**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

Epigenetics in Cancer: The Myelodysplastic Syndrome as a

(Tan & Wei, 2011).

subset of patients.

**implications** 

during the initiation and disease progression (Vigna et al., 2011).

Model to Study Epigenetic Alterations as Diagnostic and Prognostic Biomarkers 31

slow (but with increased tendency) to AML progression represents a prototype of the multistep concept in leukemogenesis with accumulation of cellular and molecular defects

Recent studies have revealed that DNA methylation and histone modification may be controlled by Polycomb-group (PcG) proteins, which may give new clues toward understanding the epigenetic mechanisms of MDS. PcG family members, such as EZH2, RING1 and BMI-1, are essential for the self-renewal and proliferation of normal cells. However, the induced over expression of these proteins can drive tumorigenesis. In MDS patients, the expression of EZH2, RING1 and BMI 1 were positively correlated with the IPSS prognostic scoring system, suggesting that the over expression of each of these three genes is a negative prognostic indicator (Xu et al., 2011). The EZH2 expression level was positively correlated with a reduction of peripheral blood cells, which was likely to reflect the severity of ineffective hematopoiesis. Molecular analyses of EZH2 showed that deletions, missense and frameshift mutations strongly suggest that EZH2 is a tumor suppressor gene in MDS pathogenesis (Nikoloski et al., 2010). Other mutations have been identified in the genes that regulate endogenous methylation networks within cells including IDH1/2, TET2 and DNMT3. The relevance of these lesions in being able to predict response to epigenetic modulators and their correlation with epigenetic signatures in MDS are beginning to emerge

In MDS, aberrant silencing due to promoter hypermethylation involves genes encoding cell adhesion molecules, cell cycle regulation and tumor suppressor genes possibly leading to dysregulation of hematopoiesis. It has been shown in MDS a high prevalence of methylation for the tumor suppressor genes *p15INK4B*, cadherin 1 (CDH1), death associated protein kinase (DAPK) and suppressor of cytokine signaling (SOCS-1). Some methylation patterns in specific genes in MDS can predict poor prognosis even in early stage of the disease (Aggerholm et al., 2006; Bejar et al., 2011; Vigna et al., 2011). Hence, epigenetic changes have been implicated as potential mechanisms in the pathogenesis and progression of MDS, which has already resulted in promising therapeutic approaches in a

**3.4 Methylation changes in myelodysplastic syndrome: Diagnostic and therapeutical** 

Different studies in MDS showed the importance of methylation changes during the clinical evolution of disease to AML. So, the identification in the diagnosis of these alterations is important for risk group stratification. Aberrant DNA methylation is view as a poor prognostic feature in MDS. For example, p15*INK4B* and p16*INK4A* genes are members of cyclin dependent kinase inhibitors family which controls the progression of cell cycle from G1 to S phase. The products of these genes regulate RB function by modulating the complexes of cyclin D-CDK4/6 which can phosphorylate and inactive the RB protein, and set up an important pathway for inhibiting cell growth (Serrano et al., 1996; Shimamoto et al., 2005). In addition, p15*INK4B* has been suggested to act as a regulator of proliferation and differentiation in myelo-monocytic and megakaryocytic lineages by arresting the cell cycle (Sakashita et al., 2001; Teofili et al., 2000). Therefore, the silencing of these genes via aberrant methylation is a critical event in leukaemogenesis. In MDS, aberrant methylation of p15*INK4B*

and tumor suppressor genes possibility involved in the development and progression of disease. The cytogenetics evaluation of a bone marrow sample from patients with MDS has become an integral part of clinical care. The clonal cytogenetic alterations can be detected in 30-50% of adult patients with primary MDS. In pediatric patients this incidence is 50-70% of the cases. These changes range from a single numerical or structural changes to complex genomic lesions involving three or more different chromosomes. The most frequent chromosomal abnormalities in MDS are: del(5q), del(7q)/-7, +8, del(11q), del(12p), del(17p), del(20q) and loss of Y chromosome (Bejar et al., 2011; Fernandez et al., 2000; Haase, 2008).

The frequency of cytogenetic abnormalities increases with the severity of disease as well as the risk of leukemic transformation. In this group, unfavorable chromosomal abnormalities are frequently found as complex abnormalities or karyotypes including monosomy 7 or trisomy 8 (Bacher, 2010; Fernandez et al., 2000). A normal karyotype is found in 30-60% of patients with MDS. This group of patients is almost certainly genetic heterogeneous, probably the leukemogenic alterations occurred at the molecular level and were not detectable with standard cytogenetic methods (Greenberg et al., 1997; Onley & Le Beau, 2009).

In MDS, some studies suggest some genes involved with specific chromosome alterations, as the del(5q). The 5q syndrome represents a distinct clinical entity characterized by a del(5q) as the sole karyotypic abnormality. The 5q syndrome occurs commonly in women. The initial laboratory findings are usually a macrocytic anemia with a normal or elevated count. The diagnosis is usually RA. On bone marrow examination, abnormalities in the megakaryocytic lineage (particularly micromegakaryocytes) are prominent. These patients have a favorable prognosis, with low rates of leukemic transformation and a relatively long survival of several years. The loss of a single copy of the RPS14 gene may be involved in the MDS 5q- pathogenesis. The RPS14 is an essential component of the 40S subunit of ribosomes and ribosomes synthesis is impaired in CD34 + cells from 5q syndrome patients (Onley & Le Beau, 2009).

The role of cytogenetic analysis in MDS is an important factor for establishing the diagnosis, prognosis and therapeutic plan and the follow up of altered clinical behavior of the disease. The chromosomal abnormalities have not only provided insights into prognosis but also into the molecular pathogenesis of this heterogeneous disease. The type of chromosomal abnormality (unbalanced, most commonly the result of the loss of a whole chromosome or a deletion of a part of a chromosome) in primary MDS indicates that the main class of genes involved in the pathogenesis of this disease is the tumor suppressor genes. The mechanism involved in the inactivating tumor suppressor genes are deletions, mutations and epigenetic alterations as the DNA methylation.

Three main epigenetic events regulate tumor-associated genes: 1) the aberrant hypermethylation of tumor suppressor genes, 2) post-translational modifications of histones and 3) post-transcriptional modifications by regulatory miRNA. The underlying causes of the pathogenesis of MDS remain to be fully elucidated. Knudson model of the "two hits" provides the basis of the concept of a multistep pathogenesis in the development of MDS, where loss or inactivation of only one allele is not sufficient to result in the development of tumors or expansion of a malignant clone. In fact, MDS in early stages with its relatively

and tumor suppressor genes possibility involved in the development and progression of disease. The cytogenetics evaluation of a bone marrow sample from patients with MDS has become an integral part of clinical care. The clonal cytogenetic alterations can be detected in 30-50% of adult patients with primary MDS. In pediatric patients this incidence is 50-70% of the cases. These changes range from a single numerical or structural changes to complex genomic lesions involving three or more different chromosomes. The most frequent chromosomal abnormalities in MDS are: del(5q), del(7q)/-7, +8, del(11q), del(12p), del(17p), del(20q) and loss of Y chromosome (Bejar et

The frequency of cytogenetic abnormalities increases with the severity of disease as well as the risk of leukemic transformation. In this group, unfavorable chromosomal abnormalities are frequently found as complex abnormalities or karyotypes including monosomy 7 or trisomy 8 (Bacher, 2010; Fernandez et al., 2000). A normal karyotype is found in 30-60% of patients with MDS. This group of patients is almost certainly genetic heterogeneous, probably the leukemogenic alterations occurred at the molecular level and were not detectable with standard cytogenetic methods (Greenberg et al., 1997; Onley & Le Beau,

In MDS, some studies suggest some genes involved with specific chromosome alterations, as the del(5q). The 5q syndrome represents a distinct clinical entity characterized by a del(5q) as the sole karyotypic abnormality. The 5q syndrome occurs commonly in women. The initial laboratory findings are usually a macrocytic anemia with a normal or elevated count. The diagnosis is usually RA. On bone marrow examination, abnormalities in the megakaryocytic lineage (particularly micromegakaryocytes) are prominent. These patients have a favorable prognosis, with low rates of leukemic transformation and a relatively long survival of several years. The loss of a single copy of the RPS14 gene may be involved in the MDS 5q- pathogenesis. The RPS14 is an essential component of the 40S subunit of ribosomes and ribosomes synthesis is impaired in CD34 + cells from 5q syndrome patients (Onley & Le

The role of cytogenetic analysis in MDS is an important factor for establishing the diagnosis, prognosis and therapeutic plan and the follow up of altered clinical behavior of the disease. The chromosomal abnormalities have not only provided insights into prognosis but also into the molecular pathogenesis of this heterogeneous disease. The type of chromosomal abnormality (unbalanced, most commonly the result of the loss of a whole chromosome or a deletion of a part of a chromosome) in primary MDS indicates that the main class of genes involved in the pathogenesis of this disease is the tumor suppressor genes. The mechanism involved in the inactivating tumor suppressor genes are deletions, mutations and epigenetic

Three main epigenetic events regulate tumor-associated genes: 1) the aberrant hypermethylation of tumor suppressor genes, 2) post-translational modifications of histones and 3) post-transcriptional modifications by regulatory miRNA. The underlying causes of the pathogenesis of MDS remain to be fully elucidated. Knudson model of the "two hits" provides the basis of the concept of a multistep pathogenesis in the development of MDS, where loss or inactivation of only one allele is not sufficient to result in the development of tumors or expansion of a malignant clone. In fact, MDS in early stages with its relatively

al., 2011; Fernandez et al., 2000; Haase, 2008).

2009).

Beau, 2009).

alterations as the DNA methylation.

slow (but with increased tendency) to AML progression represents a prototype of the multistep concept in leukemogenesis with accumulation of cellular and molecular defects during the initiation and disease progression (Vigna et al., 2011).

Recent studies have revealed that DNA methylation and histone modification may be controlled by Polycomb-group (PcG) proteins, which may give new clues toward understanding the epigenetic mechanisms of MDS. PcG family members, such as EZH2, RING1 and BMI-1, are essential for the self-renewal and proliferation of normal cells. However, the induced over expression of these proteins can drive tumorigenesis. In MDS patients, the expression of EZH2, RING1 and BMI 1 were positively correlated with the IPSS prognostic scoring system, suggesting that the over expression of each of these three genes is a negative prognostic indicator (Xu et al., 2011). The EZH2 expression level was positively correlated with a reduction of peripheral blood cells, which was likely to reflect the severity of ineffective hematopoiesis. Molecular analyses of EZH2 showed that deletions, missense and frameshift mutations strongly suggest that EZH2 is a tumor suppressor gene in MDS pathogenesis (Nikoloski et al., 2010). Other mutations have been identified in the genes that regulate endogenous methylation networks within cells including IDH1/2, TET2 and DNMT3. The relevance of these lesions in being able to predict response to epigenetic modulators and their correlation with epigenetic signatures in MDS are beginning to emerge (Tan & Wei, 2011).

In MDS, aberrant silencing due to promoter hypermethylation involves genes encoding cell adhesion molecules, cell cycle regulation and tumor suppressor genes possibly leading to dysregulation of hematopoiesis. It has been shown in MDS a high prevalence of methylation for the tumor suppressor genes *p15INK4B*, cadherin 1 (CDH1), death associated protein kinase (DAPK) and suppressor of cytokine signaling (SOCS-1). Some methylation patterns in specific genes in MDS can predict poor prognosis even in early stage of the disease (Aggerholm et al., 2006; Bejar et al., 2011; Vigna et al., 2011). Hence, epigenetic changes have been implicated as potential mechanisms in the pathogenesis and progression of MDS, which has already resulted in promising therapeutic approaches in a subset of patients.

#### **3.4 Methylation changes in myelodysplastic syndrome: Diagnostic and therapeutical implications**

Different studies in MDS showed the importance of methylation changes during the clinical evolution of disease to AML. So, the identification in the diagnosis of these alterations is important for risk group stratification. Aberrant DNA methylation is view as a poor prognostic feature in MDS. For example, p15*INK4B* and p16*INK4A* genes are members of cyclin dependent kinase inhibitors family which controls the progression of cell cycle from G1 to S phase. The products of these genes regulate RB function by modulating the complexes of cyclin D-CDK4/6 which can phosphorylate and inactive the RB protein, and set up an important pathway for inhibiting cell growth (Serrano et al., 1996; Shimamoto et al., 2005). In addition, p15*INK4B* has been suggested to act as a regulator of proliferation and differentiation in myelo-monocytic and megakaryocytic lineages by arresting the cell cycle (Sakashita et al., 2001; Teofili et al., 2000). Therefore, the silencing of these genes via aberrant methylation is a critical event in leukaemogenesis. In MDS, aberrant methylation of p15*INK4B*

Epigenetics in Cancer: The Myelodysplastic Syndrome as a

such -7, del(16q) and +8 (Qin et al., 2011).

al., 2011).

investigation (Vigna et al., 2011).

Model to Study Epigenetic Alterations as Diagnostic and Prognostic Biomarkers 33

Some studies have shown that reduced methylation overtime was correlated with better clinical response for patients during decitabine treatment. But, further studies of methylation dynamics, both before and after treatment, will be useful to determine the ability of epigenetic biomarkers to direct the treatment and may predict for the success (Shen et al., 2010). However, in some clinical trials, it was found that a number of patients does not respond to decitabine initially (primary resistance) and most patients, who initially respond to decitabine treatment, eventually relapse (secondary resistance) despite continued therapy. Clinical response to hypomethylating drugs *in vivo* is complex and may involve differentiation and immune activating components. Cytogenetic analysis showed that MDS patients after relapse using decitabine showed evolution in 20% patients with abnormalities

The understanding of the epigenetic changes characteristic of the malignant phenotype also permit the development of drugs that are able to target other regulators of chromatin conformation that contribute to aberrant gene transcription and dysregulated cell growth. The histone deacetylase inhibitors (HDAC) belong to one class of therapeutics developed using this paradigm. HDAC inhibitors modulate gene expression by inhibiting the deacetylation of histone lysine tails, relaxing the chromatin structure by decreasing the interaction between positively charged lysine tails of histones and negatively charged DNA. Although responses using HDAC inhibitors alone in MDS have been modest, preclinical data drives clinical trials in which they are utilized in combination with DNA methyltransferase inhibitors. Combination therapy offers the possibility of hematologic improvement and remission to MDS patients with previously untreatable disease (Vigna et

DNA methyltransferase inhibitors (hypomethylating agents) have emerged as options for the treatment of patients with MDS. These drugs lead to the progressive loss of methylation and reversal of gene silencing. In addition to their differentiation-inducing activity, these agents also have direct cytotoxic effects (Gurion et al., 2009). Currently available DNA methylation blocks all DNMTs. One of the main problems in using DNMT-Is in therapy is activation of cancer-promoting genes as well as other disease-promoting genes by hypomethylation. Recent studies suggest that there might be differences in the target specificity of different DNMTs. It is important to characterize the cancer related genes regulated by each of the DNMTs and develop DNMT gene-specific inhibitors. Moreover, treatment duration and maintenance therapy of using these agents require further

**3.5 DNA methylation alterations as diagnostic and prognostic biomarkers** 

biomarkers that may aid the diagnosis and the prognosis.

Epigenetic transcriptional silencing of genes required for proliferation and differentiation of the hematopoetic cells are likely to contribute to the leukemogenic event underlying MDS. Altered DNA methylation patterns of some genes are not only of importance to our understanding of the molecular pathogenesis of the MDS, but may also serve as novel indicators for the diagnosis, the prognosis and the prediction of response to therapy. We can see in table 3 some genes that are methylated in MDS and are suggested as possible

gene has been related to more aggressive subtypes of disease and it is a possible biomarker of disease evolution (Quesnel et al., 1998; Rodrigues et al., 2010).

Another gene methylated in MDS is the death-associated protein kinase (DAP-kinase), a proapoptotic serine/threonine kinase. The analysis of the methylation status of DAP-kinase in bone marrow samples from patients with MDS at the time of initial diagnosis showed that hypermethylation of DAP-kinase was significantly correlated to loss of DAP-kinase expression. Alteration in the apoptotic response due to the loss of DAP-kinase function may be an early event in the transformation pathway to secondary leukemia via myelodysplasia (Wu et al., 2011).

The mechanistic bases of CIMP (CpG island methylator phenotype) in MDS remains unknown. One proposed mechanisms involves aberrant recruitment of DNA methyltransferases to CpG islands and/or loss of methylation protection. Another possible explanation is that hypermethylation may not be directly linked to the methylation machinery, but rather reflects environmental exposures (Shen et al., 2010).

The reversible character of epigenetic alterations (in contrast to genetic changes) was an important point for the development of therapeutic strategies evolving various epigenetic components for anticancer therapy. So, in MDS, the characterization in the diagnosis of epigenetic biomarkers of disease evolution may indicate the use of hypomethylating drugs in this group of patients.

A large number of treatments has been used in adults and children with primary MDS, with the goal of eliminating the cytopenias as well as to recover hematopoiesis. One of the therapies used is the support that involves blood transfusions, antibiotics, growth factors alone or in combination, cyclosporin or anti-lymphocyte globulin (ATG) are also used in patients with hypocellular bone marrow. In more advanced MDS subtypes (RAEB, RAEB-t), in some cases, it has been used the chemotherapy. The allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapeutic option for patients with MDS, however, its use is limited to patients up to 55 years old and patients who have histocompatible donors (Giralt et al., 2005; Kindwall-Keller & Isola, 2009). For children with MDS, allogeneic HSCT is considered as the best treatment option (Niemeyer & Kratz, 2008).

With the delineation of the characteristics that drive the biological phenotype of MDS, new drugs introduced in the treatment of this disease have shown a great therapeutic potential, such as the hypomethylant agents, called methyltransferase inhibitors (IMT). The representatives of this class include azacitidine (5-azacytidine) and decitabine (5-aza 2 'deoxycytidine). Both are incorporated into DNA and then irreversibly bind and inhibit the action of DNA methyltransferase. This interaction results initially in a semi-methylated DNA. However, after further cell cycles, it becomes completely unmethylated. The action of these drugs leads to reactivation of epigenetically repressed genes, such as tumor suppressor genes. Initial results showed that patients with higher-risk MDS have an increased time to AML transformation and an increase of survival time (Atallah et al., 2007; Fenaux et al., 2009; Silverman & Mufti, 2005). The decitabine and azacytidine are approved for treatment of patients with int-2 and high-risk MDS. Demethylating agents seem to be the best choice for elderly patients with MDS, even in case of high risk cytogenetic changes in karyotype, like monosomy 7 (Gurion et al., 2010; Szmigielska-Kaplon & Robak, 2011).

gene has been related to more aggressive subtypes of disease and it is a possible biomarker

Another gene methylated in MDS is the death-associated protein kinase (DAP-kinase), a proapoptotic serine/threonine kinase. The analysis of the methylation status of DAP-kinase in bone marrow samples from patients with MDS at the time of initial diagnosis showed that hypermethylation of DAP-kinase was significantly correlated to loss of DAP-kinase expression. Alteration in the apoptotic response due to the loss of DAP-kinase function may be an early event in the transformation pathway to secondary leukemia via myelodysplasia

The mechanistic bases of CIMP (CpG island methylator phenotype) in MDS remains unknown. One proposed mechanisms involves aberrant recruitment of DNA methyltransferases to CpG islands and/or loss of methylation protection. Another possible explanation is that hypermethylation may not be directly linked to the methylation

The reversible character of epigenetic alterations (in contrast to genetic changes) was an important point for the development of therapeutic strategies evolving various epigenetic components for anticancer therapy. So, in MDS, the characterization in the diagnosis of epigenetic biomarkers of disease evolution may indicate the use of hypomethylating drugs

A large number of treatments has been used in adults and children with primary MDS, with the goal of eliminating the cytopenias as well as to recover hematopoiesis. One of the therapies used is the support that involves blood transfusions, antibiotics, growth factors alone or in combination, cyclosporin or anti-lymphocyte globulin (ATG) are also used in patients with hypocellular bone marrow. In more advanced MDS subtypes (RAEB, RAEB-t), in some cases, it has been used the chemotherapy. The allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapeutic option for patients with MDS, however, its use is limited to patients up to 55 years old and patients who have histocompatible donors (Giralt et al., 2005; Kindwall-Keller & Isola, 2009). For children with MDS, allogeneic HSCT is considered as the best treatment option (Niemeyer & Kratz, 2008). With the delineation of the characteristics that drive the biological phenotype of MDS, new drugs introduced in the treatment of this disease have shown a great therapeutic potential, such as the hypomethylant agents, called methyltransferase inhibitors (IMT). The representatives of this class include azacitidine (5-azacytidine) and decitabine (5-aza 2 'deoxycytidine). Both are incorporated into DNA and then irreversibly bind and inhibit the action of DNA methyltransferase. This interaction results initially in a semi-methylated DNA. However, after further cell cycles, it becomes completely unmethylated. The action of these drugs leads to reactivation of epigenetically repressed genes, such as tumor suppressor genes. Initial results showed that patients with higher-risk MDS have an increased time to AML transformation and an increase of survival time (Atallah et al., 2007; Fenaux et al., 2009; Silverman & Mufti, 2005). The decitabine and azacytidine are approved for treatment of patients with int-2 and high-risk MDS. Demethylating agents seem to be the best choice for elderly patients with MDS, even in case of high risk cytogenetic changes in karyotype, like monosomy 7 (Gurion et al., 2010; Szmigielska-Kaplon & Robak, 2011).

machinery, but rather reflects environmental exposures (Shen et al., 2010).

of disease evolution (Quesnel et al., 1998; Rodrigues et al., 2010).

(Wu et al., 2011).

in this group of patients.

Some studies have shown that reduced methylation overtime was correlated with better clinical response for patients during decitabine treatment. But, further studies of methylation dynamics, both before and after treatment, will be useful to determine the ability of epigenetic biomarkers to direct the treatment and may predict for the success (Shen et al., 2010). However, in some clinical trials, it was found that a number of patients does not respond to decitabine initially (primary resistance) and most patients, who initially respond to decitabine treatment, eventually relapse (secondary resistance) despite continued therapy. Clinical response to hypomethylating drugs *in vivo* is complex and may involve differentiation and immune activating components. Cytogenetic analysis showed that MDS patients after relapse using decitabine showed evolution in 20% patients with abnormalities such -7, del(16q) and +8 (Qin et al., 2011).

The understanding of the epigenetic changes characteristic of the malignant phenotype also permit the development of drugs that are able to target other regulators of chromatin conformation that contribute to aberrant gene transcription and dysregulated cell growth. The histone deacetylase inhibitors (HDAC) belong to one class of therapeutics developed using this paradigm. HDAC inhibitors modulate gene expression by inhibiting the deacetylation of histone lysine tails, relaxing the chromatin structure by decreasing the interaction between positively charged lysine tails of histones and negatively charged DNA. Although responses using HDAC inhibitors alone in MDS have been modest, preclinical data drives clinical trials in which they are utilized in combination with DNA methyltransferase inhibitors. Combination therapy offers the possibility of hematologic improvement and remission to MDS patients with previously untreatable disease (Vigna et al., 2011).

DNA methyltransferase inhibitors (hypomethylating agents) have emerged as options for the treatment of patients with MDS. These drugs lead to the progressive loss of methylation and reversal of gene silencing. In addition to their differentiation-inducing activity, these agents also have direct cytotoxic effects (Gurion et al., 2009). Currently available DNA methylation blocks all DNMTs. One of the main problems in using DNMT-Is in therapy is activation of cancer-promoting genes as well as other disease-promoting genes by hypomethylation. Recent studies suggest that there might be differences in the target specificity of different DNMTs. It is important to characterize the cancer related genes regulated by each of the DNMTs and develop DNMT gene-specific inhibitors. Moreover, treatment duration and maintenance therapy of using these agents require further investigation (Vigna et al., 2011).

### **3.5 DNA methylation alterations as diagnostic and prognostic biomarkers**

Epigenetic transcriptional silencing of genes required for proliferation and differentiation of the hematopoetic cells are likely to contribute to the leukemogenic event underlying MDS. Altered DNA methylation patterns of some genes are not only of importance to our understanding of the molecular pathogenesis of the MDS, but may also serve as novel indicators for the diagnosis, the prognosis and the prediction of response to therapy. We can see in table 3 some genes that are methylated in MDS and are suggested as possible biomarkers that may aid the diagnosis and the prognosis.

Epigenetics in Cancer: The Myelodysplastic Syndrome as a

mathematical formulas and theorems.

we recommend the book of Zar, 2010.

distribution: a probability distribution.

**4.1 Descriptive statistics and inferential statistics** 

research.

**4. Statistical analysis of epigenetics biomarkers** 

Model to Study Epigenetic Alterations as Diagnostic and Prognostic Biomarkers 35

Biomarkers have become important tools for diagnosis and treatment of a wide range of illnesses, including cancer. Early detection of cancer through biomarkers will allow for the development of new therapeutic procedures in order to increase survival rate of patients diagnosed with cancer. To help the evaluation of new biomarkers for medical practice, we use statistical methods. In this section, we shall discuss statistical techniques for biomarkers evaluation in the myelodysplastic syndrome (MDS). While the application of the methods presented is on biomarkers in MDS, the content of this section can be applied to any medical

The main mathematical concept necessary to understand statistical methods is *probability.* Although earlier work on the subject was done by the Italian mathematician Giralamo Cardano (1501-1576), the investigation of probability as a branch of Mathematics sprang about 1654 with two great French mathematicians: Blaise Pascal (1623-1662) and Pierre Fermat (1601–1665). Both Pascal and Fermat were interested in predicting outcomes in the games of chance popular among the French nobility of the mid-seventeenth century. Of course, we shall not do a discourse on probability. But we need to say that the theory of probability underlies the procedures in inferential statistics, which is very useful to medicine and other disciplines in the health field. In our exposition, we will try to avoid

Statistics is a branch of Mathematics. The word "statistics" derives from the Latin word *status*, meaning "manner of standing" or "position". Statistics were first used by tax assessors to collect information for determining assets and assessing taxes. Nowadays, the application of statistics is broad and includes business, marketing, economics, agriculture, education, medicine and others. Statistics applied to medicine and other health disciplines is called biostatistics or biometrics. For those who would like to review or study this subject

Statistics is divided into two branches: descriptive and inferential. The goal of descriptive statistics is to organize and summarize data. And the goal of inferential statistics is to draw inferences and reach conclusions about a population, when only a sample from the population has been studied. A population is a complete set of observations, patients,

To organize data and summarize their main characteristics we can use *tables, graphs* and *quantitative indices*. Tables are often used to present qualitative and quantitative data. Graphs are used widely to provide a visual display data. The bar diagram, histogram and frequency polygon are three graphic formats that are commonly used to present medical data. A table or a graph in which all values of a variable of interest are displayed with their corresponding frequency is called a *frequency distribution,* or simply a *distribution.* We shall see in the next subsection that in inferential statistics we are interested in a special type of

Quantitative indices are numbers that describe the center and the variation of a distribution. Quantitative indices that describe the center of a distribution are referred to as *measures of* 

measurements, and so forth. A sample is a subset of a certain population.


Table 3. Methylation alterations in genes involved in the pathogenesis of MDS.
