**1. Introduction**

18 Biomarker

Stark A, Kheradpour P, Parts L, Brennecke J, Hodges E, Hannon GJ, Kellis M. Systematic

Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic

Théry, C, Zitvogel, L and Amigorena, S. Exosomes: composition, biogenesis and function.

Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer

van Niel G, Raposo G, Candalh C, Boussac M, Hershberg R, Cerf-Bensussan N, Heyman M.

Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y,

Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone R, Iorio M, Roldo C,

defines cancer gene targets.Proc Natl Acad Sci U S A. 2006.14;103(7):2257-61 Wang H, Ach RA, Curry B. Direct and sensitive miRNA profiling from low-input total RNA.

White V, Scarpini C, Barbosa-Morais NL, Ikelle E, Carter S, Laskey RA, Miller R, Coleman

Xi Y, Formentini A, Chien M, Weir DB, Russo JJ, Ju J, Kornmann M, Ju J. Prognostic Values of microRNAs in Colorectal Cancer . Biomark Insights. 2006;2:113-121 Zhou H, Huang X, Cui H, Luo X, Tang Y, Chen S, Wu L, Shen N. miR-155 and its star-form

plasmacytoid dendritic cells. Blood. 2010.23;116(26):5885-94.

biomarkers of ovarian cancer. Gynecol Oncol. 2008;110(1):13-21

131(5):1400-7.

;121(2):337-49

RNA. 2007;13:151–159.

2009 ;18(7):2006-13

Genome Res. 2007;17(12):1865-79

Nat Rev Immunol. 2002; 2: 569–579

cells. Nat Cell Biol. 2007; 9(6):654-9

development. N Engl J Med. 1988; 319(9):525-32

prospective study of patients undergoing colonoscopy. Gastroenterology.2006;

discovery and characterization of fly microRNAs using 12 Drosophila genomes.

of mRNAs and microRNAs is a novel mechanism of genetic exchange between

Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology. 2001

White R, Smits AM, Bos JL. Genetic alterations during colorectal-tumor

Ferracin M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM. A microRNA expression signature of human solid tumors

N. Isolation of stool-derived mucus provides a high yield of colonocytes suitable for early detection of colorectal carcinoma. Cancer Epidemiol Biomarkers Prev.

partner miR-155\* cooperatively regulate type I interferon production by human

Epigenetics is characterized as hereditary changes in gene activity and expression that occur without alteration in DNA genomic sequence. It is known that epigenetics corresponds basically by two majority modifications: DNA methylation and histone modifications. Epigenetics events are reversible without primary DNA base sequence changes, resulting in possible modulation of the gene expression. The accurate DNA modifications and chromatin changes are important to normal embryonic development, to correct tissue cells differentiation, to precise cell cycle progression and cell death control. However, since epigenetics is also crucial to regulate gene expression, uncontrolled and/or incorrect modifications can unbalance the genetic expression profile and result in cellular transformation from normal to malignant cells.

The development of cancer cell is frequently associated with sequential of genetic and/or epigenetics hits, resulting in loss- or gain-of-function in genes, which leads to cell transformation. At a glance, aberrant global levels of histone modifications as well as incorrect methylation gene promoter may lead to the silencing of tumor suppressor genes and the activation of proto-oncogenes. Recently, many studies have revealed how epigenetics regulation has an implication in the identification of new biomarkers and the development of new therapies at several types of cancers. Moreover, nowadays, a series of identified epigenetics changes have been used as markers for cancer progression and for given prognostic value.

The field of cancer epigenetics is evolving rapidly in many aspects. In myelodysplastic syndrome (MDS), some research groups have been showed the importance to study epigenetic alterations as new diagnostic, prognostic and risk stratification biomarkers. The

Epigenetics in Cancer: The Myelodysplastic Syndrome as a

Island.

cancer (Worn & Gulberg, 2002).

Model to Study Epigenetic Alterations as Diagnostic and Prognostic Biomarkers 21

Fig. 1. DNA methylation. (A) Methyl radical transference from SAM (S-adenosyl-l-

(Robertson et al., 1999; Tabby & Issa, 2010; Worn & Gulberg, 2002).

methionine) 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

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

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

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

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.

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 epigenetic biomarkers.
