**2. The role of epigenetics in cancer**

Epigenetics alterations have been growing as a promisor tool to understand cancer development, for better clinical therapy management, to identify new cancer biomarkers, which can help in monitoring disease evolution. It is known that epigenetics corresponds basically by two majority modifications: DNA methylation and chromatin modifications.

DNA methylation is a covalent modification of the cytosine ring 5' position of a CpG dinucleotide, whereby a methyl group is deposited on carbon 5 of that ring using Sadenosyl methionine as a methyl donor. This transfer of methyl group is a replicationdependent reaction catalyzed by DNA methyltrasferases (DNMTs) (Figure 1).

Humans DNA methyltransferases are represented basically by three proteins: DNMT1, DNMT3A and DNMT3B. In general, DNMT1 are preferentially responsible for the methylation of one strand of DNA using as reference the other strand already methylated, mechanisms known as *de novo* methylation. This DNA hemi-methylation activity is important to maintain the methylation profile of genomic DNA cells during cellular

MDS comprises a heterogeneous group of clonal bone marrow disorders characterized by varying degrees of pancytopenia, morphological and functional abnormalities of hematopoietic cells and increased risk of transformation into acute myeloid leukemia. This hematologic malignancy became a model to study the genetics and epigenetics changes involved in development stages of leukemia and it is considered a model study to tumorigenesis. MDS is viewed as a disease of adults, particularly the elderly. Pediatric MDS is an uncommon disorder, accounting for less than 5% of hematopoietic malignancies. Some studies in children showed that MDS appears with distinct clinical and laboratory characteristics when compared with adults, which may reflect special biological issues of MDS during childhood. There are different pathways involved in the pathogenesis of MDS. Due to the MDS heterogeneity, little is known about the molecular basis of MDS in adults and mainly in pediatric patients. Identification of the underlying genetic and epigenetic alterations in MDS may promote proper classification and prognostication of disease and, eventually, the development of new therapies. An important point in the epigenetic studies is the introduction of new forms of treatment for MDS patients. It is well documented that hematopoetic stem cell transplantation (HSCT) is, until now, the only curative treatment for MDS, both in adults and in children, but relapse after HSCT is the major cause of treatment failure in advanced stages. Other important factors in HSCT are the necessity of histocompatibility of donor cells and the age of the patients, sometimes limiting the use of this treatment. Thus, it is extremely important detecting biomarkers of disease evolution, especially those involved in epigenetic modifications, because news forms of treatment, as

the use of hypomethylation agents, can be introduced as a better treatment option.

epigenetic biomarkers.

**2. The role of epigenetics in cancer** 

This chapter will review the advances in the study of epigenetics in cancer, the discovery of new epigenetic biomarkers and the development of therapeutic strategies using hypomethylation drugs. We will focus the advances in the epigenetic field using the myelodysplastic syndrome as a model, since it was demonstrated the importance of epigenetics alterations in the pathogenesis of this disease. Finally, we will describe the importance of statistical methods to aid the analysis of new diagnostic and prognostic

Epigenetics alterations have been growing as a promisor tool to understand cancer development, for better clinical therapy management, to identify new cancer biomarkers, which can help in monitoring disease evolution. It is known that epigenetics corresponds basically by two majority modifications: DNA methylation and chromatin modifications.

DNA methylation is a covalent modification of the cytosine ring 5' position of a CpG dinucleotide, whereby a methyl group is deposited on carbon 5 of that ring using Sadenosyl methionine as a methyl donor. This transfer of methyl group is a replication-

Humans DNA methyltransferases are represented basically by three proteins: DNMT1, DNMT3A and DNMT3B. In general, DNMT1 are preferentially responsible for the methylation of one strand of DNA using as reference the other strand already methylated, mechanisms known as *de novo* methylation. This DNA hemi-methylation activity is important to maintain the methylation profile of genomic DNA cells during cellular

dependent reaction catalyzed by DNA methyltrasferases (DNMTs) (Figure 1).

Fig. 1. DNA methylation. (A) Methyl radical transference from SAM (S-adenosyl-lmethionine) to 5th carbon of aromatic ring of cytosine nucleotide mediated by DNMT (DNA methyltransferase). (B) DNA sequence indicating that only cytosines before guanine will be methylate (red arrows). This cytosine/guanine in the methylation studies is also known as CpG and the concentration of CpG in some region of DNA sequence is known as CpG Island.

division. DNA hemi-methylation promoted by DNMT1 is crucial for initial stages of embryonic development and cell survival. In other hand, DNMT3A and DNMT3B play essential role for DNA hemi-methylation or unmethylated with the same level. Catalytic methyl-transferase of DNMT3A or DNMT3B is mainly promoted in cytosine preceded by guanine at the CpG dinucleotide. DNA methylation is a no random phenomenon. It normally occurs at the CG rich DNA sequences (the CpG islands) at promoter regions (Robertson et al., 1999; Tabby & Issa, 2010; Worn & Gulberg, 2002).

DNA methylation is frequently associated to transcriptional gene repression. It has been suggested that repression occurs by physically interfering in transcriptional factors binding at gene promoter regions, modified by 5'-methylcitosine or by recruiting methylated-DNA binding domain (MBD) proteins that block an original site of transcriptional factor*.* In addition, MBD proteins are frequently found associated with histone deacetylases. Physiological DNA methylation has been shown important to regulate genetic expression during embryonic development, genomic imprinting, X chromosome inactivation and cancer (Worn & Gulberg, 2002).

Chromatin is defined by a DNA and DNA-associated proteins, known as histones, in which genomic eukaryotic DNA is packaged. The basic unit of chromatin is called nucleosome, which is composed of a small DNA sequence, approximately 147 bases pairs, wrapped on

Epigenetics in Cancer: The Myelodysplastic Syndrome as a

Begemann, 2007).

Jerónimo et al., 2011).

Model to Study Epigenetic Alterations as Diagnostic and Prognostic Biomarkers 23

found to control the activities of homeotic genes, which determine segmentation and structures body during development (Ingham, 1985). Several PcG orthologues genes were found in humans (van Lohuizen et al., 1991). PcG proteins are subdivided into two classes designed as Polycomb Repressive Complex: PRC1 and PRC2. The number of PcG proteins suggests a greater complexity of functions on the chromatin regulating. In this context, PcG have been reported to act in a myriad of histone modifications such as, ubiquitylation, sumoylation and methylation (Margueron & Reinberg, 2005). PcG proteins perform a critical role in gene regulation. PCR2 and PCR1 are considered to be involved in the initialization and maintenance of the repression of the gene transcription, respectively. PCR2 comprises the core components enhancer of zeste-2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12), while PCR1 consists in a ring finger protein 1 (RING1), B lymphoma Mo-MLV insertion region 1 (BMI1) and chromobox homologue 2/4/8 (CBX2/4/8). EZH2 is the catalytic subunit of PRC2. It is a highly conserved histone methyltransferase that targets lysine 27 of histone H3. This methylated H3-K27 is usually associated with silencing of genes involved in differentiation. In addition, EZH2 is required for DNA methylation of EZH2- target promoters, serving as a recruitment platform DNA methyltransferases. SUZ12 is a recently identified PcG protein that, together with EED, is essential to maintaining the repressive function of PRC2. RING1 catalyzes the mono ubiquitylation of histone H2A at lysine 119. The H2AK 119 ubiquitylation likely increases chromatin compaction and, thus, interferes with the access or action of transcription factors. BMI 1 is mostly detected in stem cells and progenitors and takes part in stem cell proliferation and self-renewal (Bantignies & Cavalli, 2006; Levine et al., 2004; Rajasekhar &

Cancer development is a consequence of multi-step molecular and cellular events that transform normal to malignant cell. During this process genetic and epigenetic alterations are involved. In recent years, several studies have indicated how epigenetics regulation has an implication in the identification of new biomarkers and the development of new therapies in a majority of cancers. Several evidences of oncogenes and tumor supressor genes DNA methylation indicated the importance of these epigenetics changes under expression control. DNA hypomethylation was directly related to the overexpression of *Raf*, *c-Myc*, *c-Fos*, *c-H-Ras* and *c-K-ras* oncogenes and tumor liver formation (Rao et al., 1989). Hypermethylation of tumor suppressor genes has also been demonstrated. For example, DNA methylation interferes in the expression of key cell cycle checkpoints genes: *p16INK4A, p15INK4B, Rb, p14ARF*. These are the most studied genes used to correlate methylation and cell cycle control in several cancer types (Esteller, 2011). DNA or chromatin epigenetic alterations have been directly related to molecular changes to cancer development. Some epigenenetic modifications have been usefulness as biomarkers to aid in the clinicaltherapeutic decision. For instance, *APC* and *GSTP1* gene methylation and H3K4me/H3K4me2/H3K18Ac chromatin modifications have been used to predict response to therapy and prognostic information in prostate cancer (Henrique & Jerónimo, 2004;

The potential reversibility of epigenetics states offers exciting opportunities for new cancer drugs that can reactivate epigenetically silenced tumor-suppressor genes. Blocking either DNA methyltransferases or histone deacetylase activity could potentially inhibit or reverse the process of epigenetic silencing (Kelley et al., 2010). DNA demethylating drugs (DMI), as

protein octamer of the four core Histones (H2A, H2B, H3, and H4) and linker Histone H1. Epigenetics regulation involving chromatin comprises the post-translational modifications of histone protein tails. Depending on the kind of histone alteration, chromatin becomes compressed or weakens, which results in repression or permission to gene expression respectively. The histone complexes can be heterogeneous post-translational modified; it comprises methylation, acetylation, phosphorylation and ubiquitinylation. These distinct levels of combinatory histone modifications possibilities lead to regulation of gene transcription, a process called "histone coding". These histone changes are promoted by a series of distinct proteins, such as histone acetyl transferases (HAT), histone deacetylases (HDAC) and Polycomb group (PcG) proteins (Marks & Dokmanovic, 2005). The HAT and HDAC catalyze the transference of acetyl radical to histone tails (Kleff et al., 1995). Commonly, chromatin acetylation promotes the transcription factors access to DNA consensus sequences. Therefore, chromatin acetylation is frequently related to increase of transcriptional gene activity (Figure 2). Acetylation is not a random event and occurs in H3 and H4 tails, mainly on lysine residue, such as H3K4 and H3K14 (Agalioti et al., 2002).

Fig. 2. Chromatin structure and histone tails modifications. Two mainly chromatin modifications, acetylation (AC) and methylation (MET). Acetylation is direct related to loose of chromatin compression which allows transcriptional factors access to DNA molecule and transcript its target gene. Methylation of histone tails is associated to chromatin compression and, as consequence, block the transcriptional factors access.

Among chromatin modifiers, Polycomb Group (PcG) proteins have been established as classical players of epigenetics regulation. PcG genes were discovered at experiments of mutations in *Drosophila* development (Lewis, 1978). In these studies, PcG proteins were

protein octamer of the four core Histones (H2A, H2B, H3, and H4) and linker Histone H1. Epigenetics regulation involving chromatin comprises the post-translational modifications of histone protein tails. Depending on the kind of histone alteration, chromatin becomes compressed or weakens, which results in repression or permission to gene expression respectively. The histone complexes can be heterogeneous post-translational modified; it comprises methylation, acetylation, phosphorylation and ubiquitinylation. These distinct levels of combinatory histone modifications possibilities lead to regulation of gene transcription, a process called "histone coding". These histone changes are promoted by a series of distinct proteins, such as histone acetyl transferases (HAT), histone deacetylases (HDAC) and Polycomb group (PcG) proteins (Marks & Dokmanovic, 2005). The HAT and HDAC catalyze the transference of acetyl radical to histone tails (Kleff et al., 1995). Commonly, chromatin acetylation promotes the transcription factors access to DNA consensus sequences. Therefore, chromatin acetylation is frequently related to increase of transcriptional gene activity (Figure 2). Acetylation is not a random event and occurs in H3 and H4 tails, mainly on lysine residue, such as H3K4 and H3K14 (Agalioti et al., 2002).

Fig. 2. Chromatin structure and histone tails modifications. Two mainly chromatin

and, as consequence, block the transcriptional factors access.

modifications, acetylation (AC) and methylation (MET). Acetylation is direct related to loose of chromatin compression which allows transcriptional factors access to DNA molecule and transcript its target gene. Methylation of histone tails is associated to chromatin compression

Among chromatin modifiers, Polycomb Group (PcG) proteins have been established as classical players of epigenetics regulation. PcG genes were discovered at experiments of mutations in *Drosophila* development (Lewis, 1978). In these studies, PcG proteins were found to control the activities of homeotic genes, which determine segmentation and structures body during development (Ingham, 1985). Several PcG orthologues genes were found in humans (van Lohuizen et al., 1991). PcG proteins are subdivided into two classes designed as Polycomb Repressive Complex: PRC1 and PRC2. The number of PcG proteins suggests a greater complexity of functions on the chromatin regulating. In this context, PcG have been reported to act in a myriad of histone modifications such as, ubiquitylation, sumoylation and methylation (Margueron & Reinberg, 2005). PcG proteins perform a critical role in gene regulation. PCR2 and PCR1 are considered to be involved in the initialization and maintenance of the repression of the gene transcription, respectively. PCR2 comprises the core components enhancer of zeste-2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12), while PCR1 consists in a ring finger protein 1 (RING1), B lymphoma Mo-MLV insertion region 1 (BMI1) and chromobox homologue 2/4/8 (CBX2/4/8). EZH2 is the catalytic subunit of PRC2. It is a highly conserved histone methyltransferase that targets lysine 27 of histone H3. This methylated H3-K27 is usually associated with silencing of genes involved in differentiation. In addition, EZH2 is required for DNA methylation of EZH2- target promoters, serving as a recruitment platform DNA methyltransferases. SUZ12 is a recently identified PcG protein that, together with EED, is essential to maintaining the repressive function of PRC2. RING1 catalyzes the mono ubiquitylation of histone H2A at lysine 119. The H2AK 119 ubiquitylation likely increases chromatin compaction and, thus, interferes with the access or action of transcription factors. BMI 1 is mostly detected in stem cells and progenitors and takes part in stem cell proliferation and self-renewal (Bantignies & Cavalli, 2006; Levine et al., 2004; Rajasekhar & Begemann, 2007).

Cancer development is a consequence of multi-step molecular and cellular events that transform normal to malignant cell. During this process genetic and epigenetic alterations are involved. In recent years, several studies have indicated how epigenetics regulation has an implication in the identification of new biomarkers and the development of new therapies in a majority of cancers. Several evidences of oncogenes and tumor supressor genes DNA methylation indicated the importance of these epigenetics changes under expression control. DNA hypomethylation was directly related to the overexpression of *Raf*, *c-Myc*, *c-Fos*, *c-H-Ras* and *c-K-ras* oncogenes and tumor liver formation (Rao et al., 1989). Hypermethylation of tumor suppressor genes has also been demonstrated. For example, DNA methylation interferes in the expression of key cell cycle checkpoints genes: *p16INK4A, p15INK4B, Rb, p14ARF*. These are the most studied genes used to correlate methylation and cell cycle control in several cancer types (Esteller, 2011). DNA or chromatin epigenetic alterations have been directly related to molecular changes to cancer development. Some epigenenetic modifications have been usefulness as biomarkers to aid in the clinicaltherapeutic decision. For instance, *APC* and *GSTP1* gene methylation and H3K4me/H3K4me2/H3K18Ac chromatin modifications have been used to predict response to therapy and prognostic information in prostate cancer (Henrique & Jerónimo, 2004; Jerónimo et al., 2011).

The potential reversibility of epigenetics states offers exciting opportunities for new cancer drugs that can reactivate epigenetically silenced tumor-suppressor genes. Blocking either DNA methyltransferases or histone deacetylase activity could potentially inhibit or reverse the process of epigenetic silencing (Kelley et al., 2010). DNA demethylating drugs (DMI), as

Epigenetics in Cancer: The Myelodysplastic Syndrome as a

(Parker et al., 2000).

Model to Study Epigenetic Alterations as Diagnostic and Prognostic Biomarkers 25

defects result in ineffective hematopoiesis (bone marrow failure) and an increased risk of transformation into acute myeloid leukemia (AML) (Davids & Steensma, 2010; Jadersten & Hellström-Lindberg, 2008). MDS is viewed as a disease of adults, particularly the elderly. Pediatric MDS is an uncommon disorder, accounting for less than 5% of hematopoietic malignancies (Elghetany, 2007; Niemeyer & Baumann, 2008). The primary MDS presents a natural history since an indolent disease with long time of duration to a rapid progression to AML in few months (Nishino & Chang, 2005). The diagnosis is done initially by the hemogram indicating one or more cytopenias in peripheral blood like anemia, neutropenia and thrombocytopenia. The analysis is performed by the myelogram and bone marrow biopsy to identify dysplastic cells, the possible presence of blasts, characterizing later stages of the disease, and the presence of abnormal localization of immature precursors (ALIP). The bone marrow of MDS patients is usually hypercellular or normocellular, but there are a small number of cases with hypocellular bone marrow. In cases where bone marrow is hypocellular, it is recommended to perform differential diagnosis of severe aplastic anemia (SAA) and paroxysmal nocturnal hemoglobinuria (PNH). In these cases, important diagnostic tools, like the cytogenetics and the immunophenotyping, aid this diagnosis (Bennett & Orazi, 2009; Wong & So, 2002). The apparent paradox of hypercellular bone marrow and peripheral blood cytopenias was clarified by studies showing that MDS patients have increased rates of apoptosis in bone marrow in early stages of the disease

The primary MDS diagnosis is considered a difficult clinical practice, because there are several clinical manifestations which may present a clinical and histological picture quite similar to MDS, such as nutritional deficiencies, infections and congenital conditions. It is necessary a differential diagnosis, where the presence of cytogenetic clonality helps in the diagnosis of primary MDS and contributes for the prognosis (Haase et al., 2007; Olney & Le Beau, 2009; Solé et al., 2005; Tiu et al., 2011). Nevertheless, there are cases with normal karyotype, so it is important to characterize molecular biomarkers to aid the MDS diagnosis. Because the primary MDS is a disease extremely heterogeneous, the definition of prognostic factors are often difficult. Thus, in the later years, it has been extensively discussed the

**3.1 Classifications and prognostic scores systems in myelodysplastic syndrome** 

Until 1980, the MDS included a variety of hematologic abnormalities classified as syndromes or pre-leukemic states. However, these denominations were unsatisfactory, not grouping all the patients who showed an ineffective hematopoiesis and not progressed to acute leukemia, occurring complications because of the cytopenias leading to death. The term "pre-leukemia" disappeared and the term myelodysplstic syndrome became widely

In 1982, the FAB group (French, American and British group) proposed a classification for primary MDS into five subgroups: refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation (RAEB-t) and chronic myelomonocytic leukemia (CMML).

classifications and prognostic scales for adult and pediatric patients.

accepted in 1982 with the FAB classification.

**3.1.1 FAB classification** 

5-azacytidine and 5-aza-2'-deoxycytidine, have been indicated as a promising new treatment for cancer (Streseman & Lyko, 2008). Histone deacetylase inihibitors (HDACs) have also been analyzed at clinical protocols for solid tumors (breast, non-small lung cells, prostatic cancer) and mainly for hematological malignancies (myelomas, leukemias and myelodysplastic syndrome) (Ellis et al., 2009; Graham et al., 2009; Hrebackova et al., 2010; Razak et al., 2011). In figure 3, we can see that the epigenetic therapy can "re-programmed" gene expression patterns.

Fig. 3. Epigenetic therapy acting in the effects of DNMT or HDAC. Inhibitors of DNMT (*DNA methyltransferase*) or HDAC (*histone deacetylases*), as DMI (*DNMT inhibitor*) and HDI (*Histone Deacetylase Inhibitor*), respectively, induce the reprogramming expression by chromatin decompression. In summary, these inhibitors act mainly cell-cycle, apoptosis and differentiation related genes.
