**3.1.3 Classification of myelodysplastic syndrome in childhood**

Some studies have shown differences in morphological, cytogenetic, molecular and clinical manifestations of primary MDS in childhood that affect their inclusion in traditional classification systems (FAB and WHO), based mainly on adult patients. The rarity of childhood MDS and the heterogeneous nature of the disease have further contributed to the difficulties in classifying this disease (Hasle &Niemeyer, 2011). In 2003, Hasle and colleagues proposed a pediatric approach to the WHO classification of myelodysplastic syndrome: 1- MDS occurring both "de novo" and secondary, including the subtypes refractory cytopenia (RC), RAEB, and RAEB-t.; 2- a group of myelodysplastic/ myeloproliferative disorders with Juvenile Myelomonocytic Leukemia (JMML) as the most common disorder of this category; 3- myeloid leukemia of Down syndrome (DS), a disease with distinct clinical and biological features, encompassing both MDS and AML. In this classification, the minimal diagnostic criteria are: unexplained cytopenia (neutropenia,

Epigenetics in Cancer: The Myelodysplastic Syndrome as a

(Aktas et al., 2006; Fernandez et al., 2000; Sasaki et al., 2001).

**A B**

associated with the leukemogenesis process in MDS.

Model to Study Epigenetic Alterations as Diagnostic and Prognostic Biomarkers 29

In relation to cytogenetic studies, they showed a key role in the diagnosis of the suspected cases of pediatric MDS, being used to confirm the clonal nature of this disease (Sasaki et al., 2001). The monosomy 7 is the most common chromosomal abnormality in these patients (Figure 4). This alteration is associated with poor prognosis and a rapid progression to AML

Fig. 4. (A) Karyotype of bone marrow cell by GTG-banding showing: Monosomy 7. (B) FISH analysis showing monosomy 7 using the probe: LSI D7S486 spectrum orange/ CEP 7 spectrum green, Vysis, Inc. Downers Grove, USA. It may be observed in the interphases nucleus four signals characterizing normal cells (yellow arrows) and others interphases nucleus showing two signals (white arrows), confirming the loss of the chromosome 7.

The molecular mechanisms involved in MDS mainly, in childhood, are not well defined. A recent molecular study of *TP53* and *c-fms* genes showed no mutations in children with MDS. The presence of mutations in onocogene *N-ras* also occurs in a very low frequency in childhood MDS. However, mutations in *TP53*, *c-fms* and *N-ras* genes are involved in the development and evolution from MDS to AML in adult patients (Fernandez et al., 1998; Jekic et al., 2004, 2006). These results suggest that some molecular mechanisms involved in the pathogenesis of MDS in children are different from those seen in adults. It has been observed the importance of epigenetic alterations in the pathogenesis of MDS, but the majority of these studies is focused in adult patients. Few studies showed the epigenetic alterations in children (Hasegawa et al., 2005; Rodrigues et al., 2010; Vidal et al., 2007). Rodrigues and colleagues, 2010, suggested that methylation of *p15INK4B* and *p16INK4A* genes are epigenetic alterations in pediatric MDS patients and, as in adult patients, are later events

**3.3 Cytogenetics and epigenetics alterations in myelodysplastic syndrome** 

The discovery of non-random chromosomal abnormalities in primary MDS confirmed the clonality, providing a way to identify the malignant clone and point out some oncogenes

thrombocytopenia or anemia), at least bilineage morphologic myelodysplasia, acquired clonal cytogenetic abnormality in hematopoietic cells and blast cells number ≥5%. However, this classification has also been widely discussed because not all patients have chromosomal abnormalities, especially in the early stages of the disease (Niemeyer & Baumann, 2008). And in 2009, JMML was considered a myeloproliferative disorder (Hebeda & Fend, 2009).

#### **3.1.4 Prognostic score system in myelodysplastic syndrome**

Parallel to the improvement of classification systems, due to the large variability in survival within the same subgroup of primary MDS, it was necessary to develop score systems for prognostic stratification of risk groups, assisting the choice of treatment. The score system for risk groups most widely used for primary MDS is the International Prognostic Score System (IPSS) (Greenberg et al., 1997). The IPSS considers the percentage of bone marrow blasts, the number of peripheral blood cytopenias and the cytogenetic, the prognostic factors most important in relation to survival time and about the rate of leukemic transformation. The IPSS recognized four risk groups: low risk, intermediate 1, intermediate 2 and high risk. This system considers three categories for cytogenetic analysis: low risk [normal karyotypes, -Y, del(5q) and del(20q)]; high risk (alterations involving chromosome 7 and complex karyotypes) and intermediate risk (other chromosome abnormalities) .

The IPSS has gained prominence for its clinical utility due to the fact that it allows the prediction of disease progression in independent series of previously untreated patients. However, despite its importance, this system has some limitations like the risk groups in relation to karyotype. In some studies, trisomy 8, for example, is often associated with disease progression (Fernandez et al., 2000; Garcia-Manero, 2010; Solé et al., 2000). However, the IPSS classifies this chromosomal alteration with intermediate prognosis. Other important point is related to normal karyotypes that are associated, in some cases, to shorter survival when compared to some chromosomal alterations like: -X, del(5q), del(20q), +21 (Haase, 2008). So, the introduction of molecular data will help to characterize new prognostic factors and use these biomarkers to contribute in understanding the development of MDS and its evolution to AML.

#### **3.2 Pediatric and adult myelodysplastic syndrome**

Although the pediatric MDS shows dysplastic features and ineffective hematopoiesis, such as MDS in adults, clinical characteristics, the presence of constitucional genetic associated abnormalities and characterization of chromosomal changes have reflected a different biological question of MDS in childhood (Elghetany, 2007; Polychronopoulou et al., 2004). The main differences between childhood and adult MDS are: the incidence of RARS cases are extremely rare in pediatric patients and in adults consists of about 25% of cases; the monosomy 7 is the chromosomal alteration most frequent in pediatric patients and in adults is the deletion of the long arm of chromosome 5; the therapeutic possibilities in adult patients is generally limited due to advanced age and usually it is indicated a palliative therapy, whereas, in children with MDS, the main therapy indicated is curative; the allogeneic hematopoetic stem cell transplantation (Halse & Niemeyer, 2002). Some clinical features are different between adults and children with MDS and the factors that predict survival or progression in adults are of little value to children. So, the IPSS has limited value for pediatric MDS (Hasle et al., 2004).

thrombocytopenia or anemia), at least bilineage morphologic myelodysplasia, acquired clonal cytogenetic abnormality in hematopoietic cells and blast cells number ≥5%. However, this classification has also been widely discussed because not all patients have chromosomal abnormalities, especially in the early stages of the disease (Niemeyer & Baumann, 2008). And in 2009, JMML was considered a myeloproliferative disorder (Hebeda & Fend, 2009).

Parallel to the improvement of classification systems, due to the large variability in survival within the same subgroup of primary MDS, it was necessary to develop score systems for prognostic stratification of risk groups, assisting the choice of treatment. The score system for risk groups most widely used for primary MDS is the International Prognostic Score System (IPSS) (Greenberg et al., 1997). The IPSS considers the percentage of bone marrow blasts, the number of peripheral blood cytopenias and the cytogenetic, the prognostic factors most important in relation to survival time and about the rate of leukemic transformation. The IPSS recognized four risk groups: low risk, intermediate 1, intermediate 2 and high risk. This system considers three categories for cytogenetic analysis: low risk [normal karyotypes, -Y, del(5q) and del(20q)]; high risk (alterations involving chromosome 7 and complex

The IPSS has gained prominence for its clinical utility due to the fact that it allows the prediction of disease progression in independent series of previously untreated patients. However, despite its importance, this system has some limitations like the risk groups in relation to karyotype. In some studies, trisomy 8, for example, is often associated with disease progression (Fernandez et al., 2000; Garcia-Manero, 2010; Solé et al., 2000). However, the IPSS classifies this chromosomal alteration with intermediate prognosis. Other important point is related to normal karyotypes that are associated, in some cases, to shorter survival when compared to some chromosomal alterations like: -X, del(5q), del(20q), +21 (Haase, 2008). So, the introduction of molecular data will help to characterize new prognostic factors and use these biomarkers to contribute in understanding the

Although the pediatric MDS shows dysplastic features and ineffective hematopoiesis, such as MDS in adults, clinical characteristics, the presence of constitucional genetic associated abnormalities and characterization of chromosomal changes have reflected a different biological question of MDS in childhood (Elghetany, 2007; Polychronopoulou et al., 2004). The main differences between childhood and adult MDS are: the incidence of RARS cases are extremely rare in pediatric patients and in adults consists of about 25% of cases; the monosomy 7 is the chromosomal alteration most frequent in pediatric patients and in adults is the deletion of the long arm of chromosome 5; the therapeutic possibilities in adult patients is generally limited due to advanced age and usually it is indicated a palliative therapy, whereas, in children with MDS, the main therapy indicated is curative; the allogeneic hematopoetic stem cell transplantation (Halse & Niemeyer, 2002). Some clinical features are different between adults and children with MDS and the factors that predict survival or progression in adults are of little value to children. So, the IPSS has limited value

**3.1.4 Prognostic score system in myelodysplastic syndrome** 

karyotypes) and intermediate risk (other chromosome abnormalities) .

development of MDS and its evolution to AML.

for pediatric MDS (Hasle et al., 2004).

**3.2 Pediatric and adult myelodysplastic syndrome** 

In relation to cytogenetic studies, they showed a key role in the diagnosis of the suspected cases of pediatric MDS, being used to confirm the clonal nature of this disease (Sasaki et al., 2001). The monosomy 7 is the most common chromosomal abnormality in these patients (Figure 4). This alteration is associated with poor prognosis and a rapid progression to AML (Aktas et al., 2006; Fernandez et al., 2000; Sasaki et al., 2001).

Fig. 4. (A) Karyotype of bone marrow cell by GTG-banding showing: Monosomy 7. (B) FISH analysis showing monosomy 7 using the probe: LSI D7S486 spectrum orange/ CEP 7 spectrum green, Vysis, Inc. Downers Grove, USA. It may be observed in the interphases nucleus four signals characterizing normal cells (yellow arrows) and others interphases nucleus showing two signals (white arrows), confirming the loss of the chromosome 7.

The molecular mechanisms involved in MDS mainly, in childhood, are not well defined. A recent molecular study of *TP53* and *c-fms* genes showed no mutations in children with MDS. The presence of mutations in onocogene *N-ras* also occurs in a very low frequency in childhood MDS. However, mutations in *TP53*, *c-fms* and *N-ras* genes are involved in the development and evolution from MDS to AML in adult patients (Fernandez et al., 1998; Jekic et al., 2004, 2006). These results suggest that some molecular mechanisms involved in the pathogenesis of MDS in children are different from those seen in adults. It has been observed the importance of epigenetic alterations in the pathogenesis of MDS, but the majority of these studies is focused in adult patients. Few studies showed the epigenetic alterations in children (Hasegawa et al., 2005; Rodrigues et al., 2010; Vidal et al., 2007). Rodrigues and colleagues, 2010, suggested that methylation of *p15INK4B* and *p16INK4A* genes are epigenetic alterations in pediatric MDS patients and, as in adult patients, are later events associated with the leukemogenesis process in MDS.
