**Infectious Etiology of Childhood Acute Lymphoblastic Leukemia, Hypotheses and Evidence**

Abigail Morales-Sánchez and Ezequiel M. Fuentes-Pananá

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54455

## **1. Introduction**

Research on the role of infectious agents in the etiology of cancer has grown remarkably in recent decades. A causal association between infection events and the development of differ‐ ent types of cancer has been strongly suggested in epidemiologic studies, while the direct oncogenic capacity of a set of pathogens has been demonstrated in the laboratory.

It is now recognized that between 15 and 20% of all tumors are associated with infection by direct tumorigenic agents [1]. However, the transforming mechanisms of carcinogenic infec‐ tious agents are not restricted to the expression of oncogenes and their ability to modulate the expression and function of oncogenes and anti-oncogenes in target cells. Other routes of transformation have been described, in which, an agent participates through more indirect mechanisms, such as promoting immune suppression or chronic inflammation. Although, in indirect mechanisms of transformation the infectious agent usually does not reside in the cell that will form the tumor mass, it contributes to cancer development making favorable conditions for tumor initiation or growth.

One of the malignancies proposed to be etiologically related to infection is childhood acute lymphoblastic leukemia (ALL). ALL is a heterogeneous group of hematologic malignancies in which the process of differentiation and limited proliferation that characterizes normal lymphopoiesis is altered and replaced by a malignant clonal expansion of immature lym‐ phocytes. ALL is the most common type of childhood malignancy worldwide, unfortunate‐ ly, little is known about the origin of ALL, some cases are associated with genetic predisposition conferred by Down syndrome, Bloom syndrome, ataxia-telangiectasia, Nij‐ megen breakage syndrome or exposure to environmental agents such as ionizing radiation

properly cited.

© 2013 Morales-Sánchez and Fuentes-Pananá; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

or mutagenic chemicals, however these events account for less than 5% of ALL cases [2], therefore, discernible causal factors involved in cancer initiation or promotion are unknown for the bulk of primary leukemia.

Greaves' hypothesis is based on the observation of the steady increase of childhood leuke‐ mia parallel to the increase of upscale living conditions in developed countries. Since its publication, a series of epidemiological studies have been designed to test the *delayed infec‐ tion* hypothesis. Evaluation of parity, breastfeeding, improved hygiene conditions, neonatal or infant infections, vaccination against some viruses, day care attendance [9-13] among oth‐ ers, have been used as markers of exposure to infectious agents during the first years of life. As we will see next, these studies have found heterogeneous and even contradictory results. The United Kingdom Childhood Cancer Study (UKCCS), a nationwide, population based case-control study, was designed to investigate different hypotheses about risk factors in childhood cancer, one of them referred to the association between day care attendance dur‐ ing the first year of life and the risk of developing leukemia [11]. Day care attendance was used as a surrogate marker for exposure to infectious agents, assuming that as more contacts a child has, there is a larger chance for exposure to infections. Data were obtained through interviews with parents of 1286 children with ALL between 2 and 14 years of age and 3605 controls from 10 different regions of the UK. The results showed an inverse relationship be‐ tween 'social activity' and the risk of leukemia, OR=0.73 (95% confidence interval (CI): 0.62-0.87), showing also a dose-response trend. The interpretation of these findings was that early exposure to infections, indicated by day care attendance, is a protective factor against

Infectious Etiology of Childhood Acute Lymphoblastic Leukemia, Hypotheses and Evidence

http://dx.doi.org/10.5772/54455

21

childhood leukemia, thus supporting Greaves' *delayed infection* proposal [11].

no support to the *delayed infection* hypothesis.

Another study from the same UKCCS data set was published two years later. In this report, it was analyzed the relationship between neonatal infections and risk of leukemia; the data were extracted from primary-care records compiled before diagnosis and interviews with parents. According to this study, children with ALL (ages 2-5 years) had more clinically di‐ agnosed neonatal infections than their counterpart control: episodes number=3.6 (95% CI: 3.3-3.9) *vs* 3.1 (95% CI:2.9-3.2) [14]. These results contrast with the ones from the previous UKCCS study and argue that early infections are a risk factor for ALL, and therefore, give

The study by Cardwell and colleagues using hospital records of clinically diagnosed infec‐ tions in the first year of life from the UK General Practice Research Database (GPRD), com‐ pared 162 ALL cases with 2215 matched controls, no differences were found between cases and controls OR=1.05 (95%CI:0.64-1.74), then this study provided no support to Greaves' hy‐ pothesis [15]. Another large group, the Northern California study group analyzed day care attendance and parental recall of children ear infections between 294 ALL cases (ages 1-14) and 376 matched controls. Both markers were found protective, OR=0.42 (95% CI:0.18-0.99) and OR=0.32 (95% CI:0.14-0.74), respectively, but only for non-Hispanic white children, sup‐ porting Greaves' hypothesis but suggesting ethnic differences in the etiology of ALL [16]. The number of children born in a family has also been used as a marker for microbiological exposure. Dockerty and colleagues investigated the association between parity and risk of ALL in children aged 0-14 from England and Wales. They found a statistically significant protective effect for ALL in children of houses with increasing parity, OR=0.5 (95% CI: 0.3-0.8) [9]. Infante-Rivard et al also evaluated parity and day care attendance in a popula‐ tion based study (491 leukemia cases of children under 10 years old and 491 matched con‐

Several etiologic factors have been proposed to cause ALL. One of the most reported in the literature and the subject of this chapter is related to infections. Independently, Greaves, Kinlen and Smith have suggested different mechanisms by which certain events related to infection may explain at least some cases of childhood leukemia [3-5]. Interestingly, the sug‐ gested role of infectious agents in leukemogenesis varies from one hypothesis to another, fa‐ voring either direct or indirect mechanisms of transformation. It is our main goal to describe these hypotheses highlighting the type of evidence in favor and against them and providing a biological frame in which to discuss possible mechanisms of leukemogenesis by the infec‐ tious agents. Due to the large number of publications in the field, this is not intended as an in-deep and complete review of all published literature but a summary in which to set the basis for discussion.

## **2. 'Delayed infection' hypothesis and 'two-hits' minimal model by Greaves**

One of the most cited proposals on the infectious etiology of ALL is the *delayed infection* hy‐ pothesis, in which Greaves argues that some cases of the common B-ALL (CD10+ CD19+ preB cALL) observed in the peak age of 2 to 5 years could be associated with an aberrant immune response displayed by an immature immune system [3]. This hypothesis is based in the theory that early exposures to common infectious agents are required for the proper ma‐ turation of the immune system, lack of these exposures results in aberrant responses when children are finally in contact with the agent(s). In Greaves view, ALL develops in the bio‐ logical context of an aberrant immune response due to delayed infections, and thus, the in‐ fectious agents are only an indirect trigger of the leukemogenic process.

More recently, Greaves has added to his proposal the most frequent chromosomal aberra‐ tions in pre-B cALL, hyperdiplody and the translocation TEL-AML1 (also known as ETV6- RUNX1), as susceptibility factors. Molecular analysis has shown shared clonotypic TEL and AML1 breakpoints in leukemic blasts from monochorionic monozygotic identical twins [6]. The same result has been observed when comparing the patients' blood at diagnosis and their blood archived at birth (Guthrie cards) [7]. These results have supported that these ge‐ netic insults are often generated *in utero*, based on such findings, Greaves has proposed a minimal 'two-hits' model to explain the development of pre-B cALL [8]. According to this model, hyperdiploidy or the TEL-AML1 translocation originate *in utero* and provide the first oncogenic hit, which is not sufficient for the occurrence of the disease but generates a preleukemic clone. In the presence of additional postnatal oncogenic hits, this susceptible clone then evolves into a malignant leukemic clone. Such additional hits could be promoted indi‐ rectly by the aberrant immune response to infection of children growing in microbiological isolated environments.

Greaves' hypothesis is based on the observation of the steady increase of childhood leuke‐ mia parallel to the increase of upscale living conditions in developed countries. Since its publication, a series of epidemiological studies have been designed to test the *delayed infec‐ tion* hypothesis. Evaluation of parity, breastfeeding, improved hygiene conditions, neonatal or infant infections, vaccination against some viruses, day care attendance [9-13] among oth‐ ers, have been used as markers of exposure to infectious agents during the first years of life. As we will see next, these studies have found heterogeneous and even contradictory results.

or mutagenic chemicals, however these events account for less than 5% of ALL cases [2], therefore, discernible causal factors involved in cancer initiation or promotion are unknown

20 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

Several etiologic factors have been proposed to cause ALL. One of the most reported in the literature and the subject of this chapter is related to infections. Independently, Greaves, Kinlen and Smith have suggested different mechanisms by which certain events related to infection may explain at least some cases of childhood leukemia [3-5]. Interestingly, the sug‐ gested role of infectious agents in leukemogenesis varies from one hypothesis to another, fa‐ voring either direct or indirect mechanisms of transformation. It is our main goal to describe these hypotheses highlighting the type of evidence in favor and against them and providing a biological frame in which to discuss possible mechanisms of leukemogenesis by the infec‐ tious agents. Due to the large number of publications in the field, this is not intended as an in-deep and complete review of all published literature but a summary in which to set the

**2. 'Delayed infection' hypothesis and 'two-hits' minimal model by**

fectious agents are only an indirect trigger of the leukemogenic process.

One of the most cited proposals on the infectious etiology of ALL is the *delayed infection* hy‐ pothesis, in which Greaves argues that some cases of the common B-ALL (CD10+ CD19+ preB cALL) observed in the peak age of 2 to 5 years could be associated with an aberrant immune response displayed by an immature immune system [3]. This hypothesis is based in the theory that early exposures to common infectious agents are required for the proper ma‐ turation of the immune system, lack of these exposures results in aberrant responses when children are finally in contact with the agent(s). In Greaves view, ALL develops in the bio‐ logical context of an aberrant immune response due to delayed infections, and thus, the in‐

More recently, Greaves has added to his proposal the most frequent chromosomal aberra‐ tions in pre-B cALL, hyperdiplody and the translocation TEL-AML1 (also known as ETV6- RUNX1), as susceptibility factors. Molecular analysis has shown shared clonotypic TEL and AML1 breakpoints in leukemic blasts from monochorionic monozygotic identical twins [6]. The same result has been observed when comparing the patients' blood at diagnosis and their blood archived at birth (Guthrie cards) [7]. These results have supported that these ge‐ netic insults are often generated *in utero*, based on such findings, Greaves has proposed a minimal 'two-hits' model to explain the development of pre-B cALL [8]. According to this model, hyperdiploidy or the TEL-AML1 translocation originate *in utero* and provide the first oncogenic hit, which is not sufficient for the occurrence of the disease but generates a preleukemic clone. In the presence of additional postnatal oncogenic hits, this susceptible clone then evolves into a malignant leukemic clone. Such additional hits could be promoted indi‐ rectly by the aberrant immune response to infection of children growing in microbiological

for the bulk of primary leukemia.

basis for discussion.

isolated environments.

**Greaves**

The United Kingdom Childhood Cancer Study (UKCCS), a nationwide, population based case-control study, was designed to investigate different hypotheses about risk factors in childhood cancer, one of them referred to the association between day care attendance dur‐ ing the first year of life and the risk of developing leukemia [11]. Day care attendance was used as a surrogate marker for exposure to infectious agents, assuming that as more contacts a child has, there is a larger chance for exposure to infections. Data were obtained through interviews with parents of 1286 children with ALL between 2 and 14 years of age and 3605 controls from 10 different regions of the UK. The results showed an inverse relationship be‐ tween 'social activity' and the risk of leukemia, OR=0.73 (95% confidence interval (CI): 0.62-0.87), showing also a dose-response trend. The interpretation of these findings was that early exposure to infections, indicated by day care attendance, is a protective factor against childhood leukemia, thus supporting Greaves' *delayed infection* proposal [11].

Another study from the same UKCCS data set was published two years later. In this report, it was analyzed the relationship between neonatal infections and risk of leukemia; the data were extracted from primary-care records compiled before diagnosis and interviews with parents. According to this study, children with ALL (ages 2-5 years) had more clinically di‐ agnosed neonatal infections than their counterpart control: episodes number=3.6 (95% CI: 3.3-3.9) *vs* 3.1 (95% CI:2.9-3.2) [14]. These results contrast with the ones from the previous UKCCS study and argue that early infections are a risk factor for ALL, and therefore, give no support to the *delayed infection* hypothesis.

The study by Cardwell and colleagues using hospital records of clinically diagnosed infec‐ tions in the first year of life from the UK General Practice Research Database (GPRD), com‐ pared 162 ALL cases with 2215 matched controls, no differences were found between cases and controls OR=1.05 (95%CI:0.64-1.74), then this study provided no support to Greaves' hy‐ pothesis [15]. Another large group, the Northern California study group analyzed day care attendance and parental recall of children ear infections between 294 ALL cases (ages 1-14) and 376 matched controls. Both markers were found protective, OR=0.42 (95% CI:0.18-0.99) and OR=0.32 (95% CI:0.14-0.74), respectively, but only for non-Hispanic white children, sup‐ porting Greaves' hypothesis but suggesting ethnic differences in the etiology of ALL [16].

The number of children born in a family has also been used as a marker for microbiological exposure. Dockerty and colleagues investigated the association between parity and risk of ALL in children aged 0-14 from England and Wales. They found a statistically significant protective effect for ALL in children of houses with increasing parity, OR=0.5 (95% CI: 0.3-0.8) [9]. Infante-Rivard et al also evaluated parity and day care attendance in a popula‐ tion based study (491 leukemia cases of children under 10 years old and 491 matched con‐ trols) in Quebéc Canada. This group found a protective association with day care attendance, OR=0.49 (95% IC:0.31-0.77) and breast-feeding OR=0.68 (95% IC:0.49-0.95), while having older siblings was associated with increased risk of leukemia, OR=2.12 (95% IC: 1.57-2.85) [12].

an increased incidence of childhood leukemia, returning to normal numbers in subsequent years [4]. Other relevant studies of Kinlen's group are concerned with new military settle‐ ments; for instance, in post-war Britain between 1949 and 1950, when national military serv‐ ice was mandatory for all men reaching 18 years of age and the period of service was increased from 1 to 2 years. During the following years there was a significant increase of leukemia in areas with the highest proportion of military servicemen. A similar phenomen‐ on was observed in Fallon, Nevada US when there was a considerable increase in the num‐

Infectious Etiology of Childhood Acute Lymphoblastic Leukemia, Hypotheses and Evidence

http://dx.doi.org/10.5772/54455

23

Virtually every study that has been led by Kinlen's working group has shown similar re‐ sults, *i.e.* they have observed a significant increase in childhood leukemia matching largescale mixing between rural and urban populations. [22-27]. In favor of Kinlen proposal, childhood leukemia clusters were more evident when people from urban regions were mixed with people from isolated areas with low population density, and those who develop leukemia were mostly children from the most immunologically isolated. Also, the leukemia peaks were transitory coinciding with the largest flow of people, arguing against a common

Other researchers have addressed the same question. For example, Koushik and colleagues conducted an ecologic study of childhood leukemia and population mixing in Ontario, Can‐ ada. The percent of population change was employed as indicator of mixing population. In this study, 1394 leukemia cases recorded between 1978 and 1992 were included. The results showed that population growth was also associated with a high incidence of leukemia, but only in rural and not in urban areas [28]. Other studies have shown no support for the Kin‐ len's hypothesis, among them is Laplanche & de Vathaire's [29]. This study included all French communities and covered the period between 1968 and 1990 during which occurred a rapid population increase. According to the results during the mentioned period, deaths from leukemia in children or young adults under 25 years of age were slightly lower than the expected estimate and no differences in risk according to the size of population increase or region were found. Another French study carried around the nuclear reprocessing plant

Although, not all the studies carried out around areas of population mixing have correlated with clusters of childhood leukemia, it is relevant that most do. It is also important that, al‐ though the original observation was done around nuclear plants, there is evidence of a simi‐ lar phenomenon occurring in many other regions around non-nuclear sites, including military settlements. From his observations, Kinlen proposed that a common infectious agent could be responsible and adults are the main transmitters, thus population mixing

If Kinlen proposal is true, it is possible that the data against his hypothesis had different ex‐ planations: 1) the effect may be dose dependent, so, high levels of contact might be necessa‐ ry; 2) the hypothesis has been proposed for large-scale rural-urban population mixing and many studies might not reach the required population threshold, and 3) other genetic

of La Hague found no evidence of increase in childhood leukemia cases [30].

could be responsible for the leukemia cases seen even in the first year of life.

and/or environmental differences might be affecting the outcome [4, 22].

ber of trainee recruits in the nearby naval base [21].

source of a persistent chemical/radiation contaminant.

The study by Flores-Lujano evaluated the frequency of severe infections that required hospi‐ talization in the first year of life in children with Down's syndrome (DS) with or without ALL (57 cases and 218 controls aged 19 years or younger). In this study, DS children were chosen because it is known that they have an around 10 to 30 fold higher incidence of B cell ALL. They also found an association between infection an increased risk of leukemia, OR=3.45 (95% CI:1.37–8.66), which is against the Greaves*'* hypothesis [17].

In summary, many studies have explored the *delayed infection* hypotheses with heterogene‐ ous results, with some studies actually showing an increased risk given by infections in the first years of life. The lack of consistency among investigations deserves further analysis and it is beyond the aim of this chapter. Some of the variables among studies are concerned with the methodological approach, study design, statistical tests and the representativeness of the studied population, among many others that could explain the heterogeneity of the results. Many other considerations are more related to the biological aspects of the hypothesis as it is discussed in the integrated discussion with the other hypotheses concerning an infectious origin of childhood leukemia.

## **3. 'Population mixing' hypothesis by Kinlen**

In early 1980, an unusual increase in the incidence of childhood leukemia was observed in young people living in the vicinity of nuclear reprocessing plants in Cumbria, England and Dounreay, Scotland. It was thought that such increase in leukemias was the result of radio‐ active contamination, which might have caused somatic or germinal line mutations in the population [18-20]. However, in deep tests showed no evidence of radioactive leaks (Com‐ mittee on Medical Aspects of Radiation in the Environment) or many other types of popula‐ tion occupational exposures [20].

In 1988 Kinlen proposed that the observed leukemia clusters could result from the un‐ usual population mixing occurring in regions receiving the influx of workers and their families who were attracted by new jobs in nuclear plants. Disease outbreaks associated with population growth and migration had been previously documented, and Kinlen hy‐ pothesized that this was also the case for the leukemia clusters. During populations mix‐ ing, resident people would be naive to infection by different agents carried by the newcomers and vice versa, exposure to such agents would cause an abnormal response leading to the outbreak [4].

Kinlen first proved his *population mixing* hypothesis in Thurso, Scotland, an isolated rural area that received large influxes of people who had migrated to work at a nuclear plant. The results showed that during the period when the population doubled (1951-1967) there was an increased incidence of childhood leukemia, returning to normal numbers in subsequent years [4]. Other relevant studies of Kinlen's group are concerned with new military settle‐ ments; for instance, in post-war Britain between 1949 and 1950, when national military serv‐ ice was mandatory for all men reaching 18 years of age and the period of service was increased from 1 to 2 years. During the following years there was a significant increase of leukemia in areas with the highest proportion of military servicemen. A similar phenomen‐ on was observed in Fallon, Nevada US when there was a considerable increase in the num‐ ber of trainee recruits in the nearby naval base [21].

trols) in Quebéc Canada. This group found a protective association with day care attendance, OR=0.49 (95% IC:0.31-0.77) and breast-feeding OR=0.68 (95% IC:0.49-0.95), while having older siblings was associated with increased risk of leukemia, OR=2.12 (95% IC:

The study by Flores-Lujano evaluated the frequency of severe infections that required hospi‐ talization in the first year of life in children with Down's syndrome (DS) with or without ALL (57 cases and 218 controls aged 19 years or younger). In this study, DS children were chosen because it is known that they have an around 10 to 30 fold higher incidence of B cell ALL. They also found an association between infection an increased risk of leukemia,

In summary, many studies have explored the *delayed infection* hypotheses with heterogene‐ ous results, with some studies actually showing an increased risk given by infections in the first years of life. The lack of consistency among investigations deserves further analysis and it is beyond the aim of this chapter. Some of the variables among studies are concerned with the methodological approach, study design, statistical tests and the representativeness of the studied population, among many others that could explain the heterogeneity of the results. Many other considerations are more related to the biological aspects of the hypothesis as it is discussed in the integrated discussion with the other hypotheses concerning an infectious

In early 1980, an unusual increase in the incidence of childhood leukemia was observed in young people living in the vicinity of nuclear reprocessing plants in Cumbria, England and Dounreay, Scotland. It was thought that such increase in leukemias was the result of radio‐ active contamination, which might have caused somatic or germinal line mutations in the population [18-20]. However, in deep tests showed no evidence of radioactive leaks (Com‐ mittee on Medical Aspects of Radiation in the Environment) or many other types of popula‐

In 1988 Kinlen proposed that the observed leukemia clusters could result from the un‐ usual population mixing occurring in regions receiving the influx of workers and their families who were attracted by new jobs in nuclear plants. Disease outbreaks associated with population growth and migration had been previously documented, and Kinlen hy‐ pothesized that this was also the case for the leukemia clusters. During populations mix‐ ing, resident people would be naive to infection by different agents carried by the newcomers and vice versa, exposure to such agents would cause an abnormal response

Kinlen first proved his *population mixing* hypothesis in Thurso, Scotland, an isolated rural area that received large influxes of people who had migrated to work at a nuclear plant. The results showed that during the period when the population doubled (1951-1967) there was

OR=3.45 (95% CI:1.37–8.66), which is against the Greaves*'* hypothesis [17].

22 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

1.57-2.85) [12].

origin of childhood leukemia.

tion occupational exposures [20].

leading to the outbreak [4].

**3. 'Population mixing' hypothesis by Kinlen**

Virtually every study that has been led by Kinlen's working group has shown similar re‐ sults, *i.e.* they have observed a significant increase in childhood leukemia matching largescale mixing between rural and urban populations. [22-27]. In favor of Kinlen proposal, childhood leukemia clusters were more evident when people from urban regions were mixed with people from isolated areas with low population density, and those who develop leukemia were mostly children from the most immunologically isolated. Also, the leukemia peaks were transitory coinciding with the largest flow of people, arguing against a common source of a persistent chemical/radiation contaminant.

Other researchers have addressed the same question. For example, Koushik and colleagues conducted an ecologic study of childhood leukemia and population mixing in Ontario, Can‐ ada. The percent of population change was employed as indicator of mixing population. In this study, 1394 leukemia cases recorded between 1978 and 1992 were included. The results showed that population growth was also associated with a high incidence of leukemia, but only in rural and not in urban areas [28]. Other studies have shown no support for the Kin‐ len's hypothesis, among them is Laplanche & de Vathaire's [29]. This study included all French communities and covered the period between 1968 and 1990 during which occurred a rapid population increase. According to the results during the mentioned period, deaths from leukemia in children or young adults under 25 years of age were slightly lower than the expected estimate and no differences in risk according to the size of population increase or region were found. Another French study carried around the nuclear reprocessing plant of La Hague found no evidence of increase in childhood leukemia cases [30].

Although, not all the studies carried out around areas of population mixing have correlated with clusters of childhood leukemia, it is relevant that most do. It is also important that, al‐ though the original observation was done around nuclear plants, there is evidence of a simi‐ lar phenomenon occurring in many other regions around non-nuclear sites, including military settlements. From his observations, Kinlen proposed that a common infectious agent could be responsible and adults are the main transmitters, thus population mixing could be responsible for the leukemia cases seen even in the first year of life.

If Kinlen proposal is true, it is possible that the data against his hypothesis had different ex‐ planations: 1) the effect may be dose dependent, so, high levels of contact might be necessa‐ ry; 2) the hypothesis has been proposed for large-scale rural-urban population mixing and many studies might not reach the required population threshold, and 3) other genetic and/or environmental differences might be affecting the outcome [4, 22].

Similar to the Greaves' hypothesis, the identity of the infectious agent(s) involved in Kinlen model is still not known. In fact, most of the population mixing studies had failed to find an increase in a symptomatic infection in adults or children, paralleling the increase in leuke‐ mia incidence. Considering that there are viruses of recognized leukemia causality in ani‐ mals and one human's leukemia caused by a virus, Kinlen has proposed that the agent involved could be a prevalent virus causing an uncommon infection [31]. Kinlen also con‐ siders that the putative causative virus is not transmitted as a typical acute infection virus, a characteristic common of tumorigenic viruses. However, the viral family known to be in‐ volved in animal leukemia is the retroviridae, and specifically for adult humans the causa‐ tive agent is the human T cell leukemia/lymphoma virus type 1 (HTLV-1), which is endemic of areas with no recognized picks of childhood leukemia. Because both Kinlen and Greaves models fail to identify the causative agent, both hypotheses seem similar pointing out to a common mechanism of response rather than a possible direct mechanism of infection.

carries the power to trigger the chromosomal abnormalities often present in childhood leu‐ kemia, while for Greaves, the genetic insult is already present and the infection indirectly

Infectious Etiology of Childhood Acute Lymphoblastic Leukemia, Hypotheses and Evidence

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25

Several viral families fulfill Smith's criteria for a causative agent. Members of the adenovi‐ rus, herpesvirus and polyomavirus are transmitted very early pre- or post-natally, have tropism for bone marrow cells and have oncogenic potential; we know that most of the pop‐ ulation carries all these viruses asymptomatically, with only a few of them developing a re‐ lated-neoplasia. On the other hand, the retroviruses are also good candidates, as they already have been implicated in leukemias. Several transforming mechanisms have been de‐ scribed for all of these viruses, including expression of constitutively active viral signaling proteins, transcriptional activation of cellular oncogenes and/or disruption of tumor sup‐ pressor genes, and importantly, induction of genetic instability; for instance Epstein Barr Vi‐ rus (EBV or human herpesvirus-4) is associated with Burkitt's lymphoma, in which it also

Studies showing that maternal infections are associated with an increased risk of ALL sup‐ ported Smith's model. Lehtinen et al analyzed sera of the first trimester from 342 Finnish and Icelandic mothers of children with ALL, searching for antibodies against herpesvirus EBV, cytomegalovirus and HHV-6 (human herpesvirus-6). Only an increase of anti-EBV an‐ tibodies was found correlating with leukemia cases, OR=2.9 (95% CI:1.5-5.8) [33]. Because of the nature of the antibodies found, this data suggested EBV reactivation as a potential event leading development of ALL. This same group confirmed the above observation with an ad‐ ditional 304 mothers: anti-EBV reactivation antibodies, OR=1.9 (95% CI:1.2-3.0) [34]. The possible role of EBV reactivation during pregnancy is still awaiting confirmation from other groups. Naumberg's group also found a similar positive association when the mother had lower genital tract infections, OR=1.78 (95% CI:1.2-2.7), especially in children older than 4

Many other studies have shown conflicting results between viral infection during pregnancy and subsequent childhood leukemia in offspring, either by influenza virus or by other un‐ specified common infections [10, 12, 36]. On the other hand, several small studies have found an association between maternal varicella-zoster virus (causing chicken-pox) reactiva‐ tion and childhood leukemia [37, 38]. Note, however, that none of these approaches have addressed viruses with recognized oncogenic potential and that they are epidemiological

A distinct approach to explore direct transformation occurring *in utero* has been conducted through retrospective analyses of children who developed leukemia; in these studies, viral genomes have been searched in archived blood spots collected at birth with very heteroge‐ neous results. For instance, an early study found blood spots positive to adenovirus-C in two children that developed leukemia, but other groups have not reproduced such result [39]. Bogdanovic et al searched for viral genomes from herpesvirus EBV and HHV-6, polyo‐ mavirus JCV and BKV (from the patients' initials from whom the viruses were isolated) and parvovirus 19 in Guthrie cards from 54 Swedish patients, finding no association [40-42]. Par‐ vovirus B19 was another good candidate for causality since it has been associated with sev‐

studies based on the mother recalled history of infection during pregnancy.

promotes the acquisition of additional hits.

correlates with translocation of the cellular oncogene c-Myc [32].

years of age at diagnosis, OR=2.01 (95% CI:1.1-3.8) [35].

#### **4. Direct viral leukemogenesis hypothesis by Smith**

A third hypothesis regarding the infectious etiology of childhood leukemia was proposed by Smith and colleagues. According to the *delayed infection* hypothesis, children exposed to infectious agents during the first months of life (e.g. in developing countries) should have almost no leukemogenic potential, whereas children that become infected later (e.g. in afflu‐ ent societies), exposure to the same agent would be potentially leukemogenic. Smith disa‐ grees with this scenario, especially for children aged 2 and 3, which represent the larger proportion of children within the peak incidence of 2 to 5 years old, and suggested that there should be an alternative mechanism by which the infection leads to leukemia and that could explain all age-related picks of disease, including infant leukemias [5].

In his publication *Considerations on a possible viral etiology for B-precursor acute lymphoblastic leukemia of childhood* Smith proposed that the infectious process leading to leukemia occurs during intrauterine life by mother to fetus transmission [5]. *De novo* infected seronegative women or those in which the agent reactivation occurred during pregnancy were especially vulnerable to infect their fetus. This hypothesis also considers possible infections during the first year of life of children from seronegative mothers unable to passively immunize their offspring. According to Smith's hypothesis, the pathogen acts through a direct mechanism of B cell infection, initiating or complementing the process of cellular transformation togeth‐ er with additional oncogenic hits either intrauterine or postnatal.

Considering that more than 60% of cases of ALL-B are associated with chromosomal abnor‐ malities, Smith hypothesized that the agent involved should be a virus, since many viral agents present a variety of mechanisms that promote genetic instability. According to Smith's hypothesis the putative virus should have the ability to cross the placenta, to infect B lymphocytes and to have oncogenic potential. However, such agent should not have the ability to induce severe abnormalities, since ALL is not associated with other cancers or birth defects. Thus, an important difference of Smith's hypothesis is that the infection *per se* carries the power to trigger the chromosomal abnormalities often present in childhood leu‐ kemia, while for Greaves, the genetic insult is already present and the infection indirectly promotes the acquisition of additional hits.

Similar to the Greaves' hypothesis, the identity of the infectious agent(s) involved in Kinlen model is still not known. In fact, most of the population mixing studies had failed to find an increase in a symptomatic infection in adults or children, paralleling the increase in leuke‐ mia incidence. Considering that there are viruses of recognized leukemia causality in ani‐ mals and one human's leukemia caused by a virus, Kinlen has proposed that the agent involved could be a prevalent virus causing an uncommon infection [31]. Kinlen also con‐ siders that the putative causative virus is not transmitted as a typical acute infection virus, a characteristic common of tumorigenic viruses. However, the viral family known to be in‐ volved in animal leukemia is the retroviridae, and specifically for adult humans the causa‐ tive agent is the human T cell leukemia/lymphoma virus type 1 (HTLV-1), which is endemic of areas with no recognized picks of childhood leukemia. Because both Kinlen and Greaves models fail to identify the causative agent, both hypotheses seem similar pointing out to a common mechanism of response rather than a possible direct mechanism of infection.

A third hypothesis regarding the infectious etiology of childhood leukemia was proposed by Smith and colleagues. According to the *delayed infection* hypothesis, children exposed to infectious agents during the first months of life (e.g. in developing countries) should have almost no leukemogenic potential, whereas children that become infected later (e.g. in afflu‐ ent societies), exposure to the same agent would be potentially leukemogenic. Smith disa‐ grees with this scenario, especially for children aged 2 and 3, which represent the larger proportion of children within the peak incidence of 2 to 5 years old, and suggested that there should be an alternative mechanism by which the infection leads to leukemia and that

In his publication *Considerations on a possible viral etiology for B-precursor acute lymphoblastic leukemia of childhood* Smith proposed that the infectious process leading to leukemia occurs during intrauterine life by mother to fetus transmission [5]. *De novo* infected seronegative women or those in which the agent reactivation occurred during pregnancy were especially vulnerable to infect their fetus. This hypothesis also considers possible infections during the first year of life of children from seronegative mothers unable to passively immunize their offspring. According to Smith's hypothesis, the pathogen acts through a direct mechanism of B cell infection, initiating or complementing the process of cellular transformation togeth‐

Considering that more than 60% of cases of ALL-B are associated with chromosomal abnor‐ malities, Smith hypothesized that the agent involved should be a virus, since many viral agents present a variety of mechanisms that promote genetic instability. According to Smith's hypothesis the putative virus should have the ability to cross the placenta, to infect B lymphocytes and to have oncogenic potential. However, such agent should not have the ability to induce severe abnormalities, since ALL is not associated with other cancers or birth defects. Thus, an important difference of Smith's hypothesis is that the infection *per se*

**4. Direct viral leukemogenesis hypothesis by Smith**

24 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

could explain all age-related picks of disease, including infant leukemias [5].

er with additional oncogenic hits either intrauterine or postnatal.

Several viral families fulfill Smith's criteria for a causative agent. Members of the adenovi‐ rus, herpesvirus and polyomavirus are transmitted very early pre- or post-natally, have tropism for bone marrow cells and have oncogenic potential; we know that most of the pop‐ ulation carries all these viruses asymptomatically, with only a few of them developing a re‐ lated-neoplasia. On the other hand, the retroviruses are also good candidates, as they already have been implicated in leukemias. Several transforming mechanisms have been de‐ scribed for all of these viruses, including expression of constitutively active viral signaling proteins, transcriptional activation of cellular oncogenes and/or disruption of tumor sup‐ pressor genes, and importantly, induction of genetic instability; for instance Epstein Barr Vi‐ rus (EBV or human herpesvirus-4) is associated with Burkitt's lymphoma, in which it also correlates with translocation of the cellular oncogene c-Myc [32].

Studies showing that maternal infections are associated with an increased risk of ALL sup‐ ported Smith's model. Lehtinen et al analyzed sera of the first trimester from 342 Finnish and Icelandic mothers of children with ALL, searching for antibodies against herpesvirus EBV, cytomegalovirus and HHV-6 (human herpesvirus-6). Only an increase of anti-EBV an‐ tibodies was found correlating with leukemia cases, OR=2.9 (95% CI:1.5-5.8) [33]. Because of the nature of the antibodies found, this data suggested EBV reactivation as a potential event leading development of ALL. This same group confirmed the above observation with an ad‐ ditional 304 mothers: anti-EBV reactivation antibodies, OR=1.9 (95% CI:1.2-3.0) [34]. The possible role of EBV reactivation during pregnancy is still awaiting confirmation from other groups. Naumberg's group also found a similar positive association when the mother had lower genital tract infections, OR=1.78 (95% CI:1.2-2.7), especially in children older than 4 years of age at diagnosis, OR=2.01 (95% CI:1.1-3.8) [35].

Many other studies have shown conflicting results between viral infection during pregnancy and subsequent childhood leukemia in offspring, either by influenza virus or by other un‐ specified common infections [10, 12, 36]. On the other hand, several small studies have found an association between maternal varicella-zoster virus (causing chicken-pox) reactiva‐ tion and childhood leukemia [37, 38]. Note, however, that none of these approaches have addressed viruses with recognized oncogenic potential and that they are epidemiological studies based on the mother recalled history of infection during pregnancy.

A distinct approach to explore direct transformation occurring *in utero* has been conducted through retrospective analyses of children who developed leukemia; in these studies, viral genomes have been searched in archived blood spots collected at birth with very heteroge‐ neous results. For instance, an early study found blood spots positive to adenovirus-C in two children that developed leukemia, but other groups have not reproduced such result [39]. Bogdanovic et al searched for viral genomes from herpesvirus EBV and HHV-6, polyo‐ mavirus JCV and BKV (from the patients' initials from whom the viruses were isolated) and parvovirus 19 in Guthrie cards from 54 Swedish patients, finding no association [40-42]. Par‐ vovirus B19 was another good candidate for causality since it has been associated with sev‐ eral childhood hematological diseases. One should consider that, although the search for viral genomes in Guthrie cards is more stringent, the negative result does not mean that there is not increased viral infection/reactivation during pregnancy and the titer and type of antibodies are probably more reliable markers for this.

Mackenzie et al searched for human herpesvirus-4 (EBV), -6, -7 and -8 (KSHV); 20 peripheral blood or bone marrow samples were tested by Southern blot (EBV) or conventional PCR (HHV -6, -7 and -8). The authors found that seven samples were positive for some of these viruses; however, the low viral load found indicated that the viral genome was not present in every leukemia blast and therefore the result did not support that infection was part of

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27

Bender et al screened for Bovine leukemia virus (BLV) years before the publication of Smith's proposal. BLV is an exogenous retrovirus whose direct role in the genesis of bovine leukemia has been well documented. 131 samples of ALL (the article did not address a spe‐ cific subtype of leukemia) and 136 controls were screened by Southern blot for the BLV ge‐ nome. Cases and controls were negative to the virus arguing against a positive role of BLV in childhood leukemia [46]. Screening for transfusion-transmitted virus (TTV) have also

In summary, different studies have failed to identify viral agents within the leukemia cells indicative of a a viral direct leukemogenic mechanism. However, it is important to consider that these studies included only a small number of samples, 50 or less. These studies at the most suggest that if an infectious agent is involved in leukemogenesis, this would occur in a limited number of cases. A larger number of samples from more geographical regions and

The list of candidate viruses is not exhausted yet and the pathogen involved in the genesis of leukemia (if any) could still be unknown, Kaposi sarcoma associated herpesvirus (KSHV) and Merkel cell polyomavirus (MCPV) were discovered a few years ago and have already been associated with several neoplasias including the ones from which the virus were isolat‐ ed, Kaposi's sarcoma and Merkel cell carcinoma, respectively [48]. Under this idea, the study of MacKenzie et al was designed to identify undescribed members of the Herpesviri‐ dae family by a degenerate PCR, but no new herpesviruses were found in any of the 18 sam‐ ples analyzed [45]. As the individual virus "hunt" is a limited method, next generation sequencing technologies are an attractive approach to ask for the presence of known and un‐

As we learn in the previous section, childhood leukemia has been shown to be a disease of‐ ten presented in space and time clusters correlating with communities with large influx of people. Population based morbility/mortality maps are used in public health to inform us of points of an excess of cases (the cluster) relative to the expected incidence, which are then unlikely to have happened by chance and points out to possible etiological factors and the population at risk. Leukemia aggregates have been studied for decades and to date, a num‐ ber of studies have reported an unusual increase in the number of cases associated with space-time patterns, some of them have been anecdotal reports but others have been discov‐ ered through employment of formal statistical analysis. We describe next some cluster stud‐

the initial insult that preceded the malignant clonal expansion [45].

different social strata should be included for a more definitive conclusion.

**5. Space-time clustering of childhood leukemia by Alexander**

been negative [47].

known infectious agents in leukemic cells.

Based on Smith's original proposal, the notion of a direct oncogenic mechanism in the etiolo‐ gy of childhood leukemia was widened to include infections with a transforming agent oc‐ curring postnatally but prior to the onset of the disease. In this possible leukemogenic mechanism, infection is not necessarily the first oncogenic hit. To test this proposal derived from Smith's hypothesis, different viral agents have been screened directly in the leukemia blast (Table 1). One study evaluated the presence of the viral genome of polyomavirus JCV and BKV in 15 samples at diagnosis of pre-B ALL and a second study included 25 samples in which the viral genome of JCV, BKV and SV40 (simian virus 40) were searched. In both studies, the screening was performed by PCR without finding any of these viruses present in the leukemia samples [43, 44].


\* In this study, the samples were obtained at diagnosis or during treatment. BM: bone marrow, PB: peripheral blood, CSF: cerebrospinal fluid, us: unspecified.

**Table 1.** Screening for viral sequences in ALL.

Mackenzie et al searched for human herpesvirus-4 (EBV), -6, -7 and -8 (KSHV); 20 peripheral blood or bone marrow samples were tested by Southern blot (EBV) or conventional PCR (HHV -6, -7 and -8). The authors found that seven samples were positive for some of these viruses; however, the low viral load found indicated that the viral genome was not present in every leukemia blast and therefore the result did not support that infection was part of the initial insult that preceded the malignant clonal expansion [45].

eral childhood hematological diseases. One should consider that, although the search for viral genomes in Guthrie cards is more stringent, the negative result does not mean that there is not increased viral infection/reactivation during pregnancy and the titer and type of

Based on Smith's original proposal, the notion of a direct oncogenic mechanism in the etiolo‐ gy of childhood leukemia was widened to include infections with a transforming agent oc‐ curring postnatally but prior to the onset of the disease. In this possible leukemogenic mechanism, infection is not necessarily the first oncogenic hit. To test this proposal derived from Smith's hypothesis, different viral agents have been screened directly in the leukemia blast (Table 1). One study evaluated the presence of the viral genome of polyomavirus JCV and BKV in 15 samples at diagnosis of pre-B ALL and a second study included 25 samples in which the viral genome of JCV, BKV and SV40 (simian virus 40) were searched. In both studies, the screening was performed by PCR without finding any of these viruses present

**Sample Screening method N Ref.**

Nested PCR 50

Nested PCR 50

Nested PCR 50

(only for EBV) and Realtime PCR

> dot blot and Southern blot

4

4

4

47 [45]

28 [47]

[40]

[41]

[42]

B-precursor ALL 1-12 BM or PB Endpoint PCR 15 [43]

B-precursor ALL 2-5 BM Real-time PCR 25 [44]

antibodies are probably more reliable markers for this.

26 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

in the leukemia samples [43, 44].

**Virus Leukemia**

Polyomaviruses JVC and BKV

Polyomaviruses JVC, BKV and SV40

Polyomaviruses JVC and BKV

Herpesviruses EBV y HHV-6

Herpesviruses EBV, HHV-6, -7 and -8

Parvovirus B19 B-precursor ALL

CSF: cerebrospinal fluid, us: unspecified.

**Table 1.** Screening for viral sequences in ALL.

**subtype**

B-precursor ALL T-ALL

B-precursor ALL T-ALL

T-ALL

**Age (years)**

0.75-17 Archived

0.75-17 Archived

B-precursor ALL 1.5-13 BM or PB Southern blot

0.75-17 Archived

Annelovirus TT ALL us BM, PB and CFS Nested PCR,

neonatal blood spots

neonatal blood spots

neonatal blood spots

Retrovirus BLV ALL ≤16 BM and PB Southern blot 131 [46]

\* In this study, the samples were obtained at diagnosis or during treatment. BM: bone marrow, PB: peripheral blood,

Bender et al screened for Bovine leukemia virus (BLV) years before the publication of Smith's proposal. BLV is an exogenous retrovirus whose direct role in the genesis of bovine leukemia has been well documented. 131 samples of ALL (the article did not address a spe‐ cific subtype of leukemia) and 136 controls were screened by Southern blot for the BLV ge‐ nome. Cases and controls were negative to the virus arguing against a positive role of BLV in childhood leukemia [46]. Screening for transfusion-transmitted virus (TTV) have also been negative [47].

In summary, different studies have failed to identify viral agents within the leukemia cells indicative of a a viral direct leukemogenic mechanism. However, it is important to consider that these studies included only a small number of samples, 50 or less. These studies at the most suggest that if an infectious agent is involved in leukemogenesis, this would occur in a limited number of cases. A larger number of samples from more geographical regions and different social strata should be included for a more definitive conclusion.

The list of candidate viruses is not exhausted yet and the pathogen involved in the genesis of leukemia (if any) could still be unknown, Kaposi sarcoma associated herpesvirus (KSHV) and Merkel cell polyomavirus (MCPV) were discovered a few years ago and have already been associated with several neoplasias including the ones from which the virus were isolat‐ ed, Kaposi's sarcoma and Merkel cell carcinoma, respectively [48]. Under this idea, the study of MacKenzie et al was designed to identify undescribed members of the Herpesviri‐ dae family by a degenerate PCR, but no new herpesviruses were found in any of the 18 sam‐ ples analyzed [45]. As the individual virus "hunt" is a limited method, next generation sequencing technologies are an attractive approach to ask for the presence of known and un‐ known infectious agents in leukemic cells.

## **5. Space-time clustering of childhood leukemia by Alexander**

As we learn in the previous section, childhood leukemia has been shown to be a disease of‐ ten presented in space and time clusters correlating with communities with large influx of people. Population based morbility/mortality maps are used in public health to inform us of points of an excess of cases (the cluster) relative to the expected incidence, which are then unlikely to have happened by chance and points out to possible etiological factors and the population at risk. Leukemia aggregates have been studied for decades and to date, a num‐ ber of studies have reported an unusual increase in the number of cases associated with space-time patterns, some of them have been anecdotal reports but others have been discov‐ ered through employment of formal statistical analysis. We describe next some cluster stud‐ ies that have been specifically designed to test the hypothesis of the involvement of infectious agents in the development of childhood leukemia.

different hypothetical scenarios and their associated proposals depending on the type of in‐

**Interactions Hypothetical Scenery Support to**

The infection occurred *in utero* or in early infancy

Infectious Etiology of Childhood Acute Lymphoblastic Leukemia, Hypotheses and Evidence

The infection occurred before diagnosis

The infection occurred before diagnosis

No plausible according to previous results

To analyze the data, different statistical tests were used and the authors considered mobili‐ zation of children from the records of changes of residence. The results showed evidence of space-time clustering based on place of birth and time of diagnosis for the sub-groups aged 0-4 years, but no evidence based on place and time of birth, thus the results lent support to

Methodologies used to search time clusters have also been used to address seasonal varia‐ tion for childhood leukemia. According to this idea, if an infection is associated with dis‐ ease, then a seasonal pattern would be expected, either at birth or diagnostic. Perhaps the largest study of this type is the one conducted by Higgins et al, using the population based data from the UK National Registry of Childhood Tumors that included 15,835 leukemia cases from children born and diagnosed between 1953-1995. No seasonality was found in this study after leukemia classification by age, gender or immunophenotype [51]. Similar studies have been conducted in the USA, Singapore and Sweden, founding the same nega‐ tive result. In all of these studies only some temporal peaks (but no evidence of seasonality)

Many other studies have provided evidence for space-time clustering of childhood leukemia [53-56]. Some have not addressed a possible infectious explanation but correlated with pop‐ ulation mixing. An extreme example was Greece, which experienced one of the largest in‐ flux of people from rural to urban settings and presented one of the highest incidences of childhood leukemia around that time [57]. Although, these studies based on the observation of space-time clusters are considered an indirect evidence of the involvement of infectious agents in the etiology of leukemia, the identity of such agent(s) is unknown and therefore

the participation of other environmental factors cannot be presently ruled out.

Greaves and Kinlen hypotheses but they did not support Smith's [50].

Smith's hypothesis

http://dx.doi.org/10.5772/54455

29

Greaves' and Kinlen's hypothesis

Greaves' and Kinlen's hypothesis


teraction were as follows:

I Between times and places of birth

II Between times and places of diagnosis

III Between time of diagnosis and place of birth

IV Between time of birth and place of diagnosis

have been observed [52].

**Table 3.**

Alexander's work is one of the pioneering reports using rigorous statistical methods to de‐ termine the existence of spatial temporal clusters as indirect evidence of an infectious etiolo‐ gy for childhood leukemia. The analysis was performed using data obtained from the censuses of 1971 and 1981 in England, Wales and Scotland and was restricted to wards whose contribution to spatial clustering test exceeded an expected, arbitrarily established threshold, from a Poisson distribution on uniform risk of the disease. The report included 487 cases of ALL and other unspecified leukemias. The location at birth was extrapolated from the location data at diagnosis (assuming no changes in residence). The association in‐ fection-leukemia was tested from 3 hypothesis envisioned from three different scenarios based on the period of exposure and age of disease presentation:


#### **Table 2.**

To test these hypotheses, the cases were divided into series A and B, the 'susceptibles' (not exposed) and the 'infectives'. To evaluate spatial and temporal associations, the data were analyzed as pairs of cases; spatial linkage was defined based in location within the same electoral ward. Temporal linkage was an overlap of at least 3 months between the time of presumed susceptibility of the child in series A and infectivity of the child in series B.

The results of this study showed support for the hypothesis I: exposure around the time of birth leads to an increased risk of leukemia whose onset takes place at 5 years or older. At the biological level, the authors interpreted the silent and persistent infection of an agent ac‐ quired *in utero* as potentially contributing to the development of the malignancy at any time prior to its presentation. The authors exemplified the process similar to an infection by pesti‐ virus, which however, has not been associated with carcinogenic processes in animals and they are known to induce death even *in utero*. According to this paper, infections did not explain the cases in the 2-5 years old peak, which is the most common in developed coun‐ tries such as those included in this study [49].

The report of Birch et al, included 798 cases of acute leukemia diagnosed between 1954 and 1985 taken from the Manchester Children's Tumour Registry (MCTR) and aimed to evaluate various scenarios for the infectious etiology of leukemia (cluster criteria were established *a priori* as less than 5 km and less than 1 year apart). To support Greaves', Kinlen's and Smith's proposals, two working hypotheses were established: H1 is true (Greaves and Kin‐ len hypotheses) and H2 is false (Smith hypothesis). This study also considered 4 possible space-time interactions in which the potentially leukemogenic infection would occur. The different hypothetical scenarios and their associated proposals depending on the type of in‐ teraction were as follows:


#### **Table 3.**

ies that have been specifically designed to test the hypothesis of the involvement of

Alexander's work is one of the pioneering reports using rigorous statistical methods to de‐ termine the existence of spatial temporal clusters as indirect evidence of an infectious etiolo‐ gy for childhood leukemia. The analysis was performed using data obtained from the censuses of 1971 and 1981 in England, Wales and Scotland and was restricted to wards whose contribution to spatial clustering test exceeded an expected, arbitrarily established threshold, from a Poisson distribution on uniform risk of the disease. The report included 487 cases of ALL and other unspecified leukemias. The location at birth was extrapolated from the location data at diagnosis (assuming no changes in residence). The association in‐ fection-leukemia was tested from 3 hypothesis envisioned from three different scenarios

**Period of exposure Age at presentation**

II Post-natal Under 5 years

I In utero or around the time of birth 5 years or older

III Recent first exposure previous to the onset 'Childhood peak' (ages 2-4 years)

To test these hypotheses, the cases were divided into series A and B, the 'susceptibles' (not exposed) and the 'infectives'. To evaluate spatial and temporal associations, the data were analyzed as pairs of cases; spatial linkage was defined based in location within the same electoral ward. Temporal linkage was an overlap of at least 3 months between the time of

The results of this study showed support for the hypothesis I: exposure around the time of birth leads to an increased risk of leukemia whose onset takes place at 5 years or older. At the biological level, the authors interpreted the silent and persistent infection of an agent ac‐ quired *in utero* as potentially contributing to the development of the malignancy at any time prior to its presentation. The authors exemplified the process similar to an infection by pesti‐ virus, which however, has not been associated with carcinogenic processes in animals and they are known to induce death even *in utero*. According to this paper, infections did not explain the cases in the 2-5 years old peak, which is the most common in developed coun‐

The report of Birch et al, included 798 cases of acute leukemia diagnosed between 1954 and 1985 taken from the Manchester Children's Tumour Registry (MCTR) and aimed to evaluate various scenarios for the infectious etiology of leukemia (cluster criteria were established *a priori* as less than 5 km and less than 1 year apart). To support Greaves', Kinlen's and Smith's proposals, two working hypotheses were established: H1 is true (Greaves and Kin‐ len hypotheses) and H2 is false (Smith hypothesis). This study also considered 4 possible space-time interactions in which the potentially leukemogenic infection would occur. The

presumed susceptibility of the child in series A and infectivity of the child in series B.

infectious agents in the development of childhood leukemia.

28 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

based on the period of exposure and age of disease presentation:

tries such as those included in this study [49].

**Table 2.**

To analyze the data, different statistical tests were used and the authors considered mobili‐ zation of children from the records of changes of residence. The results showed evidence of space-time clustering based on place of birth and time of diagnosis for the sub-groups aged 0-4 years, but no evidence based on place and time of birth, thus the results lent support to Greaves and Kinlen hypotheses but they did not support Smith's [50].

Methodologies used to search time clusters have also been used to address seasonal varia‐ tion for childhood leukemia. According to this idea, if an infection is associated with dis‐ ease, then a seasonal pattern would be expected, either at birth or diagnostic. Perhaps the largest study of this type is the one conducted by Higgins et al, using the population based data from the UK National Registry of Childhood Tumors that included 15,835 leukemia cases from children born and diagnosed between 1953-1995. No seasonality was found in this study after leukemia classification by age, gender or immunophenotype [51]. Similar studies have been conducted in the USA, Singapore and Sweden, founding the same nega‐ tive result. In all of these studies only some temporal peaks (but no evidence of seasonality) have been observed [52].

Many other studies have provided evidence for space-time clustering of childhood leukemia [53-56]. Some have not addressed a possible infectious explanation but correlated with pop‐ ulation mixing. An extreme example was Greece, which experienced one of the largest in‐ flux of people from rural to urban settings and presented one of the highest incidences of childhood leukemia around that time [57]. Although, these studies based on the observation of space-time clusters are considered an indirect evidence of the involvement of infectious agents in the etiology of leukemia, the identity of such agent(s) is unknown and therefore the participation of other environmental factors cannot be presently ruled out.

#### **6. Integrative discussion**

Indirect evidence supports an association between infections in childhood leukemia, and three hypotheses have been proposed to explain and/or address this question with variable and even opposite results. From these hypotheses, the *delayed infection* by Greaves argues for an indirect role for infection, Smith's hypothesis for a direct causative role and Kinlen's seems to sit in the middle, favoring a direct infection of the cell that will become the leuke‐ mic blast but also an indirect mechanism of response still unexplained. In other words, for Greaves, infections in early life are protective and for Kinlen and Smith are a risk factor; for Greaves and Kinlen almost any type of infectious agents (for Kinlen mostly viral) able to trigger aberrant immune or cellular responses could be the causative agent, for Smith it would be viruses with direct oncogenic capacities.

group together several subtypes of leukemia, ethnic, stage, age and genetic insult, it is diffi‐ cult to interpret whether they support or reject the different hypothesis of the infectious ori‐

It should be noted that Greaves' hypothesis concerns the common form of B-cell ALL

observed in developed countries or in affluent communities that have improved their living standards and have become 'more hygienic' [60]. Through comparison of international re‐ ports, variations in the peaks of childhood ALL have been identified. The aforementioned peak at 2–5 years of age is reduced, or even absent, for Black Africans and for other develop‐ ing communities [61-63]. In Mexico, for example, two incidence peaks have been reported; the first occurring at 2–3 years of age and the second at 6–9 [64]. There is also the infant leu‐ kemia (of children under one year old) that is of very bad prognostic and is at least 80% pos‐

Although, the *delayed infection* and *mixing population* hypotheses exhibit several points in common, they exhibit important differences too, for instance, Greaves' hypothesis is con‐ cerned to common childhood leukemia seen in age group of 2 to 5 years, while, Kinlen has not associated the leukemia clusters with a particular subtype of disease and has interpreted his results as all types of childhood leukemia might have a common cause. This argument could weak his hypothesis since a common etiologic mechanism for the different subtypes of the disease is difficult to envision. Also, the largest increase in leukemia cases has been reported for developed countries and Kinlen has not provided an explanation of how his model of large mixing of urban and rural populations can be extrapolated to or represent an

Given the multifactorial nature of cancer, the role of other environmental and genetic factor in Kinlen's proposal is also missing. Kinlen's studies often seem to be based in the sole ac‐ tion of an infectious agent, but there are not known examples of infectious agents or onco‐

Some studies supporting the population mixing proposal have observed a specific increase of infant leukemia, which is mostly associated with MLL translocations. It has been reported that topoisomerase II inhibitors, consumed in some foods during pregnancy or present in drugs commonly used to treat cancer, are a risk factor for this type of translocation [65, 66]. There are not infectious agents known to promote MLL translocation or to inhibit topoiso‐ merase II enzyme. Therefore, how other environmental insults take part in events of popula‐ tion mixing should also be considered. In this scenario, Greaves model seems more complete, since it includes the genetic lesions that characterize childhood leukemia. Greaves usees these genetic lesions to frame a biologically plausible mechanism in which children are more vulnerable to a leukemogenic process after an untimely infection episode. Still, in Greaves model, the first and perhaps more important oncogenic hit happens by chance and

The rise of several types of diseases in recent years, mainly in developed countries, has been proposed to be associated with increasing hygienic conditions. This hygiene hypothesis

therefore his model does not provide an easy target for controlled intervention.

itive to MLL translocations, supporting different etiologies between age groups.

). This form comprises most of the ALL cases that peak at 2–5 years of age

Infectious Etiology of Childhood Acute Lymphoblastic Leukemia, Hypotheses and Evidence

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31

gin of the disease.

, CD10+

affluent or aseptic setting.

genic insults with full penetrance.

(CD19+

Based mainly in adult cancers, we now know that pathogens contribute to neoplasia through different mechanisms. The classical ones are those in which the agents infect cells and promote oncogenic transformation 'from within', through altering signaling pathways and gene expression programs (supports Smith). Indirect roles (supports Kinlen) include promotion of an inflammatory microenvironment, loss of cancer immune surveillance and a cofactor role helping the tumor through secretion of growth and angiogenic factors. The lat‐ ter one is the mechanism proposed to explain cytomegalovirus oncomodulatory role in high-grade gliomas and it is thought to be a tumor maintenance rather that an initiating mechanism [58]. From these mechanisms, a direct role would be very possible but so far multiple studies have failed to find evidence of infection by oncogenic agents in the leuke‐ mic blast. On the other hand, an inflammatory role is very unlikely because it is generally associated with chronic diseases lasting decades (e.g. *Helicobacter pylori* and hepatitis B and C virus infections). A cofactor or immune suppressive roles are possible, especially for preleukemic clones (e.g. the ones with an early chromosomal abnormality).

Considering all these mechanisms, it is important to acknowledge that the term childhood leukemia harbors many different biological entities, and it is very likely that they involve different mechanisms of origin. Examples of important known differences are the lineage origin of the leukemic blast, myeloid *vs* lymphoid or T cell *vs* B cell. Also, there are at least three recognized B cell immature developmental stages where the leukemia is originated: early proB, preB-I and large preB-II, which are recognized for the differential expression of lineage- and stage- specific antigens and are dependent on the activity of different signaling pathways and transcriptional programs [59].

As mentioned before, childhood leukemia is also associated to chromosomal abnormalities: hyperdiploidy, hypodiploidy and translocations t(12;21)(p13;q22) (TEL-AML1), t(1;19) (q23;p13) (E2A-PBX1), t(9;22)(q34;q11) (BCR-ABL) and t(4;11)(q21;q23) (MLL-AF4) are among the most common in B-ALL. These genetic abnormalities affect specific signaling pathways and favor transcriptional expression profiles related to the developmental stage of the B cell leukemic blast. Therefore, the risk and protection factors driven these known and still many unknown different childhood leukemia entities are probably different and models of the origin of the disease should be restrained to specific subtypes. Because most reports group together several subtypes of leukemia, ethnic, stage, age and genetic insult, it is diffi‐ cult to interpret whether they support or reject the different hypothesis of the infectious ori‐ gin of the disease.

**6. Integrative discussion**

would be viruses with direct oncogenic capacities.

pathways and transcriptional programs [59].

Indirect evidence supports an association between infections in childhood leukemia, and three hypotheses have been proposed to explain and/or address this question with variable and even opposite results. From these hypotheses, the *delayed infection* by Greaves argues for an indirect role for infection, Smith's hypothesis for a direct causative role and Kinlen's seems to sit in the middle, favoring a direct infection of the cell that will become the leuke‐ mic blast but also an indirect mechanism of response still unexplained. In other words, for Greaves, infections in early life are protective and for Kinlen and Smith are a risk factor; for Greaves and Kinlen almost any type of infectious agents (for Kinlen mostly viral) able to trigger aberrant immune or cellular responses could be the causative agent, for Smith it

30 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

Based mainly in adult cancers, we now know that pathogens contribute to neoplasia through different mechanisms. The classical ones are those in which the agents infect cells and promote oncogenic transformation 'from within', through altering signaling pathways and gene expression programs (supports Smith). Indirect roles (supports Kinlen) include promotion of an inflammatory microenvironment, loss of cancer immune surveillance and a cofactor role helping the tumor through secretion of growth and angiogenic factors. The lat‐ ter one is the mechanism proposed to explain cytomegalovirus oncomodulatory role in high-grade gliomas and it is thought to be a tumor maintenance rather that an initiating mechanism [58]. From these mechanisms, a direct role would be very possible but so far multiple studies have failed to find evidence of infection by oncogenic agents in the leuke‐ mic blast. On the other hand, an inflammatory role is very unlikely because it is generally associated with chronic diseases lasting decades (e.g. *Helicobacter pylori* and hepatitis B and C virus infections). A cofactor or immune suppressive roles are possible, especially for pre-

Considering all these mechanisms, it is important to acknowledge that the term childhood leukemia harbors many different biological entities, and it is very likely that they involve different mechanisms of origin. Examples of important known differences are the lineage origin of the leukemic blast, myeloid *vs* lymphoid or T cell *vs* B cell. Also, there are at least three recognized B cell immature developmental stages where the leukemia is originated: early proB, preB-I and large preB-II, which are recognized for the differential expression of lineage- and stage- specific antigens and are dependent on the activity of different signaling

As mentioned before, childhood leukemia is also associated to chromosomal abnormalities: hyperdiploidy, hypodiploidy and translocations t(12;21)(p13;q22) (TEL-AML1), t(1;19) (q23;p13) (E2A-PBX1), t(9;22)(q34;q11) (BCR-ABL) and t(4;11)(q21;q23) (MLL-AF4) are among the most common in B-ALL. These genetic abnormalities affect specific signaling pathways and favor transcriptional expression profiles related to the developmental stage of the B cell leukemic blast. Therefore, the risk and protection factors driven these known and still many unknown different childhood leukemia entities are probably different and models of the origin of the disease should be restrained to specific subtypes. Because most reports

leukemic clones (e.g. the ones with an early chromosomal abnormality).

It should be noted that Greaves' hypothesis concerns the common form of B-cell ALL (CD19+ , CD10+ ). This form comprises most of the ALL cases that peak at 2–5 years of age observed in developed countries or in affluent communities that have improved their living standards and have become 'more hygienic' [60]. Through comparison of international re‐ ports, variations in the peaks of childhood ALL have been identified. The aforementioned peak at 2–5 years of age is reduced, or even absent, for Black Africans and for other develop‐ ing communities [61-63]. In Mexico, for example, two incidence peaks have been reported; the first occurring at 2–3 years of age and the second at 6–9 [64]. There is also the infant leu‐ kemia (of children under one year old) that is of very bad prognostic and is at least 80% pos‐ itive to MLL translocations, supporting different etiologies between age groups.

Although, the *delayed infection* and *mixing population* hypotheses exhibit several points in common, they exhibit important differences too, for instance, Greaves' hypothesis is con‐ cerned to common childhood leukemia seen in age group of 2 to 5 years, while, Kinlen has not associated the leukemia clusters with a particular subtype of disease and has interpreted his results as all types of childhood leukemia might have a common cause. This argument could weak his hypothesis since a common etiologic mechanism for the different subtypes of the disease is difficult to envision. Also, the largest increase in leukemia cases has been reported for developed countries and Kinlen has not provided an explanation of how his model of large mixing of urban and rural populations can be extrapolated to or represent an affluent or aseptic setting.

Given the multifactorial nature of cancer, the role of other environmental and genetic factor in Kinlen's proposal is also missing. Kinlen's studies often seem to be based in the sole ac‐ tion of an infectious agent, but there are not known examples of infectious agents or onco‐ genic insults with full penetrance.

Some studies supporting the population mixing proposal have observed a specific increase of infant leukemia, which is mostly associated with MLL translocations. It has been reported that topoisomerase II inhibitors, consumed in some foods during pregnancy or present in drugs commonly used to treat cancer, are a risk factor for this type of translocation [65, 66]. There are not infectious agents known to promote MLL translocation or to inhibit topoiso‐ merase II enzyme. Therefore, how other environmental insults take part in events of popula‐ tion mixing should also be considered. In this scenario, Greaves model seems more complete, since it includes the genetic lesions that characterize childhood leukemia. Greaves usees these genetic lesions to frame a biologically plausible mechanism in which children are more vulnerable to a leukemogenic process after an untimely infection episode. Still, in Greaves model, the first and perhaps more important oncogenic hit happens by chance and therefore his model does not provide an easy target for controlled intervention.

The rise of several types of diseases in recent years, mainly in developed countries, has been proposed to be associated with increasing hygienic conditions. This hygiene hypothesis states that lack of early childhood exposure to microorganisms triggers the appearance of disease [67]. Although Greaves has modeled his *delayed infection* proposal in the *hygiene hy‐ pothesis*, there might be subtle differences between both hypotheses. While the *hygiene hy‐ pothesis* is highly concerned with acquisition of the human normal flora, Greaves is also concerned with pathogens that are not life threatening when acquired early. There are many examples of the latter and several diseases have been associated to delayed infection, exam‐ ples of them are EBV or cytomegalovirus-related infectious mononucleosis, measles and chickenpox. In these cases, infection in the first years of life leads to mild to no symptoms, but when acquired late leads to serious and even life threatening diseases and in the case of EBV, it has been proposed that it predisposes to lymphoma. However, the window in which these infections become dangerous are usually beyond the years of the higher incidence pick of childhood leukemia and could only explain leukemia of the teenager or young adult.

several studies failing to find an effect [70]. Moreover, the overall protection observed ar‐ gues that the same hygienic conditions driving allergies are protecting from childhood leu‐ kemia. An alternative explanation is that the molecular pathway leading to allergy and the

Infectious Etiology of Childhood Acute Lymphoblastic Leukemia, Hypotheses and Evidence

http://dx.doi.org/10.5772/54455

33

A similar approach has been proposed for parasitic infections, since the fall of this type of infection has been parallel to the increase in allergies and childhood leukemia in most devel‐ oped countries. Furthermore, many parasites drive Th1 immune responses while allergies are associated with Th2 responses providing a feasible biological frame for protection to al‐ lergies and perhaps childhood leukemia. A few studies have found a correlation between lack of infection of intestinal parasites and childhood leukemia [71]. However, many autoimmune diseases also explained by the hygiene hypothesis are triggered by Th1 responses, such as type 1 diabetes, confusing a mechanistic explanation for this phenomenon and argu‐

Different hypotheses have tried to relate the origin of childhood acute lymphoblastic leuke‐ mia to infections and epidemiological, clinical and molecular evidence have been searched to support them with highly variable results. ALL is a common term that harbors several diseases varying in their age of presentation, associated genetic lesions, cellular origin and prognosis, probably reflecting different biological origins and thus suggesting different causative factors. Hence, although some of the accumulated evidence favors one or other of the hypotheses there is not a consensus whether infections participate and this participation is through direct or indirect mechanisms of transformation. Although, the postulated mech‐ anisms differ from each other, they are not mutually exclusive. The causal factors of leuke‐ mia most probably are influenced by complex environmental and genetic interaction with some of them having greater or lesser roles in different individuals or subtypes of the dis‐ ease. New approaches and methodologies should be used to provide further data support‐ ing the role of infections. In that scenario, more direct markers of aberrant immune responses should be analyzed to support Greaves proposal. Th1/Th2/Th17 and/or regulatory immune environments should be tested as early as during pregnancy, lactation or in stored newborn blood. Next generation technologies should be used to identify novel infectious agents in ALL samples and to study the microbiota of patients. All these efforts together will result in a better understanding of the role of infectious agents in childhood ALL, their

mechanisms of leukemogenesis and will provide better points for disease control.

This work was funded by CONSEJO NACIONAL DE CIENCIA Y TECNOLOGIA (CONA‐ CYT), Grant 2010-1-141026, IMSS/FIS/PROT 895; CB-2007-1-83949; 2007-1-18-71223,

leukemogenesis pathway are mutually exclusive.

ing against a common origin for all these diseases.

**7. Conclusion**

**Acknowledgements**

IMSS/FIS/PROT 056.

Studies in mice are strongly indicative that animals grown in germ free conditions are often immunologically unsuited to fight infections and that perhaps one of the most important components of the immune instruction program is the normal flora [68]. Inoculation of pro‐ biotics in germ free mice is associated with development of a regulatory immune response in mucosa (based mainly in frequencies of regulatory T cells and levels of cytokines IL-10 and TGF-β) and equilibrated Th1/Th2/Th17 environments. Same results have been obtained after inoculation with several members of *Bifidobacterium*, which are normal residents of in‐ fant feces [69]. Animals without a normal microbiota often develop fatal responses when they are challenged with low doses of otherwise controllable pathogens. These results have supported a model in which humans have co-evolved with their flora and this flora is more than a passive passenger providing multiple benefits to the host. Several lines of evidence support that a normal microbiota is necessary for a healthy host metabolism, and also for what is now known as the microbial immunotraining.

The example of germ free animals, although extreme, points out that looking for infection mark‐ ers of childhood common pathogens might not be indicative of the normal development and equilibrium of the human microbiota and the immune system, and if it is true that leukemia is the result of an aberrant immune response, then markers of infections are not representative of homeostatic acquisition of the normal human flora. In a similar scenario, infections that may confer risk or protection for leukemia are not necessarily symptomatic, thus, data collection of infections with clinical symptoms would exclude relevant infections to normal immune system development. Also, studies that finding that early symptomatic infections are a risk factor for leukemia might only be reflecting on the antibiotics used to treat those infections and the effects that they had on the establishment of the children normal microbiota.

There are many diseases with increased incidence in affluent societies. Among the most studied are asthma, allergies and type 1 diabetes; several studies have tried to link some of these diseases to childhood leukemia. Linabery et al published a meta-analysis of the differ‐ ent studies searching for association between childhood leukemia and allergy, asthma, ecze‐ ma and hay fever. Although, this meta-analysis shows a protective effect of these diseases, OR=0.69 (95% CI:0.54-0.89), OR=0.79 (95% CI:0.61-1.02), OR=0.74 (95% CI:0.58-0.96), OR=0.55 (95% CI: 0.46-0.66), respectively, the authors observed high heterogeneity of the data with several studies failing to find an effect [70]. Moreover, the overall protection observed ar‐ gues that the same hygienic conditions driving allergies are protecting from childhood leu‐ kemia. An alternative explanation is that the molecular pathway leading to allergy and the leukemogenesis pathway are mutually exclusive.

A similar approach has been proposed for parasitic infections, since the fall of this type of infection has been parallel to the increase in allergies and childhood leukemia in most devel‐ oped countries. Furthermore, many parasites drive Th1 immune responses while allergies are associated with Th2 responses providing a feasible biological frame for protection to al‐ lergies and perhaps childhood leukemia. A few studies have found a correlation between lack of infection of intestinal parasites and childhood leukemia [71]. However, many autoimmune diseases also explained by the hygiene hypothesis are triggered by Th1 responses, such as type 1 diabetes, confusing a mechanistic explanation for this phenomenon and argu‐ ing against a common origin for all these diseases.

## **7. Conclusion**

states that lack of early childhood exposure to microorganisms triggers the appearance of disease [67]. Although Greaves has modeled his *delayed infection* proposal in the *hygiene hy‐ pothesis*, there might be subtle differences between both hypotheses. While the *hygiene hy‐ pothesis* is highly concerned with acquisition of the human normal flora, Greaves is also concerned with pathogens that are not life threatening when acquired early. There are many examples of the latter and several diseases have been associated to delayed infection, exam‐ ples of them are EBV or cytomegalovirus-related infectious mononucleosis, measles and chickenpox. In these cases, infection in the first years of life leads to mild to no symptoms, but when acquired late leads to serious and even life threatening diseases and in the case of EBV, it has been proposed that it predisposes to lymphoma. However, the window in which these infections become dangerous are usually beyond the years of the higher incidence pick of childhood leukemia and could only explain leukemia of the teenager or young adult.

32 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

Studies in mice are strongly indicative that animals grown in germ free conditions are often immunologically unsuited to fight infections and that perhaps one of the most important components of the immune instruction program is the normal flora [68]. Inoculation of pro‐ biotics in germ free mice is associated with development of a regulatory immune response in mucosa (based mainly in frequencies of regulatory T cells and levels of cytokines IL-10 and TGF-β) and equilibrated Th1/Th2/Th17 environments. Same results have been obtained after inoculation with several members of *Bifidobacterium*, which are normal residents of in‐ fant feces [69]. Animals without a normal microbiota often develop fatal responses when they are challenged with low doses of otherwise controllable pathogens. These results have supported a model in which humans have co-evolved with their flora and this flora is more than a passive passenger providing multiple benefits to the host. Several lines of evidence support that a normal microbiota is necessary for a healthy host metabolism, and also for

The example of germ free animals, although extreme, points out that looking for infection mark‐ ers of childhood common pathogens might not be indicative of the normal development and equilibrium of the human microbiota and the immune system, and if it is true that leukemia is the result of an aberrant immune response, then markers of infections are not representative of homeostatic acquisition of the normal human flora. In a similar scenario, infections that may confer risk or protection for leukemia are not necessarily symptomatic, thus, data collection of infections with clinical symptoms would exclude relevant infections to normal immune system development. Also, studies that finding that early symptomatic infections are a risk factor for leukemia might only be reflecting on the antibiotics used to treat those infections and the effects

There are many diseases with increased incidence in affluent societies. Among the most studied are asthma, allergies and type 1 diabetes; several studies have tried to link some of these diseases to childhood leukemia. Linabery et al published a meta-analysis of the differ‐ ent studies searching for association between childhood leukemia and allergy, asthma, ecze‐ ma and hay fever. Although, this meta-analysis shows a protective effect of these diseases, OR=0.69 (95% CI:0.54-0.89), OR=0.79 (95% CI:0.61-1.02), OR=0.74 (95% CI:0.58-0.96), OR=0.55 (95% CI: 0.46-0.66), respectively, the authors observed high heterogeneity of the data with

what is now known as the microbial immunotraining.

that they had on the establishment of the children normal microbiota.

Different hypotheses have tried to relate the origin of childhood acute lymphoblastic leuke‐ mia to infections and epidemiological, clinical and molecular evidence have been searched to support them with highly variable results. ALL is a common term that harbors several diseases varying in their age of presentation, associated genetic lesions, cellular origin and prognosis, probably reflecting different biological origins and thus suggesting different causative factors. Hence, although some of the accumulated evidence favors one or other of the hypotheses there is not a consensus whether infections participate and this participation is through direct or indirect mechanisms of transformation. Although, the postulated mech‐ anisms differ from each other, they are not mutually exclusive. The causal factors of leuke‐ mia most probably are influenced by complex environmental and genetic interaction with some of them having greater or lesser roles in different individuals or subtypes of the dis‐ ease. New approaches and methodologies should be used to provide further data support‐ ing the role of infections. In that scenario, more direct markers of aberrant immune responses should be analyzed to support Greaves proposal. Th1/Th2/Th17 and/or regulatory immune environments should be tested as early as during pregnancy, lactation or in stored newborn blood. Next generation technologies should be used to identify novel infectious agents in ALL samples and to study the microbiota of patients. All these efforts together will result in a better understanding of the role of infectious agents in childhood ALL, their mechanisms of leukemogenesis and will provide better points for disease control.

## **Acknowledgements**

This work was funded by CONSEJO NACIONAL DE CIENCIA Y TECNOLOGIA (CONA‐ CYT), Grant 2010-1-141026, IMSS/FIS/PROT 895; CB-2007-1-83949; 2007-1-18-71223, IMSS/FIS/PROT 056.

This chapter constitutes a partial fulfillment of the Graduate Program of Doctor Degree in Biomedical Sciences, Medicine Faculty, National Autonomous University of Mexico, Mexico City, Mexico. A Morales-Sánchez acknowledges the scholarship and financial support pro‐ vided by the National Council of Science and Technology (CONACyT) and National Auton‐ omous University of Mexico.

[9] Dockerty JD, Draper G, Vincent T, Rowan SD, Bunch KJ. Case-control study of pa‐ rental age, parity and socioeconomic level in relation to childhood cancers. Interna‐

Infectious Etiology of Childhood Acute Lymphoblastic Leukemia, Hypotheses and Evidence

http://dx.doi.org/10.5772/54455

35

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## **Author details**

Abigail Morales-Sánchez and Ezequiel M. Fuentes-Pananá\*

\*Address all correspondence to: empanana@yahoo.com

Unit of Medical Research in Clinical Epidemiology, High Specialty Medical Care Unit of the Pediatric Hospital, National Medical Center XXI Century, Mexican Institute of Social Securi‐ ty, Mexico City, Mexico

### **References**


[9] Dockerty JD, Draper G, Vincent T, Rowan SD, Bunch KJ. Case-control study of pa‐ rental age, parity and socioeconomic level in relation to childhood cancers. Interna‐ tional journal of epidemiology 2001;30(6) 1428-1437.

This chapter constitutes a partial fulfillment of the Graduate Program of Doctor Degree in Biomedical Sciences, Medicine Faculty, National Autonomous University of Mexico, Mexico City, Mexico. A Morales-Sánchez acknowledges the scholarship and financial support pro‐ vided by the National Council of Science and Technology (CONACyT) and National Auton‐

Unit of Medical Research in Clinical Epidemiology, High Specialty Medical Care Unit of the Pediatric Hospital, National Medical Center XXI Century, Mexican Institute of Social Securi‐

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[70] Linabery AM, Jurek AM, Duval S, Ross JA. The association between atopy and child‐ hood/adolescent leukemia: a meta-analysis. American Journal of Epidemiology

[71] Rivera-Luna R, Cardenas-Cardos R, Martinez-Guerra G, Ayon A, Leal C, Rivera-Or‐ tegon F. Childhood acute leukemia and intestinal parasitosis. Leukemia 1989;3(11)

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**Section 2**

**Pathophysiology of ALL**

**Pathophysiology of ALL**

**Chapter 3**

**Pathophysiology of Acute Lymphoblastic Leukemia**

The Acute lymphoblastic leukemia (ALL), it produced as a result of a process of malignant transformation of a progenitor lymphocytic cell in the B and T lineages. In ALL, the majority of the cases, the transformation affects the B lineage cells. Leukemia and other cancers share biological characteristics, as clonality. The molecular alterations that are required for the de‐ velopment of a malignant disease is a rare phenomenon when one considers the large number of target cells susceptible to this condition, in other words, a single genetic change rarely be sufficient for developing a malignant tumor. This means that a small percentage of people (1%) who develop malignant hematological disease, probably only 1 cell mutated in a critical gene for the proliferation, differentiation and survival of progenitor cells. There is evidence sup‐ porting a sequential multistep process, of alterations in several oncogenes in tumor suppressor

Most of what is known of the influence of some mutant genes of the origin of leukemia, derived from studies in molecular virology, in the gene transfection, and in the generation of leukemia in-vivo in transgenic mice. These studies are based on bacterial DNA recombinant methods. Most of the mutations in leukemia are acquired and occur in the lymphoid cell progenitor, less frequently (1% to 5% of leukemia) the mutated genes are inherited, this involved a numerical

Genetic factors of acute leukemia have been extensively studied. The results of studies of gene expression analysis of high resolution whole genome, copy number alterations of DNA, loss of heterozygosity epigenetic changes and whole genome sequencing, have allowed the rec‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Gallegos-Arreola et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

M. P. Gallegos-Arreola, C. Borjas-Gutiérrez,

A. M. Puebla-Pérez and J. R. García-González

Additional information is available at the end of the chapter

G. M. Zúñiga-González, L. E. Figuera,

genes or microRNA genes in cancerigen cells.

chromosome abnormality, for example: constitutive trisomy 21.

**Genes involved in leukemia**

http://dx.doi.org/10.5772/54652

**1. Introduction**

## **Pathophysiology of Acute Lymphoblastic Leukemia**

M. P. Gallegos-Arreola, C. Borjas-Gutiérrez, G. M. Zúñiga-González, L. E. Figuera, A. M. Puebla-Pérez and J. R. García-González

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54652

## **1. Introduction**

The Acute lymphoblastic leukemia (ALL), it produced as a result of a process of malignant transformation of a progenitor lymphocytic cell in the B and T lineages. In ALL, the majority of the cases, the transformation affects the B lineage cells. Leukemia and other cancers share biological characteristics, as clonality. The molecular alterations that are required for the de‐ velopment of a malignant disease is a rare phenomenon when one considers the large number of target cells susceptible to this condition, in other words, a single genetic change rarely be sufficient for developing a malignant tumor. This means that a small percentage of people (1%) who develop malignant hematological disease, probably only 1 cell mutated in a critical gene for the proliferation, differentiation and survival of progenitor cells. There is evidence sup‐ porting a sequential multistep process, of alterations in several oncogenes in tumor suppressor genes or microRNA genes in cancerigen cells.

#### **Genes involved in leukemia**

Most of what is known of the influence of some mutant genes of the origin of leukemia, derived from studies in molecular virology, in the gene transfection, and in the generation of leukemia in-vivo in transgenic mice. These studies are based on bacterial DNA recombinant methods. Most of the mutations in leukemia are acquired and occur in the lymphoid cell progenitor, less frequently (1% to 5% of leukemia) the mutated genes are inherited, this involved a numerical chromosome abnormality, for example: constitutive trisomy 21.

Genetic factors of acute leukemia have been extensively studied. The results of studies of gene expression analysis of high resolution whole genome, copy number alterations of DNA, loss of heterozygosity epigenetic changes and whole genome sequencing, have allowed the rec‐

© 2013 Gallegos-Arreola et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ognition of new genetic alterations, so that virtually all patients with ALL can be classified according to the specific genetic abnormality. This information has increased our knowledge of leukemogenesis, the prognosis and has served as the basis for the development of the target therapy. However, the understanding of how genetic alterations collaborate to induce leuke‐ mic transformation remains unclear.

system compared with B-cell ALL. In this sense, is known that the oncogenes and tumor sup‐ pressor genes are implicated in ALL-T are: c-MYC, NOTCH, LMO1 / 2, LYL1, TAL1 / 2, Hox11 and HOX11L2. It is clear that NOTCH activated is able to induce leukemogenesis of T cell and this is critical for the progression to ALL-T. Family members of NOTCH are transmembrane receptors that are involved in controlling the differentiation, proliferation and apoptosis in several cell types including T cells. The binding of its ligand to the extracellular domain, re‐ sulting in cleavage of the intracellular domain of NOTCH, this reaction is catalyzed by γsecretase complex, and the intracellular domain free of translocase to the nucleus, that

Pathophysiology of Acute Lymphoblastic Leukemia

http://dx.doi.org/10.5772/54652

45

The NOTCH target genes are mainly cyclina D1 and c-Myc. Both NOTCH as c-MYC regulate cell cycle progression by inducing expression of cyclins and reduced expression of p27. An important aspect is that NOTCH is able to inhibit apoptosis induced by p53 allowing the tumor regression. In the development of ALL-T there is strong evidence of pro-oncogenic function of signals transduced by NOTCH, and that modulates the activity of downstream signaling pathways, through transcriptional regulation of their target genes. Is possible regulators of signaling downstream of NOTCH especially in murine models are some intermediate signal‐ ing routes as: phosphatidylinositol 3-kinase (PI3K), Akt / protein kinase B, extracellular signalregulated kinase-1/2, and nuclear factor kB. In general, the products of oncogenes can be classified into six categories: transcription factors, chromatin remodeling, growth factors, growth factor receptors, signal transducers, and finally regulators of apoptosis. Transcription factors generally require interacting with other proteins to act, for example: Fos transcription protein dimerizes with the transcription factor Jun to form the AP1 transcription factor is really a complex, and this increases the expression of several genes control cell division, all they have

Aberrant methylation of CpG sites in promoter regions of genes has been identified in ALL cell lines, to respect it is important to note that methylation of CpG dinucleotides in position near the site of transcription initiation can silence gene expression, hypermethylation of tumor suppressor genes and hypomethylation of oncogenes can lead to various forms of cancer.

Other mechanism important in the development of the ALL is the angiogenesis and signal transducers on the binding of tyrosine kinase receptors, finally the molecules regulators of the apoptosis, where the BCL2 gene encodes for a cytoplasmic protein that is localized in the

Secondary hematological malignancies are a serious complication of cancer treatment. They usually manifest as acute leukemias and myelodysplastic syndromes. This also touch the item on secondary leukemias and its frequency is high and has increased possibly due to increas‐ ingly frequent use of genotoxic agents and by increased survival to other types of cancer. So, learn more about these could eventually help reduce its appearance. It is known that this type of leukemia may arise as a result to exposure to cytotoxic treatments (side effects of genotox‐ icity) and / or radiation therapy and as a result of other blood disorders; probably as a result

mitochondria and increases the survival of the cell by inhibiting apoptosis.

regulates transcription of genes regulated by NOTCH.

been involved in the development of leukemia.

**Secondary leukemias**

The altered genes in the leukemia can be result in loss or gain of the function through several mechanisms, for example: abnormal recombination (chromosomal, translocation, inversion, insertion) loss of genetic material (deletion) gain of genetic material (duplication) point mu‐ tation and the presence additional copies of certain chromosomes as in the case of hyperdi‐ ploidy; previous alterations favoring the activation of oncogenes, this encode proteins that control cells proliferation, apoptosis or both.

The advances in the conventional cytogenetic techniques, as the fluorescence *in situ* hybridi‐ zation, have shown the chromosomal rearrangements. In this sense, recently has been reported that the incidence of chromosomal change is related with the age, so the translocation t(9;22) (q34;q11) increases in each successive decade, up to 24% between the 40-to 49 years old, the t(4;11) (q21;q23) and t(1;19) (q23;q13) are rare in patients older than 60 years old, but t (8;14) (q24;q32) and t(14;18)(q32;q21) increased with age. The hiperdipoidia occurs in 13% of patients under 20 years old and only 5% of elderly patients. The hypodiploidy and complex karyotype (presence of more than 2 chromosomal abnormalities) also increase with age of 4% in the range of 15 to 19 years old to 16% older than 60 years old.

When an oncogen is activated by mutation, encoded protein is structurally modified so that enhances its transforming activity, thus remains on active status, continuously transmitting signals through the binding of tyrosine and treonina cinasa. These signals induce cell growth continued incessant. This mechanism of activation of ocogenes is more evident in others forms of leukemia, for example: severe myeloblastic leukemia and other myelodysplastic syndromes where the genes NRAS are mutated. There are mutations that suppress the function and are observe in tumor suppressor genes such as TP53, however, less than 3% of patients with ALL are TP53 mutations, although all cells have a resistance abnormal apoptosis induced by lack of significant proportion of p53, which is explained in large part by epigenetic medications.

By other hand, some authors have found alterations in the number of copies (ANCs) to 50 recurrent regions in ALL, some are really small and they have less than 1 Mb, however occur in genes encoding regulatory proteins of normal lymphoid development in 40% of cases of ALL progenitors B. The target most common are lymphoid transcription factor PAX5 that have deletions or amplifications until 30% of cases with ALL-B, also found in genes ANCs of tran‐ scription factor IKZF1 the IKZF3, EBF1, LEF1 and TCF3, RAG1 and RAG2.

Another important point, is the dihydrofolate reductase (DHFR) gene amplification has been considered as the most relevant in the ALL, this amplification produce cytogenetic abnormal‐ ities evident as the amplified of high DNA segment that included some hundreds of kilobases.

The ALL of T-cell type represents about 10% to 15% of the ALL in adults and the 25% in children and their clinical behavior is the most aggressive, the patients have a higher percentage of failure of remission induction, relapse rate is also higher, and had infiltration at central nervous system compared with B-cell ALL. In this sense, is known that the oncogenes and tumor sup‐ pressor genes are implicated in ALL-T are: c-MYC, NOTCH, LMO1 / 2, LYL1, TAL1 / 2, Hox11 and HOX11L2. It is clear that NOTCH activated is able to induce leukemogenesis of T cell and this is critical for the progression to ALL-T. Family members of NOTCH are transmembrane receptors that are involved in controlling the differentiation, proliferation and apoptosis in several cell types including T cells. The binding of its ligand to the extracellular domain, re‐ sulting in cleavage of the intracellular domain of NOTCH, this reaction is catalyzed by γsecretase complex, and the intracellular domain free of translocase to the nucleus, that regulates transcription of genes regulated by NOTCH.

The NOTCH target genes are mainly cyclina D1 and c-Myc. Both NOTCH as c-MYC regulate cell cycle progression by inducing expression of cyclins and reduced expression of p27. An important aspect is that NOTCH is able to inhibit apoptosis induced by p53 allowing the tumor regression. In the development of ALL-T there is strong evidence of pro-oncogenic function of signals transduced by NOTCH, and that modulates the activity of downstream signaling pathways, through transcriptional regulation of their target genes. Is possible regulators of signaling downstream of NOTCH especially in murine models are some intermediate signal‐ ing routes as: phosphatidylinositol 3-kinase (PI3K), Akt / protein kinase B, extracellular signalregulated kinase-1/2, and nuclear factor kB. In general, the products of oncogenes can be classified into six categories: transcription factors, chromatin remodeling, growth factors, growth factor receptors, signal transducers, and finally regulators of apoptosis. Transcription factors generally require interacting with other proteins to act, for example: Fos transcription protein dimerizes with the transcription factor Jun to form the AP1 transcription factor is really a complex, and this increases the expression of several genes control cell division, all they have been involved in the development of leukemia.

Aberrant methylation of CpG sites in promoter regions of genes has been identified in ALL cell lines, to respect it is important to note that methylation of CpG dinucleotides in position near the site of transcription initiation can silence gene expression, hypermethylation of tumor suppressor genes and hypomethylation of oncogenes can lead to various forms of cancer.

Other mechanism important in the development of the ALL is the angiogenesis and signal transducers on the binding of tyrosine kinase receptors, finally the molecules regulators of the apoptosis, where the BCL2 gene encodes for a cytoplasmic protein that is localized in the mitochondria and increases the survival of the cell by inhibiting apoptosis.

#### **Secondary leukemias**

ognition of new genetic alterations, so that virtually all patients with ALL can be classified according to the specific genetic abnormality. This information has increased our knowledge of leukemogenesis, the prognosis and has served as the basis for the development of the target therapy. However, the understanding of how genetic alterations collaborate to induce leuke‐

44 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

The altered genes in the leukemia can be result in loss or gain of the function through several mechanisms, for example: abnormal recombination (chromosomal, translocation, inversion, insertion) loss of genetic material (deletion) gain of genetic material (duplication) point mu‐ tation and the presence additional copies of certain chromosomes as in the case of hyperdi‐ ploidy; previous alterations favoring the activation of oncogenes, this encode proteins that

The advances in the conventional cytogenetic techniques, as the fluorescence *in situ* hybridi‐ zation, have shown the chromosomal rearrangements. In this sense, recently has been reported that the incidence of chromosomal change is related with the age, so the translocation t(9;22) (q34;q11) increases in each successive decade, up to 24% between the 40-to 49 years old, the t(4;11) (q21;q23) and t(1;19) (q23;q13) are rare in patients older than 60 years old, but t (8;14) (q24;q32) and t(14;18)(q32;q21) increased with age. The hiperdipoidia occurs in 13% of patients under 20 years old and only 5% of elderly patients. The hypodiploidy and complex karyotype (presence of more than 2 chromosomal abnormalities) also increase with age of 4% in the range

When an oncogen is activated by mutation, encoded protein is structurally modified so that enhances its transforming activity, thus remains on active status, continuously transmitting signals through the binding of tyrosine and treonina cinasa. These signals induce cell growth continued incessant. This mechanism of activation of ocogenes is more evident in others forms of leukemia, for example: severe myeloblastic leukemia and other myelodysplastic syndromes where the genes NRAS are mutated. There are mutations that suppress the function and are observe in tumor suppressor genes such as TP53, however, less than 3% of patients with ALL are TP53 mutations, although all cells have a resistance abnormal apoptosis induced by lack of significant proportion of p53, which is explained in large part by epigenetic medications. By other hand, some authors have found alterations in the number of copies (ANCs) to 50 recurrent regions in ALL, some are really small and they have less than 1 Mb, however occur in genes encoding regulatory proteins of normal lymphoid development in 40% of cases of ALL progenitors B. The target most common are lymphoid transcription factor PAX5 that have deletions or amplifications until 30% of cases with ALL-B, also found in genes ANCs of tran‐

Another important point, is the dihydrofolate reductase (DHFR) gene amplification has been considered as the most relevant in the ALL, this amplification produce cytogenetic abnormal‐ ities evident as the amplified of high DNA segment that included some hundreds of kilobases. The ALL of T-cell type represents about 10% to 15% of the ALL in adults and the 25% in children and their clinical behavior is the most aggressive, the patients have a higher percentage of failure of remission induction, relapse rate is also higher, and had infiltration at central nervous

scription factor IKZF1 the IKZF3, EBF1, LEF1 and TCF3, RAG1 and RAG2.

mic transformation remains unclear.

control cells proliferation, apoptosis or both.

of 15 to 19 years old to 16% older than 60 years old.

Secondary hematological malignancies are a serious complication of cancer treatment. They usually manifest as acute leukemias and myelodysplastic syndromes. This also touch the item on secondary leukemias and its frequency is high and has increased possibly due to increas‐ ingly frequent use of genotoxic agents and by increased survival to other types of cancer. So, learn more about these could eventually help reduce its appearance. It is known that this type of leukemia may arise as a result to exposure to cytotoxic treatments (side effects of genotox‐ icity) and / or radiation therapy and as a result of other blood disorders; probably as a result of environmental or genetic causes. In particular in this we will focus more on the former. In most cases it is suggested that the mechanism of leukemogenesis is associated with DNA damage of hematopoietic cells from bone marrow by agents such as those used in chemother‐ apy. Although the majority of secondary leukemias are acute myeloid leukemia (AML) has been reported cases of lymphoid leukemia and chronic myeloid leukemia (CML) associated with chemotherapy treatments. Finally, we intend to touch points as the classification of sec‐ ondary leukemias, its relationship to chemotherapeutic agents and ionizing radiation, etiolo‐ gy, individual susceptibility, pathogenesis, and treatment as well as their behavior in infants and adults.

In this way one can conclude that the pathophysiology of ALL involved mechanisms genetic and environmental complex at different levels, and also have a close and complex relationship. The key features in the pathophysiology of the ALL is its monoclonal origin, uncontrolled cell proliferation by sustained self-stimulation of their receptors for growth, no response to inhib‐ itory signals, and cellular longevity conditioned by decreased apoptosis.

Acute lymphoblastic leukemia (ALL) is the result of a process of malignant transformation of progenitor cell lineage of the B and T lymphocytes. (Pui et al, 2011) In most cases of ALL, this transformation affects B lineage cells. (Heltemes et al, 2011)

There is growing evidence that supports a multi-step process in leukemogenesis, ie, sequential stepsandserialnumberofalterationsinoncogenes,tumorsuppressorgenes,ormicroRNAgenes in cancer cells. (Greaves, 2002; Croce, 2008) Oncogenes are dominant genes that once mutated, from a normal cellular gene (proto), encode abnormal proteins that cannot control cell prolifera‐

5

47

Pathophysiology of Acute Lymphoblastic Leukemia

http://dx.doi.org/10.5772/54652

A suppressor gene normally inhibits cell division, and favors the growth of cancer when both alleles are mutated, i.e., they are recessive mutations, where the lack of function of both alleles

Unlike the genes involved in cancer development, microRNA genes do not encode proteins, their products are small RNA molecules (single strands of 21 to 23 nucleotides) that recognize and bind a nucleotide sequence of messenger RNA (mRNA), to the complementary microRNA sequence, and thus blocking the translation of protein from mRNA; then, their function is to

Much of what we know about the great influence of certain mutant genes, in the origin of leukemia, is derived from mouse transgenesis studies in molecular virology, with gene trans‐ fection and the generation of leukemia *in vivo*. These studies are based on bacterial recombinant

This knowledge has increased our understanding of leukemogenesis and prognosis, and ad‐ ditionally has served as foundation for the development of targeted therapy. However, the comprehension of how genetic alterations that collaborate to induce leukemic transformation

tion, apoptosis, or both, thereby contributing to cancer development. (Pierce, 2009)

is promoting the development of malignancy. (Pierce, 2009)

**Figure 1.** Cell origin and evolution of a cancer stem cell (modified of Visvader, 2011)

regulate gene expression. (Calin et al., 2002; Croce, 2008)

**2. Genes involved in the leukemias**

DNA methods. (Pui et al, 2011)

**Stem Cell**

**Common progenitor**

**Committed progenitor**

**Matured cells**

is not clear yet.(Pui et al, 2011)

In the last 3 decades, there has been a significantly improvement in treatment outcome of ALL in children, 70% to 80% of children can get cured of their disease, a situation that is different in adults with ALL, since only 30% -35% of them may heal. (Sotk et al, 2000; Mullighan and Downing, 2009)

The required molecular alterations, for the development of malignant disease, are a rare phe‐ nomenon, when it is considered the large number of target susceptible cells to such alteration (Greaves, 2002) ie, a single genetic change is unlikely to be sufficient for the development of a malignant tumor (Croce, 2008), this means that in a very low percentage of people (1%) is developed a hematologic malignancy, only 1 cell will likely experience a mutation in a critical gene for proliferation, differentiation and survival of progenitor cells (Greaves, 2002), that`s what we mean when we mention the monoclonal origin of cancer.

It is important to mention that when we refer to the origin of cancer, in this cases ALL, ref‐ erence is made to the terms: Cell Origin and Stem Cell Cancer. Actually, the concept im‐ plies that normal cells of distinct origin acquire the first mutation(s) to promote cancer, ie, that is the cell that will initiate the cancer, while the cancer stem cell will disseminate it (figure 1). (Visvader, 2011)

Malignant diseases, including acute leukemias, show a marked heterogeneity in cellular mor‐ phology, rate of proliferation, genetic lesions and, as a result, the response to treatment. The molecular mechanisms, underlying the heterogeneity of malignant neoplasia, are important points in the study of the cancer's biology. (Visvader, 2011) Above mentioned alterations may be due to somatic mutations, although alterations in the germline may predispose to familial (or hereditary) cancers. (Croce, 2008)

**Figure 1.** Cell origin and evolution of a cancer stem cell (modified of Visvader, 2011)

of environmental or genetic causes. In particular in this we will focus more on the former. In most cases it is suggested that the mechanism of leukemogenesis is associated with DNA damage of hematopoietic cells from bone marrow by agents such as those used in chemother‐ apy. Although the majority of secondary leukemias are acute myeloid leukemia (AML) has been reported cases of lymphoid leukemia and chronic myeloid leukemia (CML) associated with chemotherapy treatments. Finally, we intend to touch points as the classification of sec‐ ondary leukemias, its relationship to chemotherapeutic agents and ionizing radiation, etiolo‐ gy, individual susceptibility, pathogenesis, and treatment as well as their behavior in infants

In this way one can conclude that the pathophysiology of ALL involved mechanisms genetic and environmental complex at different levels, and also have a close and complex relationship. The key features in the pathophysiology of the ALL is its monoclonal origin, uncontrolled cell proliferation by sustained self-stimulation of their receptors for growth, no response to inhib‐

Acute lymphoblastic leukemia (ALL) is the result of a process of malignant transformation of progenitor cell lineage of the B and T lymphocytes. (Pui et al, 2011) In most cases of ALL, this

In the last 3 decades, there has been a significantly improvement in treatment outcome of ALL in children, 70% to 80% of children can get cured of their disease, a situation that is different in adults with ALL, since only 30% -35% of them may heal. (Sotk et al, 2000; Mullighan and

The required molecular alterations, for the development of malignant disease, are a rare phe‐ nomenon, when it is considered the large number of target susceptible cells to such alteration (Greaves, 2002) ie, a single genetic change is unlikely to be sufficient for the development of a malignant tumor (Croce, 2008), this means that in a very low percentage of people (1%) is developed a hematologic malignancy, only 1 cell will likely experience a mutation in a critical gene for proliferation, differentiation and survival of progenitor cells (Greaves, 2002), that`s

It is important to mention that when we refer to the origin of cancer, in this cases ALL, ref‐ erence is made to the terms: Cell Origin and Stem Cell Cancer. Actually, the concept im‐ plies that normal cells of distinct origin acquire the first mutation(s) to promote cancer, ie, that is the cell that will initiate the cancer, while the cancer stem cell will disseminate it

Malignant diseases, including acute leukemias, show a marked heterogeneity in cellular mor‐ phology, rate of proliferation, genetic lesions and, as a result, the response to treatment. The molecular mechanisms, underlying the heterogeneity of malignant neoplasia, are important points in the study of the cancer's biology. (Visvader, 2011) Above mentioned alterations may be due to somatic mutations, although alterations in the germline may predispose to familial

itory signals, and cellular longevity conditioned by decreased apoptosis.

46 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

transformation affects B lineage cells. (Heltemes et al, 2011)

what we mean when we mention the monoclonal origin of cancer.

and adults.

Downing, 2009)

(figure 1). (Visvader, 2011)

(or hereditary) cancers. (Croce, 2008)

There is growing evidence that supports a multi-step process in leukemogenesis, ie, sequential stepsandserialnumberofalterationsinoncogenes,tumorsuppressorgenes,ormicroRNAgenes in cancer cells. (Greaves, 2002; Croce, 2008) Oncogenes are dominant genes that once mutated, from a normal cellular gene (proto), encode abnormal proteins that cannot control cell prolifera‐ tion, apoptosis, or both, thereby contributing to cancer development. (Pierce, 2009) 

A suppressor gene normally inhibits cell division, and favors the growth of cancer when both alleles are mutated, i.e., they are recessive mutations, where the lack of function of both alleles is promoting the development of malignancy. (Pierce, 2009)

Unlike the genes involved in cancer development, microRNA genes do not encode proteins, their products are small RNA molecules (single strands of 21 to 23 nucleotides) that recognize and bind a nucleotide sequence of messenger RNA (mRNA), to the complementary microRNA sequence, and thus blocking the translation of protein from mRNA; then, their function is to regulate gene expression. (Calin et al., 2002; Croce, 2008)

## **2. Genes involved in the leukemias**

Much of what we know about the great influence of certain mutant genes, in the origin of leukemia, is derived from mouse transgenesis studies in molecular virology, with gene trans‐ fection and the generation of leukemia *in vivo*. These studies are based on bacterial recombinant DNA methods. (Pui et al, 2011)

This knowledge has increased our understanding of leukemogenesis and prognosis, and ad‐ ditionally has served as foundation for the development of targeted therapy. However, the comprehension of how genetic alterations that collaborate to induce leukemic transformation is not clear yet.(Pui et al, 2011)

Most mutations in leukemia are acquired, and occur *de novo* in the lymphoid progenitor cells, less frequently (1% to 5% of leukemias) the mutated genes are inherited (vgr, p53, DNA ligase), or a numeric chromosomal abnormality is involved, for example constitutive trisomy 21. (Greaves, 2002)

The Philadelphia chromosome positive (Ph+) ALL is a product of reciprocal translocation be‐ tween the long arm of chromosome 9(q34), where the oncogene *ABL1* is located, and the long arm of chromosome 22(q11), where the *BCR* gene lies, leading to the formation of the BCR-ABL1 chimeric protein (Figure 3), and as a result of this fusion the Bcr-Abl tyrosine kinase, constitutively active, is produced, which is responsible for the acute and chronic leukemia

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**Figure 3.** Philadelphia chromosome translocation (translocation between 9 and 22 chromosomes). (modified of Satter‐

This alteration is relatively rare (approximately 5%) in infants with ALL, but not in adults where its frequency range between 20% and 30%, it was the first known cytogenetic abnor‐

Despite the Ph+ ALL occurs in only about 5% of patients under 20 years of age, the incidence increases to 33% in patients over 40 years and it reaches 49% in patients over 40 years, to

A significant proportion of patients with ALL Ph+ (approximately 85%), and high-risk ALL without BCR-ABL1 fusion (~28%), have *IKZF1* gene deletion, and both situations are associated

The gene *IKZF1* is located on 7p12, and encodes the transcription factor Ikaros, which is a member of the family of transcription factors containing zinc fingers (Martinelli et al, 2009). Deletion of *IKZF1* is not observed in the chronic phase of CML, but is detected in two out of three samples analyzed during the lymphoid blast crisis. (Mullighan et al, 2009) *IKZF1* genomic alterations, causing loss expression or expression of dominant negative isoforms, are critical

Almost half of patients with BCR-ABL1 ALL and lymphoid blast crisis CML also harbor de‐ letion of *CDKN2A/B and PAX5* genes; approximately 20% of these cases have deletion of the

mality associated with chronic myeloid leukemia (CML) and Ph+ALL.

decrease the incidence to 35% in patients over 60 years. (Lee et al, 2011)

with adverse prognosis. (Martinelli et al, 2009; Cazzaniga et al, 2011)

in the pathogenesis of BCR-ABL1 ALL Ph+. (Mullighan et al, 2009)

forms. (Martinelli et al, 2009)

and James, 2003)

Acute leukemias are the most studied malignant disorders from a genetic standpoint, the re‐ sults of whole-genome studies, e.g.: gene expression analysis of high-resolution, genome-wide alterations in DNA copy number variation (CNA), loss of heterozygosity, epigenetic changes, and complete genome sequencing have favored the recognition of new genetic alterations, then virtually all patients with LLA can be classified according to the specific genetic abnor‐ mality, as shown in Figure 2, which is evident in children with ALL the high frequency of genetic abnormalities. (Mullighan et al, 2007; Pui et al, 2011)

**Figure 2.** Frequency of genetic abnormalities in childrenwith ALL. (modified of Puiet al, 2011.)

As discussed previously, the altered genes in leukemia can result in loss or gain of function, and this is achieved through various mechanisms, for example, abnormal recombination (chromosomal translocation, inversion, or insertion), loss of genetic material (deletion), gain of genetic material (duplication), or mutation. Also can be present additional copies of certain chromosomes, as in the case of hyperdiploidy. With these chromosomal alterations, the acti‐ vation of oncogenes is favored. Oncogenes can be activated by: chromosomal rearrangements, gene mutation and gene amplification. (Croce, 2008)

**i.** The demonstration of chromosomal rearrangements have been evidenced by im‐ proved conventional of cytogenetic study. The standard analysis can detect primary chromosomal abnormalities in more than 75% of all cases. (Liang et al, 2010)

Recently, it was reported that the incidence of chromosomal alterations is associated with age. (Moorman et al, 2010) In fact, age is a determining factor in the prognosis and treatment out‐ come for patients with ALL. In long term survival, the rates are close to 80% in children under 5 years of age, but will decrease to 50% or 60% in adolescents and young adults, and approx‐ imately 30% in adults of 45 to 54 years, but rarely exceed 15% in older adults. The Philadelphia chromosome (Ph) is the most common cytogenetic abnormality associated with ALL in adults. (Zuo et al, 2010; Lee et al, 2011)

The Philadelphia chromosome positive (Ph+) ALL is a product of reciprocal translocation be‐ tween the long arm of chromosome 9(q34), where the oncogene *ABL1* is located, and the long arm of chromosome 22(q11), where the *BCR* gene lies, leading to the formation of the BCR-ABL1 chimeric protein (Figure 3), and as a result of this fusion the Bcr-Abl tyrosine kinase, constitutively active, is produced, which is responsible for the acute and chronic leukemia forms. (Martinelli et al, 2009)

Most mutations in leukemia are acquired, and occur *de novo* in the lymphoid progenitor cells, less frequently (1% to 5% of leukemias) the mutated genes are inherited (vgr, p53, DNA ligase), or a numeric chromosomal abnormality is involved, for example constitutive trisomy 21.

Acute leukemias are the most studied malignant disorders from a genetic standpoint, the re‐ sults of whole-genome studies, e.g.: gene expression analysis of high-resolution, genome-wide alterations in DNA copy number variation (CNA), loss of heterozygosity, epigenetic changes, and complete genome sequencing have favored the recognition of new genetic alterations, then virtually all patients with LLA can be classified according to the specific genetic abnor‐ mality, as shown in Figure 2, which is evident in children with ALL the high frequency of

genetic abnormalities. (Mullighan et al, 2007; Pui et al, 2011)

48 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

**Figure 2.** Frequency of genetic abnormalities in childrenwith ALL. (modified of Puiet al, 2011.)

gene mutation and gene amplification. (Croce, 2008)

(Zuo et al, 2010; Lee et al, 2011)

As discussed previously, the altered genes in leukemia can result in loss or gain of function, and this is achieved through various mechanisms, for example, abnormal recombination (chromosomal translocation, inversion, or insertion), loss of genetic material (deletion), gain of genetic material (duplication), or mutation. Also can be present additional copies of certain chromosomes, as in the case of hyperdiploidy. With these chromosomal alterations, the acti‐ vation of oncogenes is favored. Oncogenes can be activated by: chromosomal rearrangements,

**i.** The demonstration of chromosomal rearrangements have been evidenced by im‐

chromosomal abnormalities in more than 75% of all cases. (Liang et al, 2010)

Recently, it was reported that the incidence of chromosomal alterations is associated with age. (Moorman et al, 2010) In fact, age is a determining factor in the prognosis and treatment out‐ come for patients with ALL. In long term survival, the rates are close to 80% in children under 5 years of age, but will decrease to 50% or 60% in adolescents and young adults, and approx‐ imately 30% in adults of 45 to 54 years, but rarely exceed 15% in older adults. The Philadelphia chromosome (Ph) is the most common cytogenetic abnormality associated with ALL in adults.

proved conventional of cytogenetic study. The standard analysis can detect primary

(Greaves, 2002)

**Figure 3.** Philadelphia chromosome translocation (translocation between 9 and 22 chromosomes). (modified of Satter‐ and James, 2003)

This alteration is relatively rare (approximately 5%) in infants with ALL, but not in adults where its frequency range between 20% and 30%, it was the first known cytogenetic abnor‐ mality associated with chronic myeloid leukemia (CML) and Ph+ALL.

Despite the Ph+ ALL occurs in only about 5% of patients under 20 years of age, the incidence increases to 33% in patients over 40 years and it reaches 49% in patients over 40 years, to decrease the incidence to 35% in patients over 60 years. (Lee et al, 2011)

A significant proportion of patients with ALL Ph+ (approximately 85%), and high-risk ALL without BCR-ABL1 fusion (~28%), have *IKZF1* gene deletion, and both situations are associated with adverse prognosis. (Martinelli et al, 2009; Cazzaniga et al, 2011)

The gene *IKZF1* is located on 7p12, and encodes the transcription factor Ikaros, which is a member of the family of transcription factors containing zinc fingers (Martinelli et al, 2009). Deletion of *IKZF1* is not observed in the chronic phase of CML, but is detected in two out of three samples analyzed during the lymphoid blast crisis. (Mullighan et al, 2009) *IKZF1* genomic alterations, causing loss expression or expression of dominant negative isoforms, are critical in the pathogenesis of BCR-ABL1 ALL Ph+. (Mullighan et al, 2009)

Almost half of patients with BCR-ABL1 ALL and lymphoid blast crisis CML also harbor de‐ letion of *CDKN2A/B and PAX5* genes; approximately 20% of these cases have deletion of the three genes. These data support the concept that it is required the alteration of several cellular pathways to induce the development of **ALL**. It has been correlated the *ALL IKZF1* focal de‐ letion with clinical response to treatment, overall response rate of relapse and disease-free survival; and it has also been shown that deletion of Ikaros gene represents the most important prognostic factor so far described, in ALL Ph+.(Mullighanh et al, 2009; Martinelli et al, 2009)

ed at 19p13.3, with gene pre-B-cell leukemia homeobox 1 (*PBX1*), located at 1q23.3, the fu‐ sion gene *TCF3 (E2A)-PBX1* encodes a chimeric protein with transforming properties. (Garg

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51

The gene encoding E2A transcription factors E12 and E47 binds enhancer elements of the gene of κ light chains of immunoglobulins, as well as some other gene regulatory ele‐

The transcriptional activator domain of the chimeric protein encoded by the fusion gene *E2A-PBX1* is provided by *E12/E47*, and the DNA binding domain is provided by the (HOX) Home‐ box *PBX1*, this protein promotes leukemogenesis by activation of several genes that are not

The t(12; 21)(p13, q22) is a consequence of gene fusion *ETV6/RUNX1* (also known as *TEL/ AML1*) and is the hallmark of one of the most common genetic subtypes of ALL of precursor of B cells in children, in whom is the most common molecular genetic alteration occurring in 20% to 25% of pediatric cases; while in adults this translocation is rare. (Pui et al, 2011)

The current model involves several steps, the fusion of these genes can occur already during fetal development and is the initial event, but is not sufficient for the neoplastic transformation (Fuka et al, 2011). Indeed, the development of ALL of infancy B cell lineage involves (at least) 2 genetic events (hits), the first of which often arises in prenatal stage. (Thomsen et al, 2011) The fusion gene that encodes a chimeric transcription factor, involves the N-terminus of the protein ETV6 and the most of the RUNX1 protein, it is believed that normally RUNX1 acts as a modulator of transcription; transcriptional repressor becomes the target genes *RUNX1*. (Fuka

Hyperdiploidy is detected in approximately 25-30% of children with ALL precursor B cells. In these patients the clinical phenotype is usually associated with low risk and good prognosis. (Grumayer et al, 2002; Pui et al, 2011) It is interesting to mention that a hyperdiploid karyotype refers to a higher number of chromosomes than the normal diploid number (e.i., greater than 46 chromosomes), but having a *chromosome* number that is not a *multiple of the haploid number* (23 chromosomes), the modal number can be located between 47-57 chromosomes (Shaffer et al, 2009). This karyotype arises through a simultaneous gain of multiple chromosomes, from

The hyperdiploidy occurs in 13% of young adults, and only 5% of elderly patients. The hypo‐ diploidy and complex karyotype (presence of more than 2 chromosomal abnormalities) also increase with age, from 4% in the range of 15 to 29 years of age and 16% older than 60 years.

**ii.** When an oncogene is activated by mutation, the encoded protein is structurally

modified in such a way that increases their transforming activity, ie, remains in the active state, continuously transmitting signals by binding of tyrosine and threonine kinase. These signals induce cell growth continued incessant. This mechanism of ac‐ tivation of oncogenes is more evident in other forms of leukemia, for example, AML and myelodysplastic syndromes (MDS) where the *NRAS* gene is mutated. There are

a diploid karyotype, during a single abnormal cell division. (Grumayer et al, 2002)

normally expressed in lymphoid tissues. (Garg et al, 2009)

et al, 2009)

et al, 2011)

(Moorman et al, 2010)

ments. (Garg et al, 2009)

Other chromosomal abnormalities associated with age are the t(4;11)(q21;q23) and t(1;19) (q23;p13), that are rare in patients older than 60 years of age, but on the other way t(8;14) (q24;q32) and t(14;18)(q32;q21) increases with age. (Moorman et al, 2010)

The translocation t(4;11)(q21;q23) leads to the formation of the *MLL-AF4* fusion gene, and is responsible for more than 50% of ALL cases in children younger than 6 months in 10-20% of older infants, in approximately 2% of children and only 10% of adults with *de novo* ALL. Chro‐ mosomal abnormality in adults with ALL is considered to be of high risk. (Marchesi et al, 2011)

The gene Mixed Lineage Leukemia (*MLL*) is frequently involved in hematological malignan‐ cies, particularly acute leukemia, both lymphoblastic and myeloblastic, it is located at 11q23, and plays an important role in the positive regulation of gene expression during early embry‐ onic development (ie it is a HOX gene) and also in hematopoiesis. (Marchesi et al, 2011)

*MLL* gene encodes a 500 kD protein containing several conserved functional domains, a target of proteolytic activity of Caspasa 1, a cleaving protein specialized in N-terminal fragments of 320 kD and C-terminal of 180 kD. This latter is responsible for methyltransferase activity in lysine 4 of histone H3 (H3K4), which mediates changes in chromatin associated with epigenetic transcriptional activation. (Milne et al, 2002; Hsieh et al, 2003)

The main chromosomal alterations that may occur with the *MLL* gene are mainly reciprocal translocations, causing fusion with other different genes, and partial tandem duplication of genes. (Schnittger et al, 2000)

Translocations in which *MLL* gene is involved can result in a chimeric protein, that fuses the MLL N-terminal with the C-terminal portion of the associated genes; the methyltransferase domain (SET domain) is invariably lost in the MLL fusion protein. This fusion of genes can alter normal cellular proliferation and differentiation processes, which favors leukemogenesis. (Ayton et al, 2001)

*MLL* gene is a target of about 104 different rearrangements, of which 64 are translocations with other genes. The proteic products of the fusions are nuclear localization signals, and play an important function as potent transcription factors. (Meyer et al, 2009)

Genes that most commonly fuses with *MLL* gene are, in order of frequency: *AF4*, *AF9*, *ENL*, *AF10*, *AF6*, *ELL*, and *AF1P*. The leukemias that express the fusion gene *MLL-AF4* are diagnosed primarily as pro-B, in pediatric and adult patients, while the fusion with genes *AF9*, *AF6*, or *AF10* are common in AML subtypes myelomonocytic or monoblastic variety. (Kohlmann et al, 2005; Moriya et al, 2012)

The t(1;19)(q23;p13) is recurrent in children and adults, and results from the fusion of gene TCF3 transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47) locat‐

ed at 19p13.3, with gene pre-B-cell leukemia homeobox 1 (*PBX1*), located at 1q23.3, the fu‐ sion gene *TCF3 (E2A)-PBX1* encodes a chimeric protein with transforming properties. (Garg et al, 2009)

three genes. These data support the concept that it is required the alteration of several cellular pathways to induce the development of **ALL**. It has been correlated the *ALL IKZF1* focal de‐ letion with clinical response to treatment, overall response rate of relapse and disease-free survival; and it has also been shown that deletion of Ikaros gene represents the most important prognostic factor so far described, in ALL Ph+.(Mullighanh et al, 2009; Martinelli et al, 2009)

Other chromosomal abnormalities associated with age are the t(4;11)(q21;q23) and t(1;19) (q23;p13), that are rare in patients older than 60 years of age, but on the other way t(8;14)

The translocation t(4;11)(q21;q23) leads to the formation of the *MLL-AF4* fusion gene, and is responsible for more than 50% of ALL cases in children younger than 6 months in 10-20% of older infants, in approximately 2% of children and only 10% of adults with *de novo* ALL. Chro‐ mosomal abnormality in adults with ALL is considered to be of high risk. (Marchesi et al, 2011)

The gene Mixed Lineage Leukemia (*MLL*) is frequently involved in hematological malignan‐ cies, particularly acute leukemia, both lymphoblastic and myeloblastic, it is located at 11q23, and plays an important role in the positive regulation of gene expression during early embry‐ onic development (ie it is a HOX gene) and also in hematopoiesis. (Marchesi et al, 2011)

*MLL* gene encodes a 500 kD protein containing several conserved functional domains, a target of proteolytic activity of Caspasa 1, a cleaving protein specialized in N-terminal fragments of 320 kD and C-terminal of 180 kD. This latter is responsible for methyltransferase activity in lysine 4 of histone H3 (H3K4), which mediates changes in chromatin associated with epigenetic

The main chromosomal alterations that may occur with the *MLL* gene are mainly reciprocal translocations, causing fusion with other different genes, and partial tandem duplication of

Translocations in which *MLL* gene is involved can result in a chimeric protein, that fuses the MLL N-terminal with the C-terminal portion of the associated genes; the methyltransferase domain (SET domain) is invariably lost in the MLL fusion protein. This fusion of genes can alter normal cellular proliferation and differentiation processes, which favors leukemogenesis.

*MLL* gene is a target of about 104 different rearrangements, of which 64 are translocations with other genes. The proteic products of the fusions are nuclear localization signals, and play an

Genes that most commonly fuses with *MLL* gene are, in order of frequency: *AF4*, *AF9*, *ENL*, *AF10*, *AF6*, *ELL*, and *AF1P*. The leukemias that express the fusion gene *MLL-AF4* are diagnosed primarily as pro-B, in pediatric and adult patients, while the fusion with genes *AF9*, *AF6*, or *AF10* are common in AML subtypes myelomonocytic or monoblastic variety. (Kohlmann et

The t(1;19)(q23;p13) is recurrent in children and adults, and results from the fusion of gene TCF3 transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47) locat‐

(q24;q32) and t(14;18)(q32;q21) increases with age. (Moorman et al, 2010)

50 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

transcriptional activation. (Milne et al, 2002; Hsieh et al, 2003)

important function as potent transcription factors. (Meyer et al, 2009)

genes. (Schnittger et al, 2000)

al, 2005; Moriya et al, 2012)

(Ayton et al, 2001)

The gene encoding E2A transcription factors E12 and E47 binds enhancer elements of the gene of κ light chains of immunoglobulins, as well as some other gene regulatory ele‐ ments. (Garg et al, 2009)

The transcriptional activator domain of the chimeric protein encoded by the fusion gene *E2A-PBX1* is provided by *E12/E47*, and the DNA binding domain is provided by the (HOX) Home‐ box *PBX1*, this protein promotes leukemogenesis by activation of several genes that are not normally expressed in lymphoid tissues. (Garg et al, 2009)

The t(12; 21)(p13, q22) is a consequence of gene fusion *ETV6/RUNX1* (also known as *TEL/ AML1*) and is the hallmark of one of the most common genetic subtypes of ALL of precursor of B cells in children, in whom is the most common molecular genetic alteration occurring in 20% to 25% of pediatric cases; while in adults this translocation is rare. (Pui et al, 2011)

The current model involves several steps, the fusion of these genes can occur already during fetal development and is the initial event, but is not sufficient for the neoplastic transformation (Fuka et al, 2011). Indeed, the development of ALL of infancy B cell lineage involves (at least) 2 genetic events (hits), the first of which often arises in prenatal stage. (Thomsen et al, 2011)

The fusion gene that encodes a chimeric transcription factor, involves the N-terminus of the protein ETV6 and the most of the RUNX1 protein, it is believed that normally RUNX1 acts as a modulator of transcription; transcriptional repressor becomes the target genes *RUNX1*. (Fuka et al, 2011)

Hyperdiploidy is detected in approximately 25-30% of children with ALL precursor B cells. In these patients the clinical phenotype is usually associated with low risk and good prognosis. (Grumayer et al, 2002; Pui et al, 2011) It is interesting to mention that a hyperdiploid karyotype refers to a higher number of chromosomes than the normal diploid number (e.i., greater than 46 chromosomes), but having a *chromosome* number that is not a *multiple of the haploid number* (23 chromosomes), the modal number can be located between 47-57 chromosomes (Shaffer et al, 2009). This karyotype arises through a simultaneous gain of multiple chromosomes, from a diploid karyotype, during a single abnormal cell division. (Grumayer et al, 2002)

The hyperdiploidy occurs in 13% of young adults, and only 5% of elderly patients. The hypo‐ diploidy and complex karyotype (presence of more than 2 chromosomal abnormalities) also increase with age, from 4% in the range of 15 to 29 years of age and 16% older than 60 years. (Moorman et al, 2010)

**ii.** When an oncogene is activated by mutation, the encoded protein is structurally modified in such a way that increases their transforming activity, ie, remains in the active state, continuously transmitting signals by binding of tyrosine and threonine kinase. These signals induce cell growth continued incessant. This mechanism of ac‐ tivation of oncogenes is more evident in other forms of leukemia, for example, AML and myelodysplastic syndromes (MDS) where the *NRAS* gene is mutated. There are mutations that suppress the function, and it is observed in tumor suppressor genes such as *TP53*, however, less than 3% of patients with ALL have *TP53* mutations, al‐ though all the cells have abnormal resistance to apoptosis induced by lack of a sig‐ nificant proportion of p53, which is explained in large part by epigenetic. (Zornoza et al, 2011)

tumor suppressor (Leong and Karsan, 2006). In the development of T-ALL there is strong evidence of pro-oncogenic function of signals transduced by Notch, and that modulates the activity of downstream signaling pathways, through transcriptional regulation of its tar‐

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Possible regulators of signaling downstream of Notch, especially in murine models, are some intermediate signaling pathways, such as phosphatidylinositol 3-kinase (PI3K), Akt /protein kinase B, extracellular signal-regulated kinase-1/2, and nuclear factor kB. (Chan et al, 2007)

**1. Transcription factors:** They generally require interacting with other proteins to act, for example: Fos transcription protein dimerizes with the transcription factor Jun to form the AP1 transcription factor which is a complex, and this increases the expression of several

**2. Chromatin remodeling:** It plays an important role in the degree of compaction of chro‐ matin and therefore in the control of gene expression, replication and chromosome seg‐ regation, by the action of two types of enzymes: the ATP-dependent enzymes, which have important role in changing the position of histones, and enzymes that modify N-terminal

Indeed, the epigenetic code is made by the pattern of histone modification, and determines in this way the interaction between nucleosomes and chromatin-associated proteins,

On this basis it is important to note that methylation of CpG-dinucleotides in position near the site of transcription initiation can silence gene expression, hypermethylation of tumor suppressor genes and hypomethylation of oncogenes can lead to various forms of cancer. Aberrant methylation of CpG sites in promoter regions of genes has been identified

In this way it has been found some improperly methylated genes that are involved in the p53 pathway suggesting that despite not having an activating mutation of this gene in ALL, there is an abnormal function of p53 mediated by epigenetic mechanisms. In fact, hypermethylation of genes involved in the TP53 pathway is an independent poor prog‐

**3. Growth factor receptors:** They are altered in many cancers. A deletion of the ligand bind‐ ing domain causes constitutive receptor activation in the absence of ligand binding sites of interaction by providing cytoplasmic proteins containing the SRC homology domain binding and other domains, this way deregulates multiple signaling pathways. Vascular endothelial growth factor (VEGF) regulates hypoxia-dependent control of gene transcrip‐ tion. VEGF activity is mediated by three tyrosine kinase receptors: VEGFR1 (FLT1),

The importance of angiogenesis and signaling pathways related to angiogenesis in the growth and expansion of cells in acute leukemia has been well established. *In vitro* in the

In general, the products of oncogenes can be classified as described below:

get genes. (Chan et al, 2007)

genes control cell division.

tails of histones. (Peterson and Workman, 2000)

in ALL cell lines. (Milani et al, 2009)

thereby determining its transcriptional capacity. (Croce, 2008)

nostic factor in patients with ALL. (Zornoza et al, 2011)

VEGFR2 (Flk1-KDR) and VEGFR3 (FLT4).

On the other hand, some authors have found change in the number of copies (CNVs) to 50 regions in ALL recurring, some are very small and have less than 1 Mb, however, occur in genes encoding regulatory proteins of normal lymphoid development up to 40% of cases of ALL stem B. The most common targets are lymphoid transcription factor PAX5, that can hold deletions or amplifications in up to 30% of cases of ALL-B, also found CNVs in transcription factor genes IKZF1, the IKZF3, EBF1 (factor Cell B early), LEF1 and TCF3, and RAG1 and RAG2 genes. (Mullighan et al, 2009)

**iii.** The most relevant gene amplification in LLA is the dihydrofolatereductase (*DHFR*). The amplification of this gene causes evident cytogenetic alterations because the am‐ plified DNA segment may involve several hundred kilobases. (Croce, 2008)

A variety of acute leukemia to consider is the T-cell ALL (ALL-T), it represents about 10% to 15% of ALL in adults and 25% of children. The clinical behavior is more aggressive, patients have a higher percentage of failure of remission, relapse rate is also higher as well, and the central nervous system infiltration compared with B-cell ALL type. (Demarest et al, 2011)

Oncogenes and tumor suppressor genes that have been implicated in T-ALL are: *c-MYC, NOTCH, LMO1 / 2, LYL1, TAL1 / 2, Hox11* and *HOX11L2*. It is clear that activated Notch is able to induce T cell leukemogenesis and is critical for the progression to T-ALL. (Demarest et al, 2008)

Members of the NOTCH family are transmembrane receptors that are critically involved in controlling the differentiation, proliferation and apoptosis in several cell types including T cells. The Notch receptor binding to its ligand exhibits a cleavage site for extracellular ADAM metalloproteinase, and a cleavage site in the transmembrane region for the γ-secretase, thus releasing the intracellular domain of Notch, which transmits this signaling to the cell nucleus, where it is associated with a DNA-binding complex. (Palomero et al, 2006; Chan et al, 2007; Sanda et al, 2010; Gomez et al, 2012)

Notch signaling cooperates with the signaling of T cell receptors (TCR) to expand the number of thymocytes undergoing β-selection. Over 50% of T-cell ALL have activating mutations in Notch1. (Gomez et al, 2012)

NOTCH target genes are mainly cyclin D1 and c-Myc. Both Notch and c-Myc regulates cell cycle progression by inducing expression of cyclins and reduced expression of *p27*. An im‐ portant aspect to point out is that Notch is able to inhibit apoptosis induced by p53. When Notch expression is suppressed, the p53 pathway is activated and leads to tumor regression. (Demarest et al, 2008; Sanda et al, 2010)

An important aspect of Notch, is that depending on the type of cells, the extracellular en‐ vironment, and the intensity of the signal, Notch can transmit signals as pro-oncogenic or tumor suppressor (Leong and Karsan, 2006). In the development of T-ALL there is strong evidence of pro-oncogenic function of signals transduced by Notch, and that modulates the activity of downstream signaling pathways, through transcriptional regulation of its tar‐ get genes. (Chan et al, 2007)

Possible regulators of signaling downstream of Notch, especially in murine models, are some intermediate signaling pathways, such as phosphatidylinositol 3-kinase (PI3K), Akt /protein kinase B, extracellular signal-regulated kinase-1/2, and nuclear factor kB. (Chan et al, 2007)

In general, the products of oncogenes can be classified as described below:

mutations that suppress the function, and it is observed in tumor suppressor genes such as *TP53*, however, less than 3% of patients with ALL have *TP53* mutations, al‐ though all the cells have abnormal resistance to apoptosis induced by lack of a sig‐ nificant proportion of p53, which is explained in large part by epigenetic. (Zornoza

On the other hand, some authors have found change in the number of copies (CNVs) to 50 regions in ALL recurring, some are very small and have less than 1 Mb, however, occur in genes encoding regulatory proteins of normal lymphoid development up to 40% of cases of ALL stem B. The most common targets are lymphoid transcription factor PAX5, that can hold deletions or amplifications in up to 30% of cases of ALL-B, also found CNVs in transcription factor genes IKZF1, the IKZF3, EBF1 (factor Cell B early), LEF1 and TCF3, and RAG1 and RAG2

52 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

**iii.** The most relevant gene amplification in LLA is the dihydrofolatereductase (*DHFR*).

plified DNA segment may involve several hundred kilobases. (Croce, 2008) A variety of acute leukemia to consider is the T-cell ALL (ALL-T), it represents about 10% to 15% of ALL in adults and 25% of children. The clinical behavior is more aggressive, patients have a higher percentage of failure of remission, relapse rate is also higher as well, and the central nervous system infiltration compared with B-cell ALL type. (Demarest et al, 2011) Oncogenes and tumor suppressor genes that have been implicated in T-ALL are: *c-MYC, NOTCH, LMO1 / 2, LYL1, TAL1 / 2, Hox11* and *HOX11L2*. It is clear that activated Notch is able to induce T cell leukemogenesis and is critical for the progression to T-ALL. (Demarest et al,

Members of the NOTCH family are transmembrane receptors that are critically involved in controlling the differentiation, proliferation and apoptosis in several cell types including T cells. The Notch receptor binding to its ligand exhibits a cleavage site for extracellular ADAM metalloproteinase, and a cleavage site in the transmembrane region for the γ-secretase, thus releasing the intracellular domain of Notch, which transmits this signaling to the cell nucleus, where it is associated with a DNA-binding complex. (Palomero et al, 2006; Chan et al, 2007;

Notch signaling cooperates with the signaling of T cell receptors (TCR) to expand the number of thymocytes undergoing β-selection. Over 50% of T-cell ALL have activating mutations in

NOTCH target genes are mainly cyclin D1 and c-Myc. Both Notch and c-Myc regulates cell cycle progression by inducing expression of cyclins and reduced expression of *p27*. An im‐ portant aspect to point out is that Notch is able to inhibit apoptosis induced by p53. When Notch expression is suppressed, the p53 pathway is activated and leads to tumor regression.

An important aspect of Notch, is that depending on the type of cells, the extracellular en‐ vironment, and the intensity of the signal, Notch can transmit signals as pro-oncogenic or

The amplification of this gene causes evident cytogenetic alterations because the am‐

et al, 2011)

genes. (Mullighan et al, 2009)

Sanda et al, 2010; Gomez et al, 2012)

(Demarest et al, 2008; Sanda et al, 2010)

Notch1. (Gomez et al, 2012)

2008)


Indeed, the epigenetic code is made by the pattern of histone modification, and determines in this way the interaction between nucleosomes and chromatin-associated proteins, thereby determining its transcriptional capacity. (Croce, 2008)

On this basis it is important to note that methylation of CpG-dinucleotides in position near the site of transcription initiation can silence gene expression, hypermethylation of tumor suppressor genes and hypomethylation of oncogenes can lead to various forms of cancer. Aberrant methylation of CpG sites in promoter regions of genes has been identified in ALL cell lines. (Milani et al, 2009)

In this way it has been found some improperly methylated genes that are involved in the p53 pathway suggesting that despite not having an activating mutation of this gene in ALL, there is an abnormal function of p53 mediated by epigenetic mechanisms. In fact, hypermethylation of genes involved in the TP53 pathway is an independent poor prog‐ nostic factor in patients with ALL. (Zornoza et al, 2011)

**3. Growth factor receptors:** They are altered in many cancers. A deletion of the ligand bind‐ ing domain causes constitutive receptor activation in the absence of ligand binding sites of interaction by providing cytoplasmic proteins containing the SRC homology domain binding and other domains, this way deregulates multiple signaling pathways. Vascular endothelial growth factor (VEGF) regulates hypoxia-dependent control of gene transcrip‐ tion. VEGF activity is mediated by three tyrosine kinase receptors: VEGFR1 (FLT1), VEGFR2 (Flk1-KDR) and VEGFR3 (FLT4).

The importance of angiogenesis and signaling pathways related to angiogenesis in the growth and expansion of cells in acute leukemia has been well established. *In vitro* in the leukemic cell, activation of VEGFR1 (FLT1) promotes cell migration and proliferation, whereas in vivo cells overexpressing FLT-1It accumulate in the bone marrow.

er therapy outcome. Currently, BCR/ABL1 expressing cells can be selectively killed with the Imatinib (or STI571, imatinib mesylate, Gleevec or Glivec; Novartis) which inhibits the excessive tyrosine kinase activity of the hybrid protein. Most of the patients achieve com‐ plete remission with this approach; however, sometimes relapse occurs mainly by muta‐ tions in the *ABL1* segment that render resistance to the Imatinib. (Mitelman et al, 2007;

Pathophysiology of Acute Lymphoblastic Leukemia

http://dx.doi.org/10.5772/54652

55

During the progression of the disease multiple genetic alterations accumulate over time being selected by their potential to give fitness advantage to the new clones. Irrespective which is the primary change, the most frequent secondary numerical chromosomal changes in ALL are +X, +6, -7, +8, and +21; whereas, the most recurrent secondary structural aberrations are

Although little is known about the etiology of leukemia, this has a multifactorial behav‐ ior with risk factors that may contribute to its development such as ionizing radiation, che‐ motherapy and chromosomal abnormalities. (Han et al, 2010) By other hand, there are three hypotheses one called delayed infection, the second population mixing and the third hy‐ giene hypothesis, (Strachan, 1989; Kinlen, 1995; Greaves, 2006) the first two suggest that the immune system deficiency in an early stage of development can cause an abnormal im‐ mune response to infections which may arise in the development of human beings. Both hypotheses are similar to third called hygiene hypothesis, which explains an increase in al‐ lergies in Western populations. (Chang et al, 2010) Although most studies support to infec‐ tions and immune system factors in the etiology of ALL, little is known about the role of genes in this etiology. The relation of immune system in the ALL is a complex process that involves the interaction of many cells that including leukocytes, epithelial barriers, comple‐ ment proteins, colexinas, pentraxins, cytokines (TNF, IL-1, chemokines, IL-2, IFN type I, IFN etc.), Th1, Th2, Treg and Th17 cells, CD28, FCGR2, GATA3, STAT4, STAT6 and may other. (Chang et al, 2010) Variations in the genes of these cells can affect their develop‐ ment and function in the immune response and therefore it may increase susceptibility to developing ALL. (Han et al, 2010; Chang et al, 2010) Moreover it was found that the CD47 molecule protects the macrophage leukemic clones to bind to a molecule on the surface of these cells. The interaction between macrophages and leukemic cells inhibits macrophage specific action which allows the cancer cell to proliferate. So that although the macro‐ phage plays an important role in the destruction of cancer cells, leukemic cells with great‐ er metabolic potential, and the potential escapes annihilating the macrophage. (Tesniere et al, 2008; Jaiswal et al, 2009) In the innate immune system, macrophages and other im‐ mune cells involved in immune surveillance protect the body permanently cell rate unex‐ pectedly mutates. In contrast, the adaptive immune system through T and B cells upon activation attempt to destroy leukemic cells; however these also evade cellular immunity. It has been shown that the human genome sequencing is useful to identify oncogenic mu‐ tations useful in predicting the diagnosis, prognosis and therapeutic choice. What has pro‐

vided important insights into the pathogenesis of leukemias. (Kalender et al, 2012)

Croce, 2008; Pui et al, 2011; Dowing et al, 2012)

**Leukemia and immunity**

dup(1q), i(7q)(q10), and der(22)t(9;22). (Johansson et al, 1994)

At the same time, the FLT-1 neutralization affects leukemia cell location (now in the bone marrow of the diaphysis), increased apoptosis, and prevents their departure to other tis‐ sues, which prolongs survival of mice inoculated. (Fragoso et al, 2006)


#### **Cytogenetics in acute lymphoblastic leukemia**

The cytogenetic studies of human neoplasia began in 1960 with the discovery by Nowell and Hungerford of the Philadelphia chromosome in individuals with chronic myeloge‐ nous leukemia. Thirteen years after, Rowley performed chromosomal banding techniques and defined the origin of the Philadelphia chromosome as the result of the chromosomal translocation t(9;22)(q34;q11). (Mitelman et al, 2007; Croce, 2008; Pui et al, 2011; Dowing et al, 2012) These findings established the beginning for the cytogenetic studies of many sol‐ id and hematologic tumors. Currently, it has been consolidated a public database (Mitel‐ man Database of Chromosome Aberrations and Gene Fusions in Cancer) containing 61,846 reported cases of cytogenetic studies and 975 different gene fusions in diverse human tu‐ mors. (Mitelman et al, 2012)

The ALL is the most common malignancy in pediatric population with a frequency of 19.7%. It is markedly different from the frequency observed in adults (1.2%). In both groups, the commitment of B-cell lineage is most frequent than the T-cell lineage. The variety of chromo‐ somal abnormalities observed during the malignant development is also different between pediatric and adult ALL. The chromosomal abnormalities most frequent in pediatric ALL are the t(12;21)(p13;q22) with the *ETV6-RUNX1* gene fusion (21%) and hiperdiploidy of >50 chro‐ mosomes (19%). In adult ALL the most recurrent chromosomal abnormalities are the t(9;22) (q34;q11) with the *BCR-ABL1* gene fusion (25%) and *MLL* (11q23) gene fusions (10%). (Dowing et al, 2012)

The chromosomal translocation t(9;22) fuses the tyrosine kinase *ABL1* (*v-abl Abelson mur‐ ine leukemia viral oncogene homolog 1*) gene located on 9q34 band with the *BCR* (*Breakpoint Cluster Region*) gene situated on 22q11 band raising the *5'-BCR/ABL1*-3' gene fusion. Vari‐ ous forms of this hybrid gene are generated depending on the breakpoint at BCR gene oc‐ curred. The e13a2 and e14a2 BCR/ABL1 transcripts code for a 210 KD protein and the e19a2 produces a 230 KD protein. These isoforms are related mainly to CML. The e1a2 tran‐ script codes for a 190 KD protein which is mostly related to ALL and a trend towards poor‐ er therapy outcome. Currently, BCR/ABL1 expressing cells can be selectively killed with the Imatinib (or STI571, imatinib mesylate, Gleevec or Glivec; Novartis) which inhibits the excessive tyrosine kinase activity of the hybrid protein. Most of the patients achieve com‐ plete remission with this approach; however, sometimes relapse occurs mainly by muta‐ tions in the *ABL1* segment that render resistance to the Imatinib. (Mitelman et al, 2007; Croce, 2008; Pui et al, 2011; Dowing et al, 2012)

During the progression of the disease multiple genetic alterations accumulate over time being selected by their potential to give fitness advantage to the new clones. Irrespective which is the primary change, the most frequent secondary numerical chromosomal changes in ALL are +X, +6, -7, +8, and +21; whereas, the most recurrent secondary structural aberrations are dup(1q), i(7q)(q10), and der(22)t(9;22). (Johansson et al, 1994)

#### **Leukemia and immunity**

leukemic cell, activation of VEGFR1 (FLT1) promotes cell migration and proliferation,

At the same time, the FLT-1 neutralization affects leukemia cell location (now in the bone marrow of the diaphysis), increased apoptosis, and prevents their departure to other tis‐

**4. Signal transducers:** They on the binding of receptor tyrosine kinases, to appropriate li‐ gand receptor, lead to reorganization and autophosphorylation of tyrosine in the intra‐ cellular portion of the molecules, this increases the activity of the receptor or receptor interaction promotes intracytoplasmic domain with other proteins such as with the SRC

**5. Regulators of apoptosis:**Regulators that finally lead to apoptosis, where the *BCL2* gene encodes for a cytoplasmic protein that is localized in the mitochondria and increases the

The cytogenetic studies of human neoplasia began in 1960 with the discovery by Nowell and Hungerford of the Philadelphia chromosome in individuals with chronic myeloge‐ nous leukemia. Thirteen years after, Rowley performed chromosomal banding techniques and defined the origin of the Philadelphia chromosome as the result of the chromosomal translocation t(9;22)(q34;q11). (Mitelman et al, 2007; Croce, 2008; Pui et al, 2011; Dowing et al, 2012) These findings established the beginning for the cytogenetic studies of many sol‐ id and hematologic tumors. Currently, it has been consolidated a public database (Mitel‐ man Database of Chromosome Aberrations and Gene Fusions in Cancer) containing 61,846 reported cases of cytogenetic studies and 975 different gene fusions in diverse human tu‐

The ALL is the most common malignancy in pediatric population with a frequency of 19.7%. It is markedly different from the frequency observed in adults (1.2%). In both groups, the commitment of B-cell lineage is most frequent than the T-cell lineage. The variety of chromo‐ somal abnormalities observed during the malignant development is also different between pediatric and adult ALL. The chromosomal abnormalities most frequent in pediatric ALL are the t(12;21)(p13;q22) with the *ETV6-RUNX1* gene fusion (21%) and hiperdiploidy of >50 chro‐ mosomes (19%). In adult ALL the most recurrent chromosomal abnormalities are the t(9;22) (q34;q11) with the *BCR-ABL1* gene fusion (25%) and *MLL* (11q23) gene fusions (10%). (Dowing

The chromosomal translocation t(9;22) fuses the tyrosine kinase *ABL1* (*v-abl Abelson mur‐ ine leukemia viral oncogene homolog 1*) gene located on 9q34 band with the *BCR* (*Breakpoint Cluster Region*) gene situated on 22q11 band raising the *5'-BCR/ABL1*-3' gene fusion. Vari‐ ous forms of this hybrid gene are generated depending on the breakpoint at BCR gene oc‐ curred. The e13a2 and e14a2 BCR/ABL1 transcripts code for a 210 KD protein and the e19a2 produces a 230 KD protein. These isoforms are related mainly to CML. The e1a2 tran‐ script codes for a 190 KD protein which is mostly related to ALL and a trend towards poor‐

whereas in vivo cells overexpressing FLT-1It accumulate in the bone marrow.

sues, which prolongs survival of mice inoculated. (Fragoso et al, 2006)

54 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

homology domains. (Croce, 2008)

survival of the cell by inhibiting apoptosis.

**Cytogenetics in acute lymphoblastic leukemia**

mors. (Mitelman et al, 2012)

et al, 2012)

Although little is known about the etiology of leukemia, this has a multifactorial behav‐ ior with risk factors that may contribute to its development such as ionizing radiation, che‐ motherapy and chromosomal abnormalities. (Han et al, 2010) By other hand, there are three hypotheses one called delayed infection, the second population mixing and the third hy‐ giene hypothesis, (Strachan, 1989; Kinlen, 1995; Greaves, 2006) the first two suggest that the immune system deficiency in an early stage of development can cause an abnormal im‐ mune response to infections which may arise in the development of human beings. Both hypotheses are similar to third called hygiene hypothesis, which explains an increase in al‐ lergies in Western populations. (Chang et al, 2010) Although most studies support to infec‐ tions and immune system factors in the etiology of ALL, little is known about the role of genes in this etiology. The relation of immune system in the ALL is a complex process that involves the interaction of many cells that including leukocytes, epithelial barriers, comple‐ ment proteins, colexinas, pentraxins, cytokines (TNF, IL-1, chemokines, IL-2, IFN type I, IFN etc.), Th1, Th2, Treg and Th17 cells, CD28, FCGR2, GATA3, STAT4, STAT6 and may other. (Chang et al, 2010) Variations in the genes of these cells can affect their develop‐ ment and function in the immune response and therefore it may increase susceptibility to developing ALL. (Han et al, 2010; Chang et al, 2010) Moreover it was found that the CD47 molecule protects the macrophage leukemic clones to bind to a molecule on the surface of these cells. The interaction between macrophages and leukemic cells inhibits macrophage specific action which allows the cancer cell to proliferate. So that although the macro‐ phage plays an important role in the destruction of cancer cells, leukemic cells with great‐ er metabolic potential, and the potential escapes annihilating the macrophage. (Tesniere et al, 2008; Jaiswal et al, 2009) In the innate immune system, macrophages and other im‐ mune cells involved in immune surveillance protect the body permanently cell rate unex‐ pectedly mutates. In contrast, the adaptive immune system through T and B cells upon activation attempt to destroy leukemic cells; however these also evade cellular immunity. It has been shown that the human genome sequencing is useful to identify oncogenic mu‐ tations useful in predicting the diagnosis, prognosis and therapeutic choice. What has pro‐ vided important insights into the pathogenesis of leukemias. (Kalender et al, 2012)

#### **Polymorphisms environmental in leukemia**

Although the clinical and biological aspects of the ALL are well documented, little is known about individual susceptibility. Polymorphic variants of several genes, diet, environmental exposure to carcinogens and individualities of immune system are potential factors that could be increase predisposition to leukemia. (Buffleret al., 2005) However some speculation exist about the mechanism of the potential agents carcinogenic that could cause such alterations to ALL origin.(Smith, 1996; Kamdar et al., 2011)

#### **Polymorphisms in the via of folate metabolism**

The genetic regulation of folate metabolism have been the focus of many investigations that may influence in the preleukemic clone origin, by the via DNA hypomethylation of key reg‐ ulatory genes, as well as uracil misincorporation into DNA leading to double-strand breaks and chromosomal aberrations.(Kamdar et al., 2011) The presence of some polymorphisms in genes involved in folate metabolism (*MTHFR, MTR, CBS, SHMT1* and *TYMS*) may cause de‐ ficiency in the enzyme activity and lead to inadequate folate metabolism and hypomethylation of DNA, which may lead to a neoplastic process. (Sharp and Little, 2004; Kamdar et al., 2011) The insufficient input of folate produces elevated plasma concentration of Homocysteine (Hcy) and adenosylmethionine (SAM) elevation, so that SAM is inhibitor methyltransferase enzyme. (Sharp et al., 2004) This inhibition may alter both, the DNA methylation process, and the reg‐ ulation of gene expression. (Sharpand Little, 2004; Lightfoot et al., 2010) The hypomethylation is associated with activation of oncogenes and neoplastic processes, whereas the hyperme‐ thylation of CpG islands in promoter regions, of some tumor suppressor genes, prevents the transcription and promotes the development of tumors. (Das and Singal, 2004) (figure 4) As‐ sociations studies have been developed to identify genetic variants associated with ALL sus‐ ceptibility, among them are: methylenetetrahydrofolatereductase (MTHFR), an enzyme that participates in Hcy and folate metabolism, plays an important role in DNA methylation and provision of nucleotides for DNA synthesis. (Robien and Ulrich, 2003)

Variations in the MTHFR gene sequence may result in enzymatic deficiency, low plasma folate levels and hyperhomocysteinemia, a risk factor for many diseases as: coronary diseases, neural tube defects, cancer, and leukemia, among others. (Lordelo et al, 2011) Association studies have been described between C677T and A1298C MTHFR polymorphisms and risk of leuke‐ mia (Skibola et al, 1999; Franco et al., 2001; Robien and Ulrich, 2003; Gallegos et al, 2008), which produces an decreased catalytic activity of MTHFR and subsequent availability of 5,10-MeTHF and SAM, have been extensively studied in relation to childhood leukemia, these findings have been inconsistent and their frequency vary among ethnic groups.(Robien and Ulrich, 2003)

A2756G was reviewed. Overall, individuals carrying MTR 2756GG genotype had a reduced cancer risk, under a recessive genetic model, in European populations. However, in Asian populations, it has a significantly high association. In stratified studies by tumor site, there

Oncogenes activation (hyphomethylation CpG isles)

16

57

http://dx.doi.org/10.5772/54652

Pathophysiology of Acute Lymphoblastic Leukemia

**DNA methylation**

**Cystathionine-β-synthase** (*CBS*; gene localized to 21q22.3), the polymorphism most studied with leukemia are T833C, that co-segregates with 844ins68, and the G919A. (Ge et al, 2011) The frequencies of these polymorphisms are variable depending of the studied populations. The CBS participate in the trans-sulfuration pathway, catalyzes the condensation of serine and Hcy to form cystathionine, an intermediate step in the synthesis of cysteine. (figure 4) The 844ins68 polymorphism was associated in Down Syndrome (DS) with leukemia myeloblasts, detecting a 4.6-fold higher rate (P < 0.001) when compared to non-DS individuals. (Ge et al, 2011) The biological function of this polymorphism is even contradictory, it has been demonstrated that carriers of 844ins68 have significantly lower total plasma Hcy levels, after a methionine load, and concluded that this polymorphism was associated with higher CBS enzyme activities. (Ge

was a *statistically significant* reduced risk with ALL. (Yu et al, 2010)

Uracil misincorporation into DNA DNA double‐strand breaks Chromosomal aberrations

**Figure 4.** Polymorphisms (MTHFR, CBS and TS) of the via of folate in the development of ALL

Response to drugs

et al, 2011)

**Methionine synthase** (MTR): Other studied polymorphisms, in association with ALL, are the missense polymorphism MTR c.2756A>G (D919G), that has been reported that alter the sus‐ ceptibility to various cancers, (Linnebank et al, 2004; Yu et al, 2010) however the results have been contradictories. MTR is a vitamin B12-dependent enzyme, which catalyzes the remethy‐ lation of Hcy to methionine and the demethylation of 5-meTHF to THF, and has influence on DNA methylation, as well as on nucleic acid synthesis (Greene, 2010) (figure 4). A meta-anal‐ ysis including 24896 cancer patients and 33862 controls, from 52 published papers, for MTR

**Figure 4.** Polymorphisms (MTHFR, CBS and TS) of the via of folate in the development of ALL

**Polymorphisms environmental in leukemia**

56 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

ALL origin.(Smith, 1996; Kamdar et al., 2011)

**Polymorphisms in the via of folate metabolism**

Although the clinical and biological aspects of the ALL are well documented, little is known about individual susceptibility. Polymorphic variants of several genes, diet, environmental exposure to carcinogens and individualities of immune system are potential factors that could be increase predisposition to leukemia. (Buffleret al., 2005) However some speculation exist about the mechanism of the potential agents carcinogenic that could cause such alterations to

The genetic regulation of folate metabolism have been the focus of many investigations that may influence in the preleukemic clone origin, by the via DNA hypomethylation of key reg‐ ulatory genes, as well as uracil misincorporation into DNA leading to double-strand breaks and chromosomal aberrations.(Kamdar et al., 2011) The presence of some polymorphisms in genes involved in folate metabolism (*MTHFR, MTR, CBS, SHMT1* and *TYMS*) may cause de‐ ficiency in the enzyme activity and lead to inadequate folate metabolism and hypomethylation of DNA, which may lead to a neoplastic process. (Sharp and Little, 2004; Kamdar et al., 2011) The insufficient input of folate produces elevated plasma concentration of Homocysteine (Hcy) and adenosylmethionine (SAM) elevation, so that SAM is inhibitor methyltransferase enzyme. (Sharp et al., 2004) This inhibition may alter both, the DNA methylation process, and the reg‐ ulation of gene expression. (Sharpand Little, 2004; Lightfoot et al., 2010) The hypomethylation is associated with activation of oncogenes and neoplastic processes, whereas the hyperme‐ thylation of CpG islands in promoter regions, of some tumor suppressor genes, prevents the transcription and promotes the development of tumors. (Das and Singal, 2004) (figure 4) As‐ sociations studies have been developed to identify genetic variants associated with ALL sus‐ ceptibility, among them are: methylenetetrahydrofolatereductase (MTHFR), an enzyme that participates in Hcy and folate metabolism, plays an important role in DNA methylation and

Variations in the MTHFR gene sequence may result in enzymatic deficiency, low plasma folate levels and hyperhomocysteinemia, a risk factor for many diseases as: coronary diseases, neural tube defects, cancer, and leukemia, among others. (Lordelo et al, 2011) Association studies have been described between C677T and A1298C MTHFR polymorphisms and risk of leuke‐ mia (Skibola et al, 1999; Franco et al., 2001; Robien and Ulrich, 2003; Gallegos et al, 2008), which produces an decreased catalytic activity of MTHFR and subsequent availability of 5,10-MeTHF and SAM, have been extensively studied in relation to childhood leukemia, these findings have been inconsistent and their frequency vary among ethnic groups.(Robien and Ulrich, 2003)

**Methionine synthase** (MTR): Other studied polymorphisms, in association with ALL, are the missense polymorphism MTR c.2756A>G (D919G), that has been reported that alter the sus‐ ceptibility to various cancers, (Linnebank et al, 2004; Yu et al, 2010) however the results have been contradictories. MTR is a vitamin B12-dependent enzyme, which catalyzes the remethy‐ lation of Hcy to methionine and the demethylation of 5-meTHF to THF, and has influence on DNA methylation, as well as on nucleic acid synthesis (Greene, 2010) (figure 4). A meta-anal‐ ysis including 24896 cancer patients and 33862 controls, from 52 published papers, for MTR

provision of nucleotides for DNA synthesis. (Robien and Ulrich, 2003)

A2756G was reviewed. Overall, individuals carrying MTR 2756GG genotype had a reduced cancer risk, under a recessive genetic model, in European populations. However, in Asian populations, it has a significantly high association. In stratified studies by tumor site, there was a *statistically significant* reduced risk with ALL. (Yu et al, 2010)

**Cystathionine-β-synthase** (*CBS*; gene localized to 21q22.3), the polymorphism most studied with leukemia are T833C, that co-segregates with 844ins68, and the G919A. (Ge et al, 2011) The frequencies of these polymorphisms are variable depending of the studied populations. The CBS participate in the trans-sulfuration pathway, catalyzes the condensation of serine and Hcy to form cystathionine, an intermediate step in the synthesis of cysteine. (figure 4) The 844ins68 polymorphism was associated in Down Syndrome (DS) with leukemia myeloblasts, detecting a 4.6-fold higher rate (P < 0.001) when compared to non-DS individuals. (Ge et al, 2011) The biological function of this polymorphism is even contradictory, it has been demonstrated that carriers of 844ins68 have significantly lower total plasma Hcy levels, after a methionine load, and concluded that this polymorphism was associated with higher CBS enzyme activities. (Ge et al, 2011)

#### **Serine Hydroxymethyltransferase 1 (SHMT1)**

Cytosolic SHMT1 regulates 5,10-MeTHF,that acts as substrate for MTHFR. The 1420C>T poly‐ morphism of this gene reduces circulating folate levels, and may mimic folate deficiency, con‐ sequently shunting 5,10-MeTHF towards DNA synthesis, and have been shown that moderate the risk of hematological malignancies. (Lightfoot et al, 2005) Folate is a component important in the development of the embryogenesis and early fetal development, via its effects on DNA methylation and synthesis. Then, the well-documented in utero origin of ALL has led to hy‐ pothesize that deficient folate intake may be important in its etiology. (Lightfoot et al, 2005; de Jonge et al, 2009)

and the environment are mostly lipophilic, so it tends to accumulate in lipophilic environments the body and are difficult to remove so they tend to trigger toxicity phenomena. (Gonzalez and Gelboin, 1994) The liver removes lipophilic xenobiotics, through a set of known reactions of biotransformation. The end result is the formation of metabolites less lipophilic and more soluble, which are easily eliminated in the urine or bile compounds. That is why these reactions are known as detoxification or deactivation. (DeAnn, 1998) In this sense, drugs are a class of compounds that are absorbed xenobiotics on the body and distributed in body fluids, tissue and organ. Where exert their pharmacological action and pharmacodynamics specified; only a small part reaches the tissue-receptor-target enzyme, while most are metabolized and elim‐ inated. (Sheweita, 2000) Moreover, the processes of biotransformation of xenobiotics are sub‐ divided into two phases: phase I metabolism, carried out by two families of enzymes oxygenases: those dependent on cytochrome P450 (CYP450) monooxygenases and the flavin (FMO). Their metabolism is characterized by the action of chemical processes of different na‐ ture mainly oxidation, oxygenation, reduction and hydrolysis, so as dealquilations and deha‐ logenations. These chemical reactions produce metabolites capable of binding covalently to endogenous molecules such as glucuronic acids, glutathione, sulfate and amino acids that generate conjugates, which are metabolized by Phase II which is characterized by solubility in generating molecules and decreased toxicity, generated by the modification of new functional groups, which transform the more polar metabolite, which facilitates their removal. In this regard, when a drug enters the body usually is modified by conjugation reactions to be easily removed. However, when there modifications in the concentrations concentration (high or low) of enzymes that perform the conjugation process and if the xenobiotic is lipophilic nature, tends to accumulate in the cell, which will lead to different processes: 1) accumulation of re‐ active metabolites adduct forming with DNA, 2) formation of a toxic compound 3) a nontoxic compound becomes a toxic derivative. This generates secondary metabolic pathways may have a carcinogenic action, toxicological, genotoxic or mutagenic in the body. (Marmiroli and Maestri, 2008) Different studies in the literature have suggested the association of polymor‐ phisms in genes involved in xenobiotic metabolism phase I and II in patients with leukemia.

Pathophysiology of Acute Lymphoblastic Leukemia

http://dx.doi.org/10.5772/54652

59

(Aydin et al, 2006; Gallegos et al,2008, Lordelo et al, 2011)

Secondary hematological malignancies represent a serious complication of cancer treatment. Usually manifiest as acute leukemia and MDS, and known more about these could eventually help reduce its appearance (Levine and Blomifield, 1992). It is known that this type of leuke‐ mias may arise as a result of exposure to cytotoxic treatments (with genotoxicity secondary effects) and/or radiotherapy (RT) and as a result of other hematological disorders (Harris et al, 1999; Brunning et al, 2001), and possibly also, product of environment or genetic causes. (Levine and Blomifield, 1992) In most cases it is proposed that the mechanism of leukemo‐ genesis is associated with DNA damage in hematopoietic cells of the bone marrow by agents such as those used in chemotherapy (CT). (Levine and Blomifield, 1992) Although most sec‐ ondary leukemias are acute AML, there have been reports of lymphoid leukemia and CML is associated with CT. (Andersen et al, 2000; Krishnan et al, 2000; Pedersen-Bjergaard et al, 2002)

**Secondary leukemias**

#### **Thymidylate Synthase (TYMS)**

Thymidylate synthase (TS) has been shown to moderate the risk of hematological malignan‐ cies. (Valiket al, 2004; Lightfoot et al, 2005) Although, it has been proposed that arise by genetics and environmental factors. (Krajinovic et al, 2004) Consistent with this paradigm, variants of genes involved in xenobiotic metabolism, DNA repair pathway and cell cycle checkpoint functions, have been shown to influence the susceptibility to ALL. (Pui, 2009) Many enzymes are involved in the folate metabolism, among which, thymidylate synthase (TS) is a crucial enzyme and hence a good candidate for studying the effect of polymorphisms in the folate metabolism gene, on the development of malignancies. TS, encoded by the *TS,* gene located on chromosome 18p11.32, plays a vital role in maintaining a balanced supply of deoxynucleo‐ tides, required for DNA synthesis and repair, by catalyzing the conversion of dUMP to dTMP. (Nazki et al, 2012)

The polymorphisms in *TYMS* gene include a 6-bp deletion (1494del6), in the 3′-untranslated region of *TS* that influences RNA levels; and a polymorphic tandem 28-bp repeat sequence within the promoter enhancer region of *TS*, where the triple repeat increases gene expression levels and reduces DNA damage. In fact, it is thought that the input of deoxynucleotides for DNA synthesis is controlled by TYMS, which has a polymorphic tandem repeat sequence within the promoter enhancer region containing a double (2R)or triple (3R) 28–bp repeat. The presence of the triple repeat leads to increased levels of gene expression and a reduction in DNA damage. (Skibola et al, 2004; Lightfoot et al, 2010) Methotrexate, an antifolic acid agent, has demonstrated to be an effective chemotherapeutic drug for the treatment of lymphoid malignancies, indicating an association between the folate metabolism and the development of such malignancies. (Hishidaet al, 2003) This increased expression may, in turn, increase the conversion of dUMP to dTMP, thereby; decreasing uracil levels and the consequent erroneous incorporation of uracil into DNA of rapidly dividing hematopoietic stem cells, and could work protectively against the development of ALL. (Skibola et al, 2004) The TS 28-bp repeat poly‐ morphism has been shown to modulate the risk of ALL in various populations, but the ob‐ tained results are controversial and require further investigation to be confirmed and clarified. (Skibola et al, 2004; deJonge et al, 2009)

#### **Polymorphisms in the xenobiotics metabolism**

An ability that man has acquired in the course of evolution is the way to metabolize foreign compounds for the body to facilitate disposal. These compounds are called xenobiotics, in food and the environment are mostly lipophilic, so it tends to accumulate in lipophilic environments the body and are difficult to remove so they tend to trigger toxicity phenomena. (Gonzalez and Gelboin, 1994) The liver removes lipophilic xenobiotics, through a set of known reactions of biotransformation. The end result is the formation of metabolites less lipophilic and more soluble, which are easily eliminated in the urine or bile compounds. That is why these reactions are known as detoxification or deactivation. (DeAnn, 1998) In this sense, drugs are a class of compounds that are absorbed xenobiotics on the body and distributed in body fluids, tissue and organ. Where exert their pharmacological action and pharmacodynamics specified; only a small part reaches the tissue-receptor-target enzyme, while most are metabolized and elim‐ inated. (Sheweita, 2000) Moreover, the processes of biotransformation of xenobiotics are sub‐ divided into two phases: phase I metabolism, carried out by two families of enzymes oxygenases: those dependent on cytochrome P450 (CYP450) monooxygenases and the flavin (FMO). Their metabolism is characterized by the action of chemical processes of different na‐ ture mainly oxidation, oxygenation, reduction and hydrolysis, so as dealquilations and deha‐ logenations. These chemical reactions produce metabolites capable of binding covalently to endogenous molecules such as glucuronic acids, glutathione, sulfate and amino acids that generate conjugates, which are metabolized by Phase II which is characterized by solubility in generating molecules and decreased toxicity, generated by the modification of new functional groups, which transform the more polar metabolite, which facilitates their removal. In this regard, when a drug enters the body usually is modified by conjugation reactions to be easily removed. However, when there modifications in the concentrations concentration (high or low) of enzymes that perform the conjugation process and if the xenobiotic is lipophilic nature, tends to accumulate in the cell, which will lead to different processes: 1) accumulation of re‐ active metabolites adduct forming with DNA, 2) formation of a toxic compound 3) a nontoxic compound becomes a toxic derivative. This generates secondary metabolic pathways may have a carcinogenic action, toxicological, genotoxic or mutagenic in the body. (Marmiroli and Maestri, 2008) Different studies in the literature have suggested the association of polymor‐ phisms in genes involved in xenobiotic metabolism phase I and II in patients with leukemia. (Aydin et al, 2006; Gallegos et al,2008, Lordelo et al, 2011)

#### **Secondary leukemias**

**Serine Hydroxymethyltransferase 1 (SHMT1)**

58 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

Jonge et al, 2009)

(Nazki et al, 2012)

(Skibola et al, 2004; deJonge et al, 2009)

**Polymorphisms in the xenobiotics metabolism**

**Thymidylate Synthase (TYMS)**

Cytosolic SHMT1 regulates 5,10-MeTHF,that acts as substrate for MTHFR. The 1420C>T poly‐ morphism of this gene reduces circulating folate levels, and may mimic folate deficiency, con‐ sequently shunting 5,10-MeTHF towards DNA synthesis, and have been shown that moderate the risk of hematological malignancies. (Lightfoot et al, 2005) Folate is a component important in the development of the embryogenesis and early fetal development, via its effects on DNA methylation and synthesis. Then, the well-documented in utero origin of ALL has led to hy‐ pothesize that deficient folate intake may be important in its etiology. (Lightfoot et al, 2005; de

Thymidylate synthase (TS) has been shown to moderate the risk of hematological malignan‐ cies. (Valiket al, 2004; Lightfoot et al, 2005) Although, it has been proposed that arise by genetics and environmental factors. (Krajinovic et al, 2004) Consistent with this paradigm, variants of genes involved in xenobiotic metabolism, DNA repair pathway and cell cycle checkpoint functions, have been shown to influence the susceptibility to ALL. (Pui, 2009) Many enzymes are involved in the folate metabolism, among which, thymidylate synthase (TS) is a crucial enzyme and hence a good candidate for studying the effect of polymorphisms in the folate metabolism gene, on the development of malignancies. TS, encoded by the *TS,* gene located on chromosome 18p11.32, plays a vital role in maintaining a balanced supply of deoxynucleo‐ tides, required for DNA synthesis and repair, by catalyzing the conversion of dUMP to dTMP.

The polymorphisms in *TYMS* gene include a 6-bp deletion (1494del6), in the 3′-untranslated region of *TS* that influences RNA levels; and a polymorphic tandem 28-bp repeat sequence within the promoter enhancer region of *TS*, where the triple repeat increases gene expression levels and reduces DNA damage. In fact, it is thought that the input of deoxynucleotides for DNA synthesis is controlled by TYMS, which has a polymorphic tandem repeat sequence within the promoter enhancer region containing a double (2R)or triple (3R) 28–bp repeat. The presence of the triple repeat leads to increased levels of gene expression and a reduction in DNA damage. (Skibola et al, 2004; Lightfoot et al, 2010) Methotrexate, an antifolic acid agent, has demonstrated to be an effective chemotherapeutic drug for the treatment of lymphoid malignancies, indicating an association between the folate metabolism and the development of such malignancies. (Hishidaet al, 2003) This increased expression may, in turn, increase the conversion of dUMP to dTMP, thereby; decreasing uracil levels and the consequent erroneous incorporation of uracil into DNA of rapidly dividing hematopoietic stem cells, and could work protectively against the development of ALL. (Skibola et al, 2004) The TS 28-bp repeat poly‐ morphism has been shown to modulate the risk of ALL in various populations, but the ob‐ tained results are controversial and require further investigation to be confirmed and clarified.

An ability that man has acquired in the course of evolution is the way to metabolize foreign compounds for the body to facilitate disposal. These compounds are called xenobiotics, in food Secondary hematological malignancies represent a serious complication of cancer treatment. Usually manifiest as acute leukemia and MDS, and known more about these could eventually help reduce its appearance (Levine and Blomifield, 1992). It is known that this type of leuke‐ mias may arise as a result of exposure to cytotoxic treatments (with genotoxicity secondary effects) and/or radiotherapy (RT) and as a result of other hematological disorders (Harris et al, 1999; Brunning et al, 2001), and possibly also, product of environment or genetic causes. (Levine and Blomifield, 1992) In most cases it is proposed that the mechanism of leukemo‐ genesis is associated with DNA damage in hematopoietic cells of the bone marrow by agents such as those used in chemotherapy (CT). (Levine and Blomifield, 1992) Although most sec‐ ondary leukemias are acute AML, there have been reports of lymphoid leukemia and CML is associated with CT. (Andersen et al, 2000; Krishnan et al, 2000; Pedersen-Bjergaard et al, 2002) The AML are hematologic malignancies characterized by the uncontrolled myeloid blast pro‐ liferation in the bone marrow and in peripheral tissues. (Sevilla et al, 2002) Differ according to the cytological, immunophenotypic and cytogenetic characteristics. (Paietta, 1995; Head, 1996) On the other hand the MDS are dis-hematopoietics processes of bone marrow, characterized by alteration in the maturation and differentiation of hematopoietic cell lines (with involve‐ ment of one, two or all three blood cell lineages) and in some cases, by the presence of bone marrow blasts, without showing acute leukemia criteria. (Bennet et al, 1982; Cheson, 1997)

terations of chromosomes 5 and 7, (-5/5q- and -7/7q-) frequently refractory to treatment. (An‐

Pathophysiology of Acute Lymphoblastic Leukemia

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61

Those in patients treated with drugs that inhibit DNA-topoisomerase II as epipodophyllotox‐ ins and anthracyclines, with a short latency period of between 2 to 3 years, without prior myelodysplasia phase and in which observed balanced translocations as 11q23 (MLL) and 21q22 (AML1/RUNX1), although there are some discrepancies. The treatment response is not

Leukemias have been associated with exposure to various agents as benzene and its metab‐ olites (phenol, hydroquinone), environmental carcinogens demonstrated relationship to in‐ creased risk of developing leukemia. (Lan et al, 2004) Other compounds studied with controversial relationship, are some agricultural pesticides, heavy metals, smoke snuff, alco‐ hol intake and exposure to cosmic rays of airlines pilots. (Larson, 2007) Ionizing radiation is a known leukemogenic agent and the main action mechanism includes the breakage of the DNA strands which can cause the aforementioned chromosomal deletions and translo‐ cations. Breaks may result from a direct effect of high doses of radiation or indirectly by free radicals generation. Furthermore, ionizing radiation can induce changes of bases in the DNA sequences, crosslinking strands, multiple damages or epigenetic alterations (Finch, 2007). In the case of RT, induced leukemia seems to start in the first 5 years after expo‐ sure, peaks at 10 years and decreases significantly after 15 years. The relative incidence of leukemias by RT is dose dependent, duration of exposure, and area of exposed bone mar‐ row. (Finch, 2007) Fractionated doses of radiation are less leukemogenic than higher sin‐ gle doses, because it allows greater efficiency of DNA repair mechanisms. The leukemogenic risk appears to be greater when low-dose exposure affects large areas of bone marrow, whereas high doses of radiation over limited areas appear to have less effect. This is attrib‐ uted to increased apoptosis induced by high doses of radiation on cells exposed. (Inskip, 1999; Smith et al, 2002) Estimates show, that patients undergoing RT for the treatment of malignancies or other non-malignant diseases has a risk of two to three times more to de‐ velop AML-PT/MDS-PT. (Smith et al, 2002) On the other hand, a younger ages at the time of exposure, greater the leukemogenic risk. The increased risk for developing AML-PT/ MDS-PT, after treatment of non Hodkin lymphomas, breast cancer, cervical cancer and ute‐ rine body and Ewing's sarcoma, is attributed to the use of RT, while associated with Hodg‐ kin's disease, ovarian cancer and testicular cancer, has been associated with the use of CT. (Levine and Blomifield, 1992; van Leeuwen et al, 1994; Inskip, 1999; Smith et al, 2002)

Many drugs used as CT in the treatment of a primary cancer, have been linked to the subse‐ quent development of AML-PT/MDS-PT, thus, alkylating agents were the first to be evidenced leukemogenic potential, (Pedersen, 2002) effect related to the cumulative dose of the drug, the effect being greater with increasing patient age (Pedersen, 2002). All alkylating agents have effect leukemogenic, such is the case of drugs such as mechlorethamine, procarbazine, chlor‐ ambucil, cyclophosphamide, melphalan, semustine, lomustine, carmustine, prednimustine, busulfan. However, although the relative risk leukemogenic of these drugs has not been de‐ finitively established, drugs such melphalan and busulfan seem to condition an increased riesk than others as cyclophosphamide for reason which are unknown. (Stott et al, 1977; Greene et

significantly different from those of patients with *de novo* AML. (Penderse, 2002)

dersen and Pedersen, 2000)

The term secondary leukemia has referred to the development of AML as result of CT treat‐ ment, particularly alkylating agents (Levine and Blomifield, 1992) or topoisomerase II inhib‐ itors, RT, or by exposure to environmental carcinogens (Harris et al, 1999; Brunning et al, 2001). Within the term of secondary acute leukemias (SAL) different entities are grouped by etiopathogenesis, prognosis and response to therapy. In general can be distinguished two groups: those that are a result of exposure to cytotoxic treatments such as CT and/or RT and those that are a result of the final evolution of other hematological disorders, such as, myeloproliferative syndromes, MDS, paroxysmal nocturnal hemoglobinuria. (Harris et al, 1999; Brunning et al, 2001)

In close relationship with these two groups of SAL also found leukemias result from environ‐ mental or occupational exposure to carcinogens, (Levine and Blomifield, 1992) or those that develop in patients with genetic disorders as chromosomal fragility syndromes such as Fan‐ coni anemia and Bloom syndrome (Popp and Bohlander, 2010)

The increase is due to the increased number of survivors of other forms of cancer, (Ng et al, 2000) is important to know more about SAL, especially AML or MDS relating to previous therapies (AML-PT, MDS-PT). The cumulative risk of developing AML-PT/MDS-PT after ten years of receiving CT for breast cancer, non-Hodgkin´s lymphoma, ovarian cancer or Hodgkin ´s disease, has been estimated at 1.5, 7.9, 8.5, and 3.8% respectively. (Bolufer et al, 2006) More‐ over, generally the cases of AML-PT/MDS-PT, the primary disease may be a solid tumor, other haematological malignancy or non-malignant disorder.

Today it is clear that AML-PT/MDS-PT can develop after exposure to cytotoxic CT with alky‐ lating agents, topoisomerase II inhibitors, and/or RT, for the treatment of other neoplasias or in treating non-malignant disorders. In this sense, has been described after the use of immu‐ nosuppressants such as azathioprine (Harris et al, 1999; Brunning et al, 2001). While the study of these entities has been important, the maximum latency between exposure to leukemogenic agent and the development of AML-PT/MDS-PT has not been established with certainty.

Due to history of exposure to certain agents and the association with some cytogenetic abnor‐ malities characteristic, two groups are recognized in AML-PT/MDS-PT: (Harris et al, 1999; Brunning et al, 2001)

Which appears as a result of the mutagenic effect of alkylating agents treatment, ionizing ra‐ diation or both, that occur after a long latency period between 5 to 7 years of exposure to cytotoxic agents and often show a phase of myelodysplasia prior to the evolution to AL, and often show a phase of myelodysplasia prior to the evolution to AL, which often produce al‐ terations of chromosomes 5 and 7, (-5/5q- and -7/7q-) frequently refractory to treatment. (An‐ dersen and Pedersen, 2000)

The AML are hematologic malignancies characterized by the uncontrolled myeloid blast pro‐ liferation in the bone marrow and in peripheral tissues. (Sevilla et al, 2002) Differ according to the cytological, immunophenotypic and cytogenetic characteristics. (Paietta, 1995; Head, 1996) On the other hand the MDS are dis-hematopoietics processes of bone marrow, characterized by alteration in the maturation and differentiation of hematopoietic cell lines (with involve‐ ment of one, two or all three blood cell lineages) and in some cases, by the presence of bone marrow blasts, without showing acute leukemia criteria. (Bennet et al, 1982; Cheson, 1997)

60 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

The term secondary leukemia has referred to the development of AML as result of CT treat‐ ment, particularly alkylating agents (Levine and Blomifield, 1992) or topoisomerase II inhib‐ itors, RT, or by exposure to environmental carcinogens (Harris et al, 1999; Brunning et al, 2001). Within the term of secondary acute leukemias (SAL) different entities are grouped by etiopathogenesis, prognosis and response to therapy. In general can be distinguished two groups: those that are a result of exposure to cytotoxic treatments such as CT and/or RT and those that are a result of the final evolution of other hematological disorders, such as, myeloproliferative syndromes, MDS, paroxysmal nocturnal hemoglobinuria. (Harris et

In close relationship with these two groups of SAL also found leukemias result from environ‐ mental or occupational exposure to carcinogens, (Levine and Blomifield, 1992) or those that develop in patients with genetic disorders as chromosomal fragility syndromes such as Fan‐

The increase is due to the increased number of survivors of other forms of cancer, (Ng et al, 2000) is important to know more about SAL, especially AML or MDS relating to previous therapies (AML-PT, MDS-PT). The cumulative risk of developing AML-PT/MDS-PT after ten years of receiving CT for breast cancer, non-Hodgkin´s lymphoma, ovarian cancer or Hodgkin ´s disease, has been estimated at 1.5, 7.9, 8.5, and 3.8% respectively. (Bolufer et al, 2006) More‐ over, generally the cases of AML-PT/MDS-PT, the primary disease may be a solid tumor, other

Today it is clear that AML-PT/MDS-PT can develop after exposure to cytotoxic CT with alky‐ lating agents, topoisomerase II inhibitors, and/or RT, for the treatment of other neoplasias or in treating non-malignant disorders. In this sense, has been described after the use of immu‐ nosuppressants such as azathioprine (Harris et al, 1999; Brunning et al, 2001). While the study of these entities has been important, the maximum latency between exposure to leukemogenic agent and the development of AML-PT/MDS-PT has not been established with certainty.

Due to history of exposure to certain agents and the association with some cytogenetic abnor‐ malities characteristic, two groups are recognized in AML-PT/MDS-PT: (Harris et al, 1999;

Which appears as a result of the mutagenic effect of alkylating agents treatment, ionizing ra‐ diation or both, that occur after a long latency period between 5 to 7 years of exposure to cytotoxic agents and often show a phase of myelodysplasia prior to the evolution to AL, and often show a phase of myelodysplasia prior to the evolution to AL, which often produce al‐

coni anemia and Bloom syndrome (Popp and Bohlander, 2010)

haematological malignancy or non-malignant disorder.

al, 1999; Brunning et al, 2001)

Brunning et al, 2001)

Those in patients treated with drugs that inhibit DNA-topoisomerase II as epipodophyllotox‐ ins and anthracyclines, with a short latency period of between 2 to 3 years, without prior myelodysplasia phase and in which observed balanced translocations as 11q23 (MLL) and 21q22 (AML1/RUNX1), although there are some discrepancies. The treatment response is not significantly different from those of patients with *de novo* AML. (Penderse, 2002)

Leukemias have been associated with exposure to various agents as benzene and its metab‐ olites (phenol, hydroquinone), environmental carcinogens demonstrated relationship to in‐ creased risk of developing leukemia. (Lan et al, 2004) Other compounds studied with controversial relationship, are some agricultural pesticides, heavy metals, smoke snuff, alco‐ hol intake and exposure to cosmic rays of airlines pilots. (Larson, 2007) Ionizing radiation is a known leukemogenic agent and the main action mechanism includes the breakage of the DNA strands which can cause the aforementioned chromosomal deletions and translo‐ cations. Breaks may result from a direct effect of high doses of radiation or indirectly by free radicals generation. Furthermore, ionizing radiation can induce changes of bases in the DNA sequences, crosslinking strands, multiple damages or epigenetic alterations (Finch, 2007). In the case of RT, induced leukemia seems to start in the first 5 years after expo‐ sure, peaks at 10 years and decreases significantly after 15 years. The relative incidence of leukemias by RT is dose dependent, duration of exposure, and area of exposed bone mar‐ row. (Finch, 2007) Fractionated doses of radiation are less leukemogenic than higher sin‐ gle doses, because it allows greater efficiency of DNA repair mechanisms. The leukemogenic risk appears to be greater when low-dose exposure affects large areas of bone marrow, whereas high doses of radiation over limited areas appear to have less effect. This is attrib‐ uted to increased apoptosis induced by high doses of radiation on cells exposed. (Inskip, 1999; Smith et al, 2002) Estimates show, that patients undergoing RT for the treatment of malignancies or other non-malignant diseases has a risk of two to three times more to de‐ velop AML-PT/MDS-PT. (Smith et al, 2002) On the other hand, a younger ages at the time of exposure, greater the leukemogenic risk. The increased risk for developing AML-PT/ MDS-PT, after treatment of non Hodkin lymphomas, breast cancer, cervical cancer and ute‐ rine body and Ewing's sarcoma, is attributed to the use of RT, while associated with Hodg‐ kin's disease, ovarian cancer and testicular cancer, has been associated with the use of CT. (Levine and Blomifield, 1992; van Leeuwen et al, 1994; Inskip, 1999; Smith et al, 2002)

Many drugs used as CT in the treatment of a primary cancer, have been linked to the subse‐ quent development of AML-PT/MDS-PT, thus, alkylating agents were the first to be evidenced leukemogenic potential, (Pedersen, 2002) effect related to the cumulative dose of the drug, the effect being greater with increasing patient age (Pedersen, 2002). All alkylating agents have effect leukemogenic, such is the case of drugs such as mechlorethamine, procarbazine, chlor‐ ambucil, cyclophosphamide, melphalan, semustine, lomustine, carmustine, prednimustine, busulfan. However, although the relative risk leukemogenic of these drugs has not been de‐ finitively established, drugs such melphalan and busulfan seem to condition an increased riesk than others as cyclophosphamide for reason which are unknown. (Stott et al, 1977; Greene et al, 1986; Krishnan et al, 2000) Although, this seems to suggest that more genotoxic and cytotoxic drugs are chosen that have less leukemogenic potential. Alkylating agents besides the afore‐ mentioned chromosomal damage can cause point mutations in some oncogenes like RAS. (Pedersen et al, 1988) However, these effects are no restricted to certain genes or chromosomal regions and possibly the selection of cells with abnormalities of chromosomes 5 and 7 come conditioned by a proliferative advantage to cells carrying these alterations. (Johansson et al, 1991; de Greef and Hagemeiger, 1996; Andersen et al, 2000)

response to CT commonly used in the treatment of AML and therefore have a significantly

Different authors have been relationship to drugs and radiation with specific emphasis on the balanced rearrangements chromosomes. (Andersen et al, 1998) Increased frequency of dicen‐ tric chromosomes in therapy-related MDS and AML compared to de novo disease is signifi‐ cantly related to previous treatment with alkylating agents and suggests a specific susceptibility to chromosome breakage at the centromere. (Andersen and Pedersen, 2000)

In this way one can conclude that the pathophysiology of acute lymphoblastic leukemia is very complex and involves various factors (genetic, immunes, environmental, and drugs) at dif‐ ferent levels, and also has a close and complex relationship. The key features in the patho‐ physiology of the ALL is its monoclonal origin, uncontrolled cell proliferation by sustained self-stimulation of their receptors for growth, no response to inhibitory signals, and cellular

1LaboratoriodeGenéticaMolecular,DivisióndeMedicinaMolecular,CIBO-IMSS,Guadalajara,

2 Laboratorio de Mutagénesis, División de Medicina Molecular, CIBO-IMSS, Guadalajara, Jal.,

3 División de Genética, CIBO-IMSS, &UMAE-Hospital de Especialidades, Servicio de Hema‐

[1] Andersen MK, Johansson B, Larsen SO, Pedersen-Bjergaard J, Chromosomal abnor‐ malities in secondary MDS and AML. (1998) Relationship to drugs and radiation with specific emphasis on the balanced rearrangements. Haematologica. 83(6):483–488.

5 Laboratorio de Inmunofarmacologia, CUCEI, UdeG, Guadalajara, Jal., México

, G. M. Zúñiga-González2

, L. E. Figuera4

Pathophysiology of Acute Lymphoblastic Leukemia

http://dx.doi.org/10.5772/54652

63

,

adverse prognosis.

**3. Conclusion**

**Author details**

M. P. Gallegos-Arreola1

A. M. Puebla-Pérez5

Jal., México

**References**

México

longevity conditioned by decreased apoptosis.

tología, CIBO-IMSS, Guadalajara, Jal., México

4 División de Genética, CIBO-IMSS, Guadalajara, Jal., México

, C. Borjas-Gutiérrez3

and J. R. García-González4

Meanwhile, drugs that act by inhibiting topoisomerase II, as the epipodophyllotoxins (etopo‐ side and teniposide) ( et al, 1999) and intercalating agents such as anthracyclines (doxorubicin, daunorubicin, idarubicin) o las anthracenediones (mitoxantrone), have been associated with balanced translocations that originates function genes. The most common affect 11q23, 21q22, inv(16) and t(15;17) (Andersen et al, 1998; Rowley and Olney, 2002). Recently high-dose CT followed by autologous hematopoietic transplantation, have been associated with the devel‐ opment of AML-PT/MDS-PT, with time of onset of 47-50 months after transplantation. (Na‐ demanee et al, 1995; Traweek et al, 1996; Krishnan et al, 2000; Pedersen et al, 2000; Gilliland and Gribben, 2002)

An important point to consider is individual susceptibility, since only a minority of patients develop secondary leukemia after exposure to CT, therefore it is suggested that differences in drug metabolism may predispose to the development of AML-PT/MDS-PT of some patients. (Bolufer et al, 2006) In this way, polymorphisms of genes encoding enzymes involved in drug metabolism could contribute to the risk of developing these pathologies. These genes could explain differences in metabolizing of these agents and condition a lower detoxification ca‐ pacity or repair of genetic damage induced by the drug. (Bolufer et al, 2006) Genes have been studied encoding cytochrome P450 (CYPs) related to phase I metabolism, glutathione S-meth‐ yltransferase (GSTT1, GSTM1, GSTP1) involved in phase II metabolism (conjugation/detoxi‐ fication), the NAD(P)H: qinone oxo-reductase 1 (NQO1) which acts on the metabolism of free radicals and oxidative stress, genes related to folate metabolism (MTHFR, TYMS, SHMT1, MTRR), also involved in DNA synthesis and genes related to DNA repair (hMLH1, hMSH2, hMSH3, RAD51, XRCC1, XRCC3, XPD, XPG, CHEK2, and ATM) that can cause genomic in‐ stability. (Bolufer et al, 2006)

The AML-PT/MDS-PT pathogenesis includes clonal alterations in the some genes function due to single mutations, chromosomal abnormalities or epigenetic phenomena. Many of the altered genes are tumor suppressor that have a recessive character and therefore, requires the loss of both alleles. The loss of a single copy of the gene can result in reduction of gene products and predispose to malignancy. Current evidence indicates that AML results from at least two mu‐ tations classes. The class I, confers proliferative advantage and/or cell survival without affect‐ ing their differentiation capacity, while the class II, prevent the normal hematopoietic cell differentiation. (Deguchi and Gilliland, 2002)

The AML-PT represent 10 to 15% of total AML and its incidence is increasing substantially in recent years, (Ng et al, 2000) the AML-PT are often associated with clonal cytogenetic abnor‐ malities similar to those found in newly diagnosed AML, but with higher incidence of poor prognosis karyotypes and have particular clinical and biological features that include a poor response to CT commonly used in the treatment of AML and therefore have a significantly adverse prognosis.

Different authors have been relationship to drugs and radiation with specific emphasis on the balanced rearrangements chromosomes. (Andersen et al, 1998) Increased frequency of dicen‐ tric chromosomes in therapy-related MDS and AML compared to de novo disease is signifi‐ cantly related to previous treatment with alkylating agents and suggests a specific susceptibility to chromosome breakage at the centromere. (Andersen and Pedersen, 2000)

## **3. Conclusion**

al, 1986; Krishnan et al, 2000) Although, this seems to suggest that more genotoxic and cytotoxic drugs are chosen that have less leukemogenic potential. Alkylating agents besides the afore‐ mentioned chromosomal damage can cause point mutations in some oncogenes like RAS. (Pedersen et al, 1988) However, these effects are no restricted to certain genes or chromosomal regions and possibly the selection of cells with abnormalities of chromosomes 5 and 7 come conditioned by a proliferative advantage to cells carrying these alterations. (Johansson et al,

Meanwhile, drugs that act by inhibiting topoisomerase II, as the epipodophyllotoxins (etopo‐ side and teniposide) ( et al, 1999) and intercalating agents such as anthracyclines (doxorubicin, daunorubicin, idarubicin) o las anthracenediones (mitoxantrone), have been associated with balanced translocations that originates function genes. The most common affect 11q23, 21q22, inv(16) and t(15;17) (Andersen et al, 1998; Rowley and Olney, 2002). Recently high-dose CT followed by autologous hematopoietic transplantation, have been associated with the devel‐ opment of AML-PT/MDS-PT, with time of onset of 47-50 months after transplantation. (Na‐ demanee et al, 1995; Traweek et al, 1996; Krishnan et al, 2000; Pedersen et al, 2000; Gilliland

An important point to consider is individual susceptibility, since only a minority of patients develop secondary leukemia after exposure to CT, therefore it is suggested that differences in drug metabolism may predispose to the development of AML-PT/MDS-PT of some patients. (Bolufer et al, 2006) In this way, polymorphisms of genes encoding enzymes involved in drug metabolism could contribute to the risk of developing these pathologies. These genes could explain differences in metabolizing of these agents and condition a lower detoxification ca‐ pacity or repair of genetic damage induced by the drug. (Bolufer et al, 2006) Genes have been studied encoding cytochrome P450 (CYPs) related to phase I metabolism, glutathione S-meth‐ yltransferase (GSTT1, GSTM1, GSTP1) involved in phase II metabolism (conjugation/detoxi‐ fication), the NAD(P)H: qinone oxo-reductase 1 (NQO1) which acts on the metabolism of free radicals and oxidative stress, genes related to folate metabolism (MTHFR, TYMS, SHMT1, MTRR), also involved in DNA synthesis and genes related to DNA repair (hMLH1, hMSH2, hMSH3, RAD51, XRCC1, XRCC3, XPD, XPG, CHEK2, and ATM) that can cause genomic in‐

The AML-PT/MDS-PT pathogenesis includes clonal alterations in the some genes function due to single mutations, chromosomal abnormalities or epigenetic phenomena. Many of the altered genes are tumor suppressor that have a recessive character and therefore, requires the loss of both alleles. The loss of a single copy of the gene can result in reduction of gene products and predispose to malignancy. Current evidence indicates that AML results from at least two mu‐ tations classes. The class I, confers proliferative advantage and/or cell survival without affect‐ ing their differentiation capacity, while the class II, prevent the normal hematopoietic cell

The AML-PT represent 10 to 15% of total AML and its incidence is increasing substantially in recent years, (Ng et al, 2000) the AML-PT are often associated with clonal cytogenetic abnor‐ malities similar to those found in newly diagnosed AML, but with higher incidence of poor prognosis karyotypes and have particular clinical and biological features that include a poor

1991; de Greef and Hagemeiger, 1996; Andersen et al, 2000)

62 Clinical Epidemiology of Acute Lymphoblastic Leukemia - From the Molecules to the Clinic

and Gribben, 2002)

stability. (Bolufer et al, 2006)

differentiation. (Deguchi and Gilliland, 2002)

In this way one can conclude that the pathophysiology of acute lymphoblastic leukemia is very complex and involves various factors (genetic, immunes, environmental, and drugs) at dif‐ ferent levels, and also has a close and complex relationship. The key features in the patho‐ physiology of the ALL is its monoclonal origin, uncontrolled cell proliferation by sustained self-stimulation of their receptors for growth, no response to inhibitory signals, and cellular longevity conditioned by decreased apoptosis.

## **Author details**

M. P. Gallegos-Arreola1 , C. Borjas-Gutiérrez3 , G. M. Zúñiga-González2 , L. E. Figuera4 , A. M. Puebla-Pérez5 and J. R. García-González4

1LaboratoriodeGenéticaMolecular,DivisióndeMedicinaMolecular,CIBO-IMSS,Guadalajara, Jal., México

2 Laboratorio de Mutagénesis, División de Medicina Molecular, CIBO-IMSS, Guadalajara, Jal., México

3 División de Genética, CIBO-IMSS, &UMAE-Hospital de Especialidades, Servicio de Hema‐ tología, CIBO-IMSS, Guadalajara, Jal., México

4 División de Genética, CIBO-IMSS, Guadalajara, Jal., México

5 Laboratorio de Inmunofarmacologia, CUCEI, UdeG, Guadalajara, Jal., México

## **References**

[1] Andersen MK, Johansson B, Larsen SO, Pedersen-Bjergaard J, Chromosomal abnor‐ malities in secondary MDS and AML. (1998) Relationship to drugs and radiation with specific emphasis on the balanced rearrangements. Haematologica. 83(6):483–488.

[2] Andersen MK, Pedersen-Bjergaard J. (2000) Increased frecuency of dicentric chromo‐ somes in terapy-related MDS and AML compared to de novo disease is significantly related to previous treatment with alkylating agents and suggests a specific suscepti‐ bility to chromosome breakage at the centromere. Leukemia. 14(1):105–111.

[16] de Greef GE, Hagemeiger A. (1996) Molecular and cytogenetic abnormalities in acute myeloid leukemia and myelodisplastic syndromes. Ballieres Clin Hematol. 9(1):1–18.

Pathophysiology of Acute Lymphoblastic Leukemia

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65

[17] de Jonge R, Tissing WJ, Hooijberg JH, Jansen G, Kaspers GJ, Lindemans J, Peters GJ, Pieters R.(2009) Polymorphisms in folate-related genes and risk of pediatric acute lym‐

[18] DeAnn J. (1998) The detoxification enzyme systems. Alternative Medicine Review. 3

[19] Deguchi K, Gilliland DG. (2002) Cooperativity between mutations in tyrosine kinases and in hematopoietic transcription factors in AML. Leukemia. 16(4):740–744.

[20] Demarest RM, Dahmane N, Capobianco AJ. (2011) Notch is oncogenic dominant in T-

[21] Demarest RM, Ratti F, Capobianco AJ. (2008) It's T-ALL about Notch. Oncogene. 27(38):

[22] Downing JR, Wilson RK, Zhang J, Mardis ER, Pui CH, Ding L, Ley TJ, Evans WE. (2012)

[23] Finch SC. (2007) Radiation-induced leukemia: Lessons from history. Best Pract Res Clin

[24] Fragoso R, Pereira T, Wu Y, Zhu Z, Cabeçadas J, Dias S. (2006) VEGFR-1 (FLT-1) acti‐ vation modulates acute lymphoblastic leukemia localization and survival within the bone marrow, determining the onset of extramedullary disease. Blood. 107(4):1608-16.

[25] Franco RF, Simões BP, Tone LG, Gabellini SM, Zago MA, Falcão RP. (2001) Themethy‐ lenetetrahydrofolatereductase C677T gene polymorphism decreases the risk of child‐

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**Chapter 4**

**Multi-Role of Cancer Stem Cell in**

Dong-qing Wang, Hai-tao Zhu, Yan-fang Liu,

Additional information is available at the end of the chapter

Rui-gen Yin, Liang Zhao, Zhi-jian Zhang, Zhao-liang Su, Yan-Zhu, Hui-qun Lu,

Juan Hong and Jie Zhang

http://dx.doi.org/10.5772/55075

incidence of ALL in the past 25 years.

**1. Introduction**

**Children Acute Lymphoblastic Leukemia**

Acute lymphoblastic leukemia (ALL) is a malignant proliferation of lymphoid precursor cells in the bone marrow blood. It is an age related tumor, with a peak between the ages of 2 and 10 and a second peak after the age of 5. Among children younger than 15 years, ALL represents 23% of cancer that was diagnosed. The children aged 2 to 3 years were a sharp peak in ALL incidence (>80 per million per year).The rates of the ALL among children aged 8 to 10 years incidence decreasing to 20 per million. Moreover, there has been a gradual increase in the

With the development of the medicine, considerable advances have been made in the treatment of childhood ALL. In the 1980's, relapsed ALL was regarded as an incurable disease. However, about 85% of childhoods ALL can hope to achieve a second remission over the last years. Meanwhile, around 40% of these can hope to achieve long term cure. On the other hand, despite optimal therapy, long term survival rate still limited to 30–40% of patients and about 15-20% children will sustain relapse. Because of the high relapse rate, refractoriness to conventional treatment protocols, the incidence of chemotherapy-related deaths, the complete remission rate, numerous challenges remained in the management of ALL, especially the children with re‐ lapsedALL.Also,thediseasemechanismismulti-factorialsandinvolvesindifferentgeneticand environmental factors. So, ALL is still a problem that clinic must face up to. However, the emergency of cancer stem cell seems give us a new direction for deeper recognize this disease.

> © 2013 Wang et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Chapter 4**
