor overall and leukemia-free survival
[51]. The prognostic effects of IDH1 and IDH2 mutations among patients with MDS in
association with IPSS-R showed an adverse prognostic effect of mutant IDH1, but not mutant
IDH2, on both overall and leukemia-free survival [52]. Mutations in the components of the
RNA splicing machinery also play an important role in the pathogenesis of MDS. Genes
involved in the spliceosome like U2AF1, SRSF2 and SF3B1, are frequently mutated in MDS.
SF3B1 is mutated in most patients with MDS with increased ring sideroblasts (84.9%) [53,
54]. In some studies patients with SF3B1 mutations had fewer cytopenias and better event-
free survival but other studies found that found that SF3B1 mutation had no additional
prognostic value in MDS [55, 56]. Mutation of any one of the following five genes has a
negative impact on the prognosis of MDS patients predicted by the IPSS: TP53, EZH2,
ETV6, RUNX1, and ASXL1 [46, 47].
At present, the most widely accepted prognostic models in MDS are based predominantly
on marrow morphology, cytogenetics and cytopenias. Patient-specific features, such as age,
performance status and comorbid illness, affect prognosis, as well as choice of therapy, but
are not considered in the IPSS or IPSS-R. Since the marrow karyotype is normal in over 50%
of MDS patients, the usefulness of the IPSS and IPSS-R is limited. Fifty percent of MDS
patients with normal karyotype will have somatic mutation in at least one of a few genes [24].
Many of these genes encode proteins involved in epigenetic modulation of gene transcription,
such as components of the RNA splicing machinery, regulators of DNA methylation, and
enzymes of histone modification.
These somatic mutations will likely soon be incorporated into the next generation of
MDS prognostic models.
Treatment
Treatment decisions are usually based on risk stratification of patients using the IPSS
prognostic classification. However as we increasingly adopt the IPSS-R and include other
point mutation data in our prognostic models, this will likely change.
ERYTHROID GROWTH FACTOR SUPPORT

Low Risk MDS
Therapy in these patients is based on the transfusion dependence of the patients. For
patients that are transfusion independent, close watchful waiting is recommended, unless they
have severe cytopenias or become symptomatic.
Several agents are approved for use in this setting with response rates from 30 to 60%
depending on study [57]. Adding G-CSF to erythropoietin increases responses rates and a
retrospective observational study showed that early introduction of this combination in
patients with low risk disease and minimally transfusion-dependent patients may have an
impact of survival [58]. This study also developed an algorithm to predict response to ESAs
[59]. The French group has also evaluated the impact of ESA on survival in a retrospective
study of 284 patients and compared it with the group of patients that formed the IPSS cohort.
In this study patients exposed to ESA had a better survival (HR for death was 0.43, 95% CI
0.250.72). Therapy should be continued for at least 3 months to judge efficacy and
continued until they start to need transfusions again [60].
LENALIDOMIDE
Low Risk MDS
Lenalidomide is approved in the US for patients with lower risk MDS, anemia and
alteration of chromosome 5 [61] but not approved in Europe due to concerns of increase
transformation to AML in patients treated with this compound. A phase II study of
lenalidomide in patients with anemia and del chromosome 5 where 148 patients received 10
mg of lenalidomide for 21 days every 4 weeks or daily showed 112 had decreased need for
transfusions (76%; 95% CI 68-82) and 99 patients (67%; 95% CI, 59 to 74) became
transfusion independent. Response was rapid with a median response time of 4.6 weeks. The
median rise of hemoglobin was 5.4 g/dL. Cytogenetic responses were observed in close to
50% patients. Predictors of response included presence of a platelet count of 100 x 109/L and
less than 4 prior units of red cells transfused. However, in this study patients with a platelet
count of less than 50, 000 were excluded [62].
In a parallel study, lenalidomide was investigated in patients without chromosome 5

alterations. In this study 214 patients received 10 mg oral lenalidomide daily or 10 mg on
days 121 of a 28-day cycle. Fifty-six (26%) patients achieved transfusion independence after
a median of 4.8 weeks of therapy. Median response duration was 41.0 weeks. Lenalidomide is
now approved for this indication at the present time [63].
Two large randomized phase III clinical trials are currently being conducted in MDS. The
AZA-004 has completed accrual and studied two different doses of lenalidomide (5 and 10
mg orally daily) versus placebo. This study was also designed to clarify the issue of
transformation to AML. This study reported that a dose of 10 mg daily was superior and that
there was no increase incidence of transformation to AML in patients treated with
lenalidomide [64]. Of importance, a recent report from the initial MDS-003 trial of
lenalidomide has indicated a longer survival for patients responding to therapy [65]. This is
additional evidence that lenalidomide can change the natural history of patients with 5q-
MDS Another Phase III is studying the role of lenalidomide in patients with lower risk
disease without an alteration of chromosome 5. In an initial study, 214 patients received
lenalidomide 10 mg orally daily or 10 mg on days 1 to 21 of a 28-day cycle. Fifty six (26%)
patients achieved transfusion independence after a median of 4.8 weeks of therapy. Median
response duration was 41 weeks [63]. Preliminary results of a phase III trial in this patient
population were presented at ASH 2014 (Santini et al., unpublished) and supported the
findings of the initial phase II trial [63]. This is not currently an approved indication of
lenolidamide.
AZANUCLEOSIDES
Low Risk MDS
Two azanuclesoides are approved for MDS: 5-azacitidine [66] and 5-aza-20-
deoxycitidine (decitabine) [67]. 5-azacitidine is approved for all subsets of MDS whereas for
those with INT-1 disease and above. There is little data in the use of these compounds in
lower risk MDS. None of them have been shown to modify the natural history of patients
with lower risk disease. Different schedules of 5-azacitidine have been explored in MD
including a 5-day schedule, a 7 day 5-2- 2 schedule (weekend off) or a 5-2-5 schedule of 10
days [68]. Oral azacitidine is currently being studied in an international phase III trial in
patients with lower risk MDS and significant cytopenias (NCT01566695).
There is relatively little data with decitabine in patients with lower risk disease. Presently
both 5-azacitidine and decitabine are used in the US in patients with lower risk disease that
are transfusion dependent and have failed or were not candidates for growth factor support or
lenalidomide. Further studies of these agents in lower risk MDS are needed.
High Risk MDS
5-Azacitidine has been studied in higher-risk MDS in two major randomized multicenter
trials: CALGB 9221 and AZA-001. In the CALGB 9221 study, 191 patients with MDS were
randomized between 5-azacitidine (75 mg/m2/day for 7 consecutive days every 28 days) and
best supportive care (BSC). Median age was 68 years. Sixty percent of the patients in the
5-azacitidine group, compared with 5% of control arm patients, responded to treatment
(P < 0.0001). The median time to leukemic transformation or death was 21 months in patients
treated with 5-azacitidine versus 12 months in the BSC arm. No significant difference in
survival was observed. A landmark analysis suggested a survival advantage for patients
initially on 5-azacitidine or who had crossed-over to 5-azacitidine within 6 months of
inclusion on study [69]. A significant improvement in quality of life was documented in
patients treated with 5-azacitidine compared with BSC [70]. AZA-001 was a randomized
study designed to test the concept that treatment with 5- azacitidine resulted in improved
survival compared with several standard of care options [66]. These included BSC, low dose
cytarabine (ara-C) or AML-like therapy. In AZA-001, 358 patients with higher-risk MDS
were randomized to either 5-azacitidine (as per CALGB9221 schedule) or to standard of care.
Median age of patients was 69 years. Median survival was significantly better in patients
treated with 5-azacitidine versus standard of care options: 24.5 months versus 15 months
Progression to AML was significantly delayed, and RBC transfusion requirements and rate of
infections were also significantly improved with 5-azacitidine. The survival advantage with 5-
azacitidine was irrespective of age (including patients older than 75 years), percent of marrow
blasts (including patients with 20%30% blasts, now classified as AML using WHO criteria)
or karyotype. This effect was significant when compared with BSC and low dose ara-C. The
number of patients treated with AML like therapy was too small to allow comparison with
5-azacitidine.
Decitabine was studied in an initial randomized comparing it to BSC. In this study the
dose of decitabine was 15 mg/m2 IV infused over 3 hours every 8 hours for 3 days (at a dose
of 135 mg/m2 per course) and repeated every 6 weeks. Although there was no clear benefit in
terms of survival in this study, the use of decitabine was associated with a complete response
rate of 9% and overall response rate of 17% [67].
These results led to the approval of decitabine in the US. A Bayesian randomized phase II
trial of three different doses and schedules of decitabine showed that a 5-day schedule of
decitabine administered daily at a dose of 20 mg/m2 was shown to be superior to a 10-day or
subcutaneous schedule [71]. A multicenter phase II trial of decitabine (ADOPT) using the 5-
day schedule confirmed the safety of this schedule [72].
In the ADOPT study the median number of courses administered was five, the CR rate
was 17% and the median survival was 19.4 months. No randomized survival study of a 5-day
schedule of decitabine has been conducted in MDS.
European investigators looked at a randomized study of decitabine using the initial 3-day
schedule. The use of decitabine at this dose and schedule was not associated with improved
survival in patients with higher risk MDS [73]. Despite all this data, the final dose and
schedule of decitabine is not fully understood. Blum et al. have indicated that a 10-day
schedule of decitabine has significant activity in AML [74].
Biomarkers of Response to Azanucleosides
Due to their capacity to induce DNA hypomethylation, the identification of methylation

patterns that would predict for response is an active area of interest.
Recently, a large-scale methylation analysis has resulted in the identification of 167

differentially methylated regions associated with response to hypomethylating agent based
therapy [75]. Of interest, most of these regions were localized in nonpromoter regions.
Two candidate biomarkers and a clinical model have been proposed. Levels of miR29b
[74] and mutations on TET2 [38] have been reported to be associated with response to
decitabine and azacitidine respectively. miR29b regulates expression levels of DNMT1 [74].
None of these two biomarkershave been confirmed in other larger studies [76].
Immune Therapy
Agents studied include antithymocyte globulin (ATG), cyclosporine, and steroids

modeled after therapy of aplastic anemia [77]. The NIH group also developed an algorithm to
predict response to these classes of agents. This model included younger age, HLADR15
positivity, and shorter duration of transfusion dependency [78]. Using this algorithm, the NIH
group reported that alemtuzumab, an antibody against CD52, has significant activity in
patients with MDS predicted to respond to immune suppressive therapy [79]. Data from a
Swiss study comparing ATG versus supportive care indicated a higher response rate, but no
survival benefit. Despite higher response rates no significant effect on survival or
transformation was observed [80].
Allogeneic Stem Cell Transplantation (AlloSCT)
AlloSCT is usually not recommended in patients with lower risk disease even if they are
young. This is based on data from Cutler et al. using a Markov mode land confirmed by
Koreth at al. [81, 82]. The anticipated early mortality with alloSCT cannot be overcome by
the potential benefit in survival of alloSCT. Using another Markov model, the investigators
analyzed the impact of reduced intensity transplant in older patients with MDS. Patients with
lower risk disease did not benefit from this less toxic transplant approach. AlloSCT is
reported to be the only curative treatment of higher-risk MDS. Results from selected studies
report prolonged DFS in about 30%50% of the patients. However its use is mainly restricted
to younger patients with an appropriate donor [83]. Different transplant modalities of
different intensities and donor sources are now in use. Current advances in transplant
technology are allowing the consideration of older patients and alternative donors. This
should result in greater number of older patients benefitting from this potentially curative
treatment modality.
There are several questions regarding alloSCT in MDS. These include timing of
transplant; and what to do with patients that achieved a complete response to
hypomethylating agent prior to alloSCT. A study from the IBMTR indicated that early
transplantation in higher-risk MDS was associated with longer life expectancy [81, 82].
Although data suggests longer survival with SCT in patients with higher risk disease, it
should be noted that curves cross close to 3 years after initiation of therapy when compared
with hypomethylating agents. It will be important to identify who are long-term survivors that
benefit the most from transplant. Other questions include whether or not alloSCT should be
preceded by a cytoreductive regimen (with chemotherapy or perhaps hypomethylating
agents). Many authors consider that when marrow blasts>10% at the time of transplant,
because of the very high relapse risk post-transplant, pretransplant therapy is required. Certain
cytogenetics and mutations have been shown to have poor outcomes with transplant, notably
monosomy 7, p53 and DNMT3A mutations have a very poor prognosis with alloSCT [84].
Although this data needs to be validated in more recent series, these results have significant
implications for the use of alloSCT in MDS, as this suggests that the current practice of
reserving transplant for poor prognostic features may not be indicated. In patients at higher
risk for relapsed post alloSCT, data from a single arm trial indicated that post-transplant
maintenance with hypomethylating agents can improve outcome [85].
INVESTIGATIONAL NEW OPTIONS FOR PATIENTS

RELAPSED/REFRACTORY MDS
Relapsed or Refractory Lower Risk MDS, Higher Risk MDS Including HMA
Failures
Patients that fail growth factors, lenalidomide or azanucleoside are candidates for clinical
trials or alloSCT. There are no drugs approved in the US for patients with low risk MDS and
HMA failure. Recently the survival of these patients was calculated to be between 14 and 17
months [86]. A number of agents are being studied for lower risk MDS. These include oral
azacitidine [87] and oral decitabine, agents that modulate TGF-B pathway such as
ACE-536 and ACE-011, [88, 89] proteasome inhibitors and antagonists of toll-like receptor
signaling [90, 91] Because the HMAs are the standard of care in front line higher risk MDS, a
number of studies are being conducted to test combinations. Agents used in combinations
include the histone deacetylase inhibitors [92] and lenalidomide [93]. So far none of these
combinations have been shown to be superior to single agent HMA. A second generation
HMA known as SGI-110, a dinucleotide form of decitabine is also in clinical trials.
At the present time there is no therapy approved for patients with higher risk MDS that
fail hypomethylating agents or relapsed after AML-like therapy or alloSCT. The group of
patients that failed hypomethylating agents has particularly poor prognosis with an estimated
survival of 46 months [94]. This is an area of active research. In a phase III study of
rigorsetib (Garcia-Manero, ASH 2014, unpublished) versus best supportive care, the study
drug was not shown to improve survival. Very low dose clofarabine may have a role in
patients with HMA failure and diploid cytogenetics (Jabbour, ASH 2014, unpublished). A
recent area of particular interest is the use of immune checkpoint inhibitors in MDS [95].
Multiple studies are investigating the role of this new class of agents in MDS. Finally, it is
becoming apparent that a fraction of patients with higher risk MDS are characterized by
targetable mutations such as Flt-3, RAS, and IDH1and IDH2. Multiple agents blocking these
mutations are currently under development [96, 97].
Gene Redirected T Cells Approaches in MDS/AML
T cells genetically redirected to recognize tumor associated antigens (TAAs) offer

promise in the management of patients with hematologic malignancies. T cells can be
genetically redirected by endowing them with a transgenic T-cell receptor (TCR) or a
chimeric antigen receptor (CAR). However, TCR redirected T cells are HLA restricted, and
TCR mispairing with the endogenous TCR could result in reduced avidity or unwanted
specificities [98]. Alternatively, CARs represent a universal platform for immune-therapy
because they are not HLA-restricted, combining the specificity of an antibody with the killing
machinery of the T cell in a single chain, [99] thus with a minimized risk of chain mispairing.
Additionally, recognizing antigens in an HLA independent fashion makes CAR T cells
intrinsically resistant to immune evasion strategies that could arise during antigen processing
or presentation [100]. Generally, CAR T cells can only recognize surface molecules, which
are often non-polymorphic and often shared between normal and tumor cells, raising justified
concerns about their safety. As a matter of fact, infusion of CAR redirected T cells has
resulted in complete remission of disease in cases of refractory leukemia, but at the expense
of frequent cytokine release syndrome (CRS), and even fatal on-target effects when targeting
TAA in solid cancers [101].
Although CAR T cells applications for AML have been investigated in vitro and in mice
models, their clinical development has been hampered by the evidence of significant
myelosuppression in the majority of the studies. Targeted antigens included CD33, CD44v6,6
CD123, Lewis (Le)-Y10 [102-106]. A clinical trial has been performed to date which used a
second generation autologous CAR T cells targeting the LeY difucosilated carbohydrate
antigen, [107] in 4 patients with AML, demonstrating in vivo expansion (~1,200 transgene
copies/1,000 cells in the peripheral blood), persistence up to 10 months, homing to bone
marrow and site of leukemia cutis and transient clinical benefit without serious adverse
events, despite the increased production of interferon-gamma detected in 2 patients. A second
clinical trial infused second generation autologous CAR T cells targeting CD33 in a patient
with refractory AML, showing mild CRS, marked decrease in AML blasts 2 weeks after
infusion, followed by florid relapse at week 9, suggesting that this strategy could be used for
patients with refractory disease to render them eligible to receive an hematopoietic stem cell
transplant [108].
CONCLUSION
The identification of risk factors, development of prognostic models and the therapies for
MDS is in rapid evolution. Although the IPSS was developed from patients receiving only
supportive care, the risk as assessed by IPSS has been shown to impact outcome with
hypomethylating agents and allogeneic hematopoietic stem cell transplant. Nonetheless,
currently used prognostic models have limited utility.
A retrospective Markov decision making analysis suggests that patients with high risk
disease should proceed to allogeneic hematopoietic stem cell transplantation, if possible [81].
However, even this data is of limited value for a variety of reasons. The analysis only applies
to patients under age 60 years who received myeloablative allogeneic hematopoietic stem cell
transplant between 1990 and 1999. The survival following allo HSCT has improved more
recently, likely due to improvement in supportive care [109]. Furthermore, the control
group received supportive care only. Azacitidine has been shown to improve survival of MDS
patients compared with other conventional care regimens including supportive care [66]. A
trial comparing hypomethylating agent therapy to reduced intensity allo HSCT using a using
a donor no donor analysis is being planned.
Currently, only del(5q) has been found to predict response to a therapy, lenalidomide,
[61] although the biologic basis of this association is still debated. We need to assess whether
mutations are associated with response to specific available therapies i.e., are predictive, not
just prognostic. In this way, we will be better able to choose treatment for our patients. True
progress in the treatment and prognosis of MDS patients will only be realized as we
understand the biologic effects of somatic mutations incorporate them into our prognostic
model along with known cytogenetic abnormalities and can effectively target the disrupted
cellular mechanisms that lead to MDS.
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Chapter 10
ENTROPY EVALUATION OF BONE MARROW BIOPSIES

IN MYELODYSPLASTIC SYNDROMES
Giorgio Bianciardi1, and Pietro Luzi2,

1
Department of Medical Biotechnology, Anatomia Patologica,
Siena University, Siena, Italy
2
Department of Medical Sciences, Surgical and Neuroscience,
Siena University, Siena, Italy
ABSTRACT
New data on fractal analysis of the bone marrow in myelodysplastic syndromes are
recently arising in order to perform differential diagnosis and prognosis in those diseases.
Here we report the use of Entropy, or Information Dimension, to evaluate bone marrow
biopsies in healthy subjects, in refractory anemia, in refractory anemia with excess blasts
(-1 & -2) and in acute myeloid leukemia. Entropy is statistically increased in the patients
compared with the normal condition (p < 0.01). Interestingly, entropy increases with the
severity of the lesions (p < 0.01). Evaluation of Entropy of the histologic images appears
to be able to add objective information relating to the differential diagnosis in
myelodysplastic syndromes.
Keywords: fractal analysis, entropy, myelodysplastic syndromes, differential diagnosis,

human pathology
INTRODUCTION
In biology complex structures have no characteristic length: often they have fractal, or,
more generally, scaling, properties. Fractality is a geometric concept related to highly
+ 39-577-233245. giorgio.bianciardi@unisi.it.
Dept. of Medical Sciences, Surgical and Neuroscience, Siena University, retired.

244 Giorgio Bianciardi and Pietro Luzi
irregular shapes, very complex objects that defy conventional measures, such as length,
perimeter and area and, mathematically, originates from simple iterated function [1] with non-
integer, or fractional, dimensions, and a property known as self-similarity. A natural fractal is
an object composed of subunits that resembles the larger scale structure, so maintaining the
same, at least statistically, complexity. Its present in a lot of biological structures [2-4]. The
meaning of these fractal structures in the human body is profound. The self-similar tracheo-
bronchial tree provides an enormous surface area for exchange of gases at the vascular-
alveolar interface, coupling pulmonary and cardiac functions [5]. For the vasculature, fractal
branching provide a rich network for distribution of nutrients and oxygen, as well for the
collection of metabolic waste products [6]. Fractal structures serve function, e.g., the fractal
organization of connective tissue in the aortic leaflets relating to the efficient distribution of
mechanical forces [7]. They can be quantified by indexes like as the fractal dimension, D,
measure of geometric complexity and of the space-filling properties of a structure or the
information dimension or entropy [2].
In healthy, the local fractal dimension, geometric complexity as well entropy, assumes
numerical values approaching the one of the Diffusion-limited aggregation (DLA), the
process whereby particles undergoing a random walk due to Brownian motion cluster
together to form aggregates of such particles [9]: retina vessels, microvascular oral mucosa,
muscle cell tissue, prostatic tissue and so on [10-13]. In healthy subject, also the bone marrow
present a fractal dimension value approaching the DLA process, as showed by Naeim et al.
[14], suggesting that the spatial organization of the bone marrow is dependent on the
diffusion of cytokines. In pathology, the local fractal dimension value, geometric complexity
or entropy, increases in comparison to healthy subjects [15].
In the present paper we have applied fractal analysis to study myelodysplastic syndromes
(MDS), in order to verify if the fractal approach (evaluation of entropy of the histological
images) may be able to distinguish among the different categories of MDS.
MATERIALS AND METHODS

Samples
70 bone marrow (BM) biopsy specimens were taken from the iliac crest of 70 subjects.
The diagnosis was based on the classification of the World Health Organization [16]: 20
samples of Refractory anemia (RA), 20 samples of Refractory anemia with excess blasts
(RAEB-1, RAEB-2), 10 cases of acute myeloid leukemia (AML), and 20 samples of normal
bone marrow.
The BM biopsy specimens were retrieved from the archive of Pathological Anatomy of
the University of Siena. The samples, approximately 1 cm in length, were prepared by routine
procedures of fixation, decalcification, dehydration and paraffin embedding. The sections
were prepared with a thickness of about 5 m, stained with hematoxylin and eosin, evaluated
at conventional light microscopy, using the 10x objective lens (Figure 1).
Entropy Evaluation of Bone Marrow Biopsies in Myelodysplastic Syndromes 245
Figure 1. Bone marrow in a healthy subject. Lipid droplets built the fractal structure of the bone marrow
at histological level, approaching the value of a Diffusion limited aggregation process. Hematoxylin-
eosin, original magnification, X 100.
Image Analysis
Image analysis was performed on a Kontron Elektronik Imaging System (KS-400: Zeiss).
The images were digitized with a resolution of 768X576 pixels at 24 bit (Matrox frame
grabber, JVC TK-C1381 color video camera). The contours were extracted and converted to a
single pixel outline (Figure 2).
Figure 2. Contours of the bone marrow histological images in a healthy subject (left), in Refractory
anemia (middle), in acute myeloid leukemia (right) - JMicroVision software.
Figure 3. Log-log plot of bone marrow, healthy subject (left), Refractory Anemia (middle), Refractory Anemia with Excess Blasts (right). The slope of the
straight line is the entropy value of the image. Note the increase of the exponent (slope) with the increase of the severity of the disease. Benoit 1.3 software.
Information Dimension (Entropy, D1) Evaluation
To evaluate the information of the patterns (Entropy, D1, the amount of randomness
present in the images), information dimension, D1, a robust estimate from a finite amount of
data that gives the probability of finding a point in the image, is calculated [17, 18]. The
skeletonized image is covered with boxes of linear size, d (as above), but now keeping track
of the mass, mi (the amount of pixels) in each box, and calculated the information entropy
from the summation of the number of points in the i-th box divided by the total number of
points in the set multiplied for its logarithm. The slope of the log-log plot of information
entropy vs. 1/box side length represented the information dimension of the distribution
(Figure 3). The log-log plot used to calculate the information dimensions shows a line from
400 to 10 pixels (0.4-0.01 mm) with high correlation coefficient, always above a value equal
to 0.99, thus justifying our fractal approach. Both the methodologies are always validated by
measuring computer-generated Euclidean and fractal shapes of known fractal dimensions. In
our experience inter- and intra-observer errors of the entire procedure are < 3%.
STATISTICAL TESTS
The t-test of linear regression was used to determine the linearity of the Log-log plot (to
ensure that the structure evaluated be fractal) and to analyze the increase among the classes.
The Mann-Whitney test was used to compare the entropy values in order to assess the
differences between the groups.
RESULTS
The normal bone marrow and RA as well RAEB bone marrows present mean entropy
values that differ from the topological dimension (p < 0.001), while AML has values
superimposable to the topological dimension.
The values of entropy of the bone marrow histological images statistically recognize
three different groups (Table 1):
a) normal bone marrow (D1 = 1.82 0.1) and RA (D1 = 1.84 0.1)
b) RAEB-1, -2 (D1 = 1.94 0.06)
c) AML (D1 = 1.98 0.05). These last values are superimposable to the topological
dimension (D = 2)
The increase of the entropy value reached the significance (Graph 1, p < 0.01).
Graph 1. In the bone marrow, mean entropy, measure of the disorder in the image, increase from
healthy subjects, to refractory anemia (RA), refractory anemia with excess blasts (RAEB), acute
myeloid leukemia (AML), p < 0.01.
Table 1. Bone marrow. Entropy evaluation in hematoxylin-eosin-stained

histological images
D1 SD
Healthy subjects 1.82 0.1
Refractory Anemia 1.84 0.1
Refractory Anemia with Excess of Blasts 1.94 0.06 P < 0.001
Acute Myeloid Leukemia 1.98 0.05 P < 0.01
Entropy evaluation distinguishes three diagnostic classes.
CONCLUSION
Myelodysplastic syndromes (MDS) arises from transformed immature hematopoietic
cells as a result of the accumulation of multiple genetic and epigenetic changes in
hematopoietic stem cells and committed progenitors [19, 20]. Primary MDS as well
secondary MDS may evolve in a fatal acute leukemia. Different subtypes of MDS possess
different transformation rate towards acute myeloid leukemia, ranging from 10% to over 60%
[21]. So, quantitative, precise, methods to perform differential diagnosis should be desirable
to assess the prognosis of the patient [22].
It is well known that myelodysplastic syndromes are associated with topographic
alterations. The more common are abnormal localization of immature precursors and, often,
the evidence of an inflammatory response, such as the presence of lymphoid aggregates and
patchy or diffuse fibrosis, causing the altered morphological structure of the bone marrow.
Entropy evaluation permits to objectively characterize the altered morphology, as shown at
histological level. The greater the severity of the disease and higher was the entropy index (p
< 0.01), meaning the decrease of fractal structure of the bone marrow tissue and the increase
of randomness in the structure of bone marrow. When acute leukemia is present, the value of
fractal dimension overlaps the topological value (D = 2), demonstrating the complete lack of
fractal structure. These results are relevant to the differential diagnosis. Three different
diagnostic classes emerged from fractal analysis: the mean entropy values of Refractory
Anemia with Excess Blasts was statistically different from the one of Refractory Anemia, the
latter presented higher mean values than in healthy subjects. In effect, it is well known the
highest rate of progression to acute leukemia and decreased survival of patients in the first
than the second and, on the other hand, Refractory Anemia has a low tendency to develop into
a more severe disease: the clustering in three classes deriving from the fractal analysis
appears in agreement with prognostic data.
We show in this work that the evaluation of entropy of the bone marrow histological
images is able to add objective information regarding diagnosis and prognosis of patients in
myelodysplastic syndromes, also confirming our previous results obtained by the use of
another fractal index, the mass dimension [23]. We emphasize that the analysis is inexpensive
and not time consuming (1 minute, on average, to skeletonize the image, 30 seconds to
perform the fractal analysis by a PC). It may be performed using cheaper ready-made fractal
analysis software, such as Benoit 1.3 [24] and image analysis may be performed using free
software such as JMicroVision [25].
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Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale
and important changes. Blood. 2009, 114 (5), 937-51.
[17] Falconer, K. J. Fractal Geometry: Mathematical Foundations and Applications. John
Wiley & Sons Ltd, New York, 2003.
[18] Pitsianis, N., Bleris, G. & Argyrakis, P. Information dimension in fractal structures.
Physical Review B. 1989, 39, 7097-7100.
[19] Shih A. H., Levine R. L. Molecular biology of myelodysplastic syndromes. Semin
Oncol. 2011, 38(5), 613-620.
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biology and clinical implications of the precursors to leukemia stem cells. Stem Cells
Transl Med 2013, 2(2), 143-150.
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myelodysplastic syndrome that accounts for events not considered in the original
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INDEX
# B
5q- MDS, 54, 65, 90, 199, 210, 217, 230 biopsy, 10, 131, 244
5q- syndrome, ix, xiv, 41, 43, 51, 52, 53, 54, 55, 56, BM blasts, xi, 90, 116, 117, 118, 125, 127, 131, 132,
57, 58, 59, 61, 62, 63, 65, 66, 68, 69, 70, 71, 72, 224
73, 74, 75, 77, 79, 81, 82, 83, 84, 111, 119, 188,
193, 197, 202, 203, 206, 208, 209, 210, 211, 214,
215, 218 C
casein kinase 1, x, xiv, 52, 55, 67, 85, 100, 188,

A 203, 205
cell cycle, vii, xii, xiv, 2, 4, 10, 12, 36, 55, 61, 64,
acute myeloid leukemia, ix, xi, xii, xiii, xv, 4, 14, 15, 68, 69, 73, 77, 91, 141, 143, 149, 187, 188, 200,
20, 22, 23, 24, 25, 28, 51, 58, 72, 73, 85, 88, 92, 206
104, 105, 107, 108, 109, 110, 112, 113, 136, 138, cereblon, x, xiii, xiv, 52, 67, 82, 100, 187, 188, 189,
141, 158, 159, 163, 164, 165, 168, 169, 170, 171, 202, 203, 205, 206, 216
172, 175, 176, 185, 188, 217, 219, 220, 235, 236, chemokines, viii, 27, 28, 34, 36, 201
237, 238, 241, 242, 243, 244, 245, 248, 249 conceptual development, 39, 102, 157
alloSCT, 232, 233 cost-effectiveness, 182
anti-innate immune therapies, ix, 28 cullin-4, 203
Aortitis, 31, 44 cytarabine, xiii, 148, 175, 176, 177, 182, 184, 185,
apoptosis, vii, viii, xii, xiv, 2, 8, 20, 25, 27, 29, 32, 186, 231
34, 36, 37, 38, 40, 42, 45, 46, 47, 48, 55, 56, 57, cytarabine ocfosfate, xiii, 175, 176, 177, 184, 185
59, 63, 64, 68, 69, 70, 73, 74, 75, 76, 91, 98, 141, cytokines, viii, 8, 12, 27, 28, 29, 31, 32, 34, 35, 40,
145, 146, 151, 152, 153, 188, 198, 200, 202, 206, 54, 68, 71, 133, 153, 201, 207, 244
215
ara-C, 148, 178, 182, 183, 184, 231
ASXL1, x, xii, 4, 11, 87, 88, 89, 90, 94, 95, 96, 99, D
100, 101, 102, 107, 109, 134, 140, 141, 151, 168,
decitabine, xii, 12, 32, 142, 146, 147, 148, 149, 150,
206, 227, 228, 238
151, 152, 153, 155, 156, 157, 165, 166, 167, 168,
azacitidine, xiii, 31, 32, 90, 107, 133, 138, 139, 142,
169, 170, 171, 172, 199, 230, 231, 232, 233, 239,
146, 147, 148, 149, 150, 151, 153, 154, 155, 156,
240, 241
157, 158, 159, 164, 165, 166, 167, 168, 169, 170,
del(5q), ix, xiii, xiv, 30, 31, 39, 51, 52, 53, 54, 55,
171, 172, 175, 176, 182, 184, 198, 213, 227, 230,
63, 64, 65, 66, 67, 68, 70, 73, 78, 79, 81, 82, 83,
231, 232, 233, 235, 238, 239, 241
84, 94, 98, 100, 101, 107, 111, 113, 119, 172,
187, 188, 190, 191, 192, 193, 194, 195, 196, 197,
198, 199, 200, 201, 202, 203, 205, 206, 207, 208,
252 Index
209, 211, 212, 213, 214, 215, 216, 217, 218, 223, geometric complexity, 244
224, 227, 228, 235, 238, 239
del5q MDS, 188, 198
differential diagnosis, xv, 243, 249 H
DNA methylation, vii, xii, 2, 4, 7, 10, 19, 22, 25, 32,
haematopoietic, 25, 71, 113, 135, 208
64, 89, 91, 92, 104, 105, 141, 142, 143, 144, 146,
haploinsufficiency, ix, 51, 52, 54, 55, 56, 57, 58, 61,
147, 150, 152, 155, 159, 160, 162, 165, 167, 171,
62, 63, 64, 65, 69, 73, 74, 76, 78, 79, 80, 83, 119,
172, 228
136, 194, 206, 210
DNA methyltransferases, xii, 91, 104, 105, 142, 155,
HDAC inhibitors, xiii, 142, 143, 156, 162
159, 171, 172
healthy controls, 204
DNMT3A, x, 4, 19, 87, 88, 89, 90, 91, 92, 95, 99,
hemopoietic differentiation, 71
102, 105, 108, 134, 143, 144, 145, 151, 155, 156,
higher-risk MDS, xii, xiii, xiv, 68, 100, 142, 146,
162, 163, 227, 228, 233, 238
147, 148, 153, 156, 173, 175, 176, 177, 182, 183,
dual specificity phosphatases, xiv, 67, 187, 188
184, 198, 219, 230, 232
dysplastic megakaryopoiesis, 71
high-throughput second generation sequencing, xi,
88
E histone acetylation, xii, 141, 142
histone deacetylases, xiii, 142, 143, 162
E3 ubiquitin ligase CRL4cereblon, xiv, 188, 203 histone methylation, 142, 144
EKLF, 65, 66, 71, 79, 80, 81 histone methyltransferases, 94, 142, 144
entropy, vii, xv, 243, 244, 247, 248, 249 hypoplastic erythropoiesis, 71
Entropy, xv, 243, 247, 249
Epigenetic and Cellular Therapies, 219
epigenetics, vii, xii, 105, 108, 141, 142, 160, 161, I
162, 165, 171
IDH1/2, 88, 156, 206, 228
erythroid cells, 56, 64, 65, 71, 80, 83
immune defects, 113
erythroid Krppel-like factor (EKLF), 65, 66, 71, 79,
immunomodulatory agents, 188
80, 81
inflammatory signaling, 28, 41, 215, 241
erythroid lineage, 56, 69, 71, 81
Information Dimension, xv, 243, 247, 250
erythropoiesis, ix, xiii, xiv, 8, 51, 54, 59, 69, 70, 76,
innate immune signaling, x, 28, 35, 42, 52, 58, 80,
80, 84, 133, 148, 166, 187, 188, 192, 194, 198,
82, 215
200, 201, 207, 210, 212, 213, 214, 239, 241
innate immunity, 29, 35, 42, 47, 52, 59
Etoposide (EP), xiii, 175, 177, 178, 180, 181, 182,
International Prognostic Scoring System, xi, 10, 29,
183, 184, 185, 186, 239
38, 83, 89, 111, 116, 118, 119, 124, 125, 127,
EZH2, x, xii, 4, 11, 87, 88, 89, 90, 94, 95, 96, 101,
128, 135, 137, 138, 139, 142, 149, 152, 176, 189,
108, 134, 141, 144, 145, 151, 161, 162, 163, 206,
222, 237, 251
227, 228, 238
F K
karyotype, 11, 18, 22, 31, 33, 55, 56, 57, 65, 79, 88,
FAB criteria, 176, 222
89, 90, 94, 101, 102, 103, 104, 112, 118, 119,
FLi-1, x, 52, 58, 59, 65, 66, 67, 71, 78, 79, 80
120, 133, 136, 137, 148, 151, 180, 181, 188, 189,
fractal analysis, xv, 243, 244, 249, 250, 251
191, 193, 205, 206, 207, 217, 224, 226, 227, 228,
fractal dimension, 244, 248, 249
231, 237, 241
French-American-British (FAB) criteria, 176
Friend leukemia virus integration 1 (FLi-1), x, 52,
59, 65, 71 L
L1210 ascites tumors, 182

G
lenalidomide, vii, viii, x, xiii, xiv, 2, 18, 48, 52, 54,
66, 67, 68, 70, 71, 81, 82, 83, 84, 90, 98, 100,
GATA2 mutation, 113
101, 111, 113, 119, 136, 156, 171, 172, 187, 188,
G-CSF, viii, 12, 18, 27, 178, 182, 213, 229, 239
Index 253
189, 190, 191, 192, 193, 194, 195, 196, 197, 198, non-responders, xii, xiv, 142, 152, 188, 193, 194,
199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 203, 204
209, 210, 211, 212, 213, 214, 215, 216, 217, 218, NRAS, xi, 4, 11, 82, 88, 89, 90, 107, 111
229, 230, 233, 235, 239, 241
lenalidomide therapy (LT), 68, 82, 101, 196, 197,
198, 203, 204, 216, 218 P
long arms, 202, 204
p53, ix, xi, xiv, 8, 51, 52, 54, 55, 56, 57, 58, 59, 60,
61, 62, 64, 67, 68, 69, 70, 71, 73, 74, 75, 76, 77,
M 78, 79, 81, 82, 83, 85, 88, 98, 110, 111, 188, 193,
194, 200, 203, 205, 209, 210, 214, 217, 233
macrocytic anemia, ix, 51, 52, 56, 71, 73, 188 post alloSCT, 90, 233
MDM2, ix, xiv, 51, 60, 61, 67, 71, 76, 77, 78, 81, 82, prognostic models, 219
85, 188, 200, 214
MDS with chromosome 5, xiii, 187, 204
MDS with isolated del(5q), ix, 51, 70, 126, 188, 197, R
204, 205, 222
refractory anemia, 3, 30, 222, 244, 245, 247, 249
MDS-5q- syndrome, 71
refractory anemia with excess blasts, 30, 222, 244,
medium- and small sized vessel vasculitis, 31
249
megakaryocytes, 71
research organization, 157
MEP, 66, 71
ribosomopathies, x, 52, 56, 58, 63, 69, 73, 84, 194,
microenvironment, ix, 28, 29, 32, 36, 37, 38, 43, 47,
210, 211
48, 54, 90, 153, 169, 199, 201, 202, 207
RPS14, ix, 51, 52, 53, 55, 56, 57, 58, 62, 63, 67, 68,
miR-145, ix, 43, 51, 52, 58, 59, 63, 65, 68, 75, 76,
69, 70, 73, 74, 75, 82, 84, 194, 200, 201, 206,
77, 82, 201, 215
210, 214
monocytosis, 90, 221
RUNX1, xi, 4, 11, 65, 79, 88, 89, 90, 97, 101, 102,
mutations, vii, x, xii, xiv, 3, 4, 8, 11, 17, 19, 20, 21,
107, 110, 134, 151, 227, 228, 238
22, 23, 24, 25, 52, 54, 56, 61, 64, 69, 70, 73, 74,
76, 79, 82, 83, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, S
107, 108, 109, 110, 111, 112, 113, 114, 134, 140,
144, 150, 151, 152, 156, 160, 161, 168, 169, 193, sideroblasts, 3, 4, 10, 11, 30, 90, 96, 99, 101, 112,
203, 205, 206, 209, 216, 217, 219, 221, 227, 228, 117, 159, 221, 222, 223, 228, 239
229, 232, 233, 235, 237, 238, 239 SPAC, 176, 177, 178, 180, 181, 182, 183, 184
myelodysplastic syndromes, vii, viii, xii, xiii, xiv, xv, SPAC + EP therapy, 178, 180, 184
2, 11, 19, 20, 21, 22, 23, 24, 25, 27, 28, 31, 39, SPARC, xiv, 53, 55, 68, 73, 82, 187, 188, 206, 218
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 66, 72, spliceosomal gene aberrations, 88
73, 74, 79, 80, 81, 82, 83, 84, 85, 87, 88, 91, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 115, 116, 135, 136, 137, 138, 139, 141, 148, T
157, 158, 159, 160, 161, 164, 165, 166, 167, 168,
169, 171, 172, 175, 176, 184, 185, 186, 187, 195, TET2, x, xii, 4, 87, 88, 89, 90, 91, 92, 93, 94, 96, 97,
202, 208, 209, 210, 211, 212, 213, 214, 215, 216, 99, 101, 102, 104, 106, 107, 113, 134, 141, 144,
217, 218, 219, 220, 223, 235, 236, 237, 238, 239, 150, 156, 160, 168, 206, 227, 228, 232, 237, 238
240, 241, 243, 244, 249, 250, 251 TET2 mutation, 90, 94, 101, 104, 106, 107, 113, 150,
myeloid deep-sequencing panel, 88 160, 168, 228, 237, 238
thrombocythemia, 71
thrombocytopenia, xi, 11, 12, 30, 32, 38, 63, 73, 88,
N 90, 101, 102, 122, 127, 137, 150, 155, 190, 191,
194, 195, 196, 197, 199, 205, 213, 217, 221, 226
NF-B, viii, 27, 28, 34, 35, 37, 59, 143, 201, 215 Toll-like receptors, viii, 27, 28, 39, 42
non5q-, 204 TP53, ix, xi, xii, xiv, 4, 11, 24, 51, 68, 69, 70, 82, 83,
non-del(5q), xiii, 70, 81, 187, 191, 194, 198, 200, 84, 88, 89, 90, 98, 99, 100, 101, 102, 111, 134,
201, 203, 205, 206, 207, 212, 217
254 Index
142, 151, 168, 188, 193, 197, 205, 206, 209, 217,
228
U
transcription factor Fli-1, 52
ubiquitin ligase, ix, xiv, 35, 51, 52, 60, 67, 96, 97,
transcription factors, viii, 27, 47, 65, 71, 79, 80, 83,
100, 104, 188, 203, 205
144, 214
UTX, x, 87, 88, 95, 108, 144, 161, 162, 206

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Chapter 9 The Old and the New-Integrating Prognostic Models and

Myelodysplastic syndrome (MDS) is a family of clonal haematopoietic stem cells

FROM RISK FACTORS TO PROGNOSIS IN

Ana Bela Sarmento-Ribeiro1,2,3,4,, MD, PhD,

DEFINITION AND EPIDEMIOLOGY

of malignancies, currently included in the World Health Organization (WHO) Classification

ETIOLOGY AND RISK FACTORS

Table 1. Mutations in SMD

Gene Frequency in MDS (%) MDS associated risk

Accumulating epidemiological evidence has suggested that folate intake is associated

associated death domain (FADD), cellular FADD-like interleukin-1 beta-converting enzyme

Aberrant methylation patterns are another common mechanism involved in human

gene codes an inhibitory protein of CDK-1 (cyclin-dependent kinase 1), frequently

DIAGNOSIS, CLASSIFICATION AND PROGNOSTIC FACTORS

diagnostic and prognostic significance as multiparametric flow cytometry can detect

Refractory anemia RA Anemia; <1% peripheral Unilineage erythroid dysplasia 10-20%

Refractory neutropenia RN Neutropenia; <1% Unilineage granulocytic <1%

Refractory RT Thrombocytopenia; <1% Unilineage megakaryocytic <1%

Refractory anemia with RAEB-2 Cytopenia(s); 5%-19% Unilineage or multilineage

Karyotype Good: normal, isolated Intermediate: all Poor: abnormal - -

ANC, x10 9 /L 0.8 < 0.8

Total score Median Overal survival

1.5 Very low 8.7 Not reached

Gene Prognosis of patients carrying mutation vs. wild type

ASXL1 Worse OS [HR, 1.38 (95% CI, 1.001.89); P = 0.006]

EZH2 a Worse OS [HR, 2.13 (95% CI, 1.363.33); P = 0.001]

Lenalidomide is a similar compound to thalidomide but it is safer and is not associated

Table 6. MDS treatment options

Best supportive care: transfusions, antibiotics

Hematopoietic growth factors: erythropoietin, darbepoetin,

Transcriptional modifying therapy

Immunomodulatory agents: lenalidomide,* ATG, CsA, thalidomide

Low-dose chemotherapy (low-dose cytarabine)

Intensive (AML-like) chemotherapy [such as cytarabine (7 days) + an anthracycline (3 days)]

Allogeneic blood (PPSCs) or marrow cell transplantation

and its relation to transfusional iron overload: an observational cross-sectional study.

L., Nascimento Costa, J. M., Sarmento-Ribeiro, A. B. (2015). Polymorphisms of DNMTs

Tefferi A., Vardiman J. W. Myelodysplastic syndromes. N Engl J Med. 2009 Nov;

THE INFLAMMATORY AND AUTOIMMUNE NATURE

Keywords: MDS, innate immune signaling, inflammatory signaling, cytokines, chemokines,

A detailed understanding of these mechanisms, which contribute to the pathogenesis of

Table 1. World Health Organization MDS Classification

Classification Blood findings Bone marrow findings

No Auer rods <5% blasts in marrow

MDS associated with isolated Anemia Normal to increased

Adapted according Nybakken and Bagg [17].

THE ASSOCIATION BETWEEN AUTOIMMUNE DISEASES

Table 2. Autoimmune diseases associated with MDS

Type of autoimmune manifestation in MDS Examples

T-CELL AND B-CELL ABNORMALITIES IN MDS

THE INVOLVEMENT OF REGULATORY T-CELLS (TREG)

NATURAL KILLER CELLS IN MDS

intermediate stages of MDS progression. NK cells become more dysfunctional as MDS

ABNORMAL LEVELS OF INFLAMMATORY CYTOKINES AND

TOLL-LIKE RECEPTOR SIGNALING AND

BONE MARROW NICHE AND ITS INVOLVEMENT IN MDS

ISBN: (eBook)