**1.4.1 Patient-related factors**

Age, comorbidities, performance status and genetic variation in the drug metabolism are the main prognostic factors related to patients with AML. Increasing age is an important independent adverse prognostic factor (Appelbaum et al., 2006). Nonetheless, calendar age alone should not be a reason for not offering potentially curative therapy to an older patient because age is not the most important prognostic factor for either TRM or resistance to therapy. Currently all patients under the age of 60 are candidates to receive standard intensive chemotherapy and according to prognostic factors stem cell transplantation or intensive chemotherapy as postremission therapy. It has to be stressed that age as a factor is not only dependent on so-called "calendar age". Recently many older patients with a good clinical status have been successfully treated with intensive chemotherapy. Attention should be given to a careful evaluation and documentation of comorbidities. Comorbidity scoring is a current field of investigation and should contribute to a better definition of the patient considered "unfit" for intensive chemotherapy (Piccirillo et al., 2004; Sorror et al., 2005).

#### **1.4.2 AML-related factors**

According to the AML working party of European Leukemia Net, several important and independent prognostic factors have been recognized: white blood cell counts, existence of prior MDS or AML with MDS features, previous cytotoxic therapy for another malignancy, and cytogenetic and molecular abnormalities in leukemic cells (Döhner et al., 2010).

#### **1.4.2.1 Cytogenetics**

Chromosome abnormalities are detected in approximately 55% of adult AML. Although, there is a diversity of cytogenetic entities of AML, the karyotype of the leukemic cells is the strongest prognostic factor for response to induction therapy and for survival for AML patients

Immunophenotyping of the Blast Cells in Correlations with

characterized(Freeman et al., 2008).

5 years survival rates of up to 40%. (Appelbaum et al, 2001).

**1.5 Treatment** 

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 483

The parallel progress of the development of the diagnostic techniques improvement of the classification and therapeutical approach lead acute leukemia which were for the first time described before 150 years, and in the 1970s were still fatal disease nowadays to have overall

Despite heterogeneity of the disease, with the exception of acute promyelocytic leukemia, this disease has been treated with a "one size fits all" approach. Although, the vast majority of AML patients can be individually characterized on the basis of the distinct chromosomal aberrations and molecular markers, treatment of AML is still based on quite unspecific cytotoxic therapy. For almost 40 years, the use of continuous infusion of cytarabine combined with another agent, usually an anthracycline, the "3+7" regimen, has been the mainstay of therapy (Yates et al, 1973). Response rates for induction with standard chemotherapy ranged from 70% to 80% for adults aged less than 60years which are enrolled in clinical trials and average in 50% for patients older than 60 years (Lichtman et al.,2010) . Alternative consolidation therapies by applying additional chemotherapy, autologous stem cell transplantation (autoSCT) or alloSCT based on the initial cytogenetic and molecular studies are available (Cornelissen J.J.et al.(2007); Fernandez H.F.,2010; Koreth et al, 2009) As more sophisticated molecular techniques have become available, it is clear that it is still possible to detect residual disease when all morphological and functional criteria for remission are met. Techniques such as "real–time"-quantitative polymerase chain reaction (RQ-PCR) are capable of detection at a level of 1 in 104 or 1 in 105 residual cells, but such markers are available for only a minority of cases in which the molecular lesion has been

AlloSCT is the most effective antileukemic modality that is characterized by immune mediated graft-versus-leukemia effect of the transplanted cells that reduce the risk of relapse considerably and improve the relapse-free survival but also is associated with the increased risk of death and morbidity. Therefore, the alloSCT advantage has to be carefully balanced against the excess mortality (ranging between 10% and 40%) and morbidity due to transplantrelated complications, such as infection and graft-versus-host disease that are typically connected with alloSCT and can diminish all of the benefit of a reduced risk of relapse. (Cornelissen et al., 2007). For this reason, allogeneic stem cell transplantation is usually avoided in a type of AML that has a pattern of cytogenetics with a relatively favorable prognosis, such as AML with the chromosomal translocations t(8;21) or inv(16)/t(16;16)(Marcucci et al., 2000; Perea et al.,2006). In the latter subtypes the risk of relapse is in the order of 35% to 40% or less. By contrast, a transplant is treatment of choice for all other patient whose leukemia cells bear a cytogenetic or molecular abnormality that predicts a high or intermediate risk of relapse after chemotherapy (Löwenberg et al., 2008b; Koreth et al, 2009). Exception could be done for two additional genotypically defined subsets of AML that are categorized as low-risk within the large category of CN-AML. Each of those entities has a risk of relapse of about 35%. The first genotype is defined by the presence of 'favorable' mutations in *NPM1* and the absence of concurrent 'unfavorable' *FLT3*-internal tandem duplications (*NPM1*mut /*FLT3*-ITDneg).61 This genotype accounts for approximately 16% of all newly diagnosed patients younger than 60 years old. There is no demonstrable benefit from transplantation in patients with *NPM1*mut/*FLT3*-ITDneg AML. The second subset of AML with 'favorable' mutations in the transcription factor gene *CEBPA* (*CEBPA*mut) could not be analyzed in this way because of a lack of statistical power due to a limited number of cases. The latter low-risk subtype *CEBPA*mut accounts for 8% of all AML. Nevertheless, the

(Swerdlow et al., 2008). Younger adult patients are commonly categorized into 3 risk groups, favorable, intermediate, or adverse. The favorable group is represented by the reciprocal translocations t(15;17)/PML/RAR, t(8;21)/AML1/ETO, and inv(16)/CBFMYH11, which are associated with a favorable prognosis In contrast, AML associated with t(9;11)(p22;q23) shows an inferior prognosis. These subgroups represent the first hierarchy of the WHO classification of AML, emphasizing that these subtypes represent distinct biologic entities. The second subgroup of AML patients shows a normal karyotype and an intermediate prognosis. However, from molecular aspects, this subgroup is very heterogeneous. Lately, there are emerging data suggesting that two genetics entities from this group, the AML with mutation of NPM1 gene without FLT3 mutations and AML with mutation in CEBPA gene should be moved from the intermediate prognosis group to the favorable group of AML. Those two entities are also added to the new WHO classification (Schlenk et al., 2008). The third (prognostically unfavorable) subgroup includes mostly unbalanced karyotypes characterized by a gain or loss of larger chromosomal regions. Within these, an especially complex aberrant karyotype, which occurs in 10% to 12% of patients and that is defined by ≥3 chromosomal anomalies shows a very unfavorable prognosis. Cytogenetics is further helpful to delineate patients with therapy related AML (t-AML), who are classified as third hierarchy in the WHO classification, and developed either after treatment with alkylating agents often associated with cytogenetic aberrations involving 5q-, −7, or p53 and complex aberrant karyotype or are showing MLL/11q23 or other balanced cytogenetic aberrations which are in frequent association to previous treatment with topoisomerase II inhibitors One striking observation is the increasing incidence of adverse versus favorable cytogenetic abnormalities with increasing age. This, at least in part, contributes to the poorer outcome of AML in older adults. (Byrd et al., 2002; Schlenk et al., 2008, Swerdlow et al., 2008)

#### **1.4.2.2 Molecular genetics**

Nowadays, considerable progress has been made in elucidating the molecular pathogenesis of acute leukemias that resulted in identification of new molecular diagnostic and prognostic markers. Gene mutations and deregulated gene expression have been identified that allow us to interpret the genetic diversity within defined cytogenetic groups, in particular the large and heterogeneous group of patients with CN-AML. Risk stratification by molecular markers in patients from the former group of AML plays an increasing role at diagnosis. The most relevant markers, which can be detected alone or in coincidence with other mutation are NPM1 mutations that is detected in approximately 40% of cases, MLL-PTD in 6%, NRAs in 8-10%, CEBPA in 10% and FLT3-TKD mutations in 6% of CN-AML. Thus, a rather of limited number of markers further subclassifies more than 85% of CN-AML. Data from the literature suggest that those markers have different prognosis regarding the outcome of the disease and they all indicate that in the near future molecular screening may allow " targeted allogeneic stem cell transplantation (alloSCT)" in AML patients with normal karyotype (Schlenk et al., 2008; Koreth et al, 2009).

There is a growing list of the new genetic abnormalities with clinical value that are being investigated. These genetic events perturb diverse cellular pathways and functions, and they often confer a profound impact upon the clinical phenotype of the disease and treatment response.

It is anticipated that advances in molecular technology will reveal additional markers that will result in more precise classification of this heterogeneous complex of disorders (Löwenberg B., 2008b; Haferlach T., 2008)

#### **1.5 Treatment**

482 Advances in Cancer Therapy

(Swerdlow et al., 2008). Younger adult patients are commonly categorized into 3 risk groups, favorable, intermediate, or adverse. The favorable group is represented by the reciprocal translocations t(15;17)/PML/RAR, t(8;21)/AML1/ETO, and inv(16)/CBFMYH11, which are associated with a favorable prognosis In contrast, AML associated with t(9;11)(p22;q23) shows an inferior prognosis. These subgroups represent the first hierarchy of the WHO classification of AML, emphasizing that these subtypes represent distinct biologic entities. The second subgroup of AML patients shows a normal karyotype and an intermediate prognosis. However, from molecular aspects, this subgroup is very heterogeneous. Lately, there are emerging data suggesting that two genetics entities from this group, the AML with mutation of NPM1 gene without FLT3 mutations and AML with mutation in CEBPA gene should be moved from the intermediate prognosis group to the favorable group of AML. Those two entities are also added to the new WHO classification (Schlenk et al., 2008). The third (prognostically unfavorable) subgroup includes mostly unbalanced karyotypes characterized by a gain or loss of larger chromosomal regions. Within these, an especially complex aberrant karyotype, which occurs in 10% to 12% of patients and that is defined by ≥3 chromosomal anomalies shows a very unfavorable prognosis. Cytogenetics is further helpful to delineate patients with therapy related AML (t-AML), who are classified as third hierarchy in the WHO classification, and developed either after treatment with alkylating agents often associated with cytogenetic aberrations involving 5q-, −7, or p53 and complex aberrant karyotype or are showing MLL/11q23 or other balanced cytogenetic aberrations which are in frequent association to previous treatment with topoisomerase II inhibitors One striking observation is the increasing incidence of adverse versus favorable cytogenetic abnormalities with increasing age. This, at least in part, contributes to the poorer outcome of AML in older adults. (Byrd et

Nowadays, considerable progress has been made in elucidating the molecular pathogenesis of acute leukemias that resulted in identification of new molecular diagnostic and prognostic markers. Gene mutations and deregulated gene expression have been identified that allow us to interpret the genetic diversity within defined cytogenetic groups, in particular the large and heterogeneous group of patients with CN-AML. Risk stratification by molecular markers in patients from the former group of AML plays an increasing role at diagnosis. The most relevant markers, which can be detected alone or in coincidence with other mutation are NPM1 mutations that is detected in approximately 40% of cases, MLL-PTD in 6%, NRAs in 8-10%, CEBPA in 10% and FLT3-TKD mutations in 6% of CN-AML. Thus, a rather of limited number of markers further subclassifies more than 85% of CN-AML. Data from the literature suggest that those markers have different prognosis regarding the outcome of the disease and they all indicate that in the near future molecular screening may allow " targeted allogeneic stem cell transplantation (alloSCT)" in AML

There is a growing list of the new genetic abnormalities with clinical value that are being investigated. These genetic events perturb diverse cellular pathways and functions, and they often confer a profound impact upon the clinical phenotype of the disease and treatment

It is anticipated that advances in molecular technology will reveal additional markers that will result in more precise classification of this heterogeneous complex of disorders

patients with normal karyotype (Schlenk et al., 2008; Koreth et al, 2009).

al., 2002; Schlenk et al., 2008, Swerdlow et al., 2008)

(Löwenberg B., 2008b; Haferlach T., 2008)

**1.4.2.2 Molecular genetics** 

response.

The parallel progress of the development of the diagnostic techniques improvement of the classification and therapeutical approach lead acute leukemia which were for the first time described before 150 years, and in the 1970s were still fatal disease nowadays to have overall 5 years survival rates of up to 40%. (Appelbaum et al, 2001).

Despite heterogeneity of the disease, with the exception of acute promyelocytic leukemia, this disease has been treated with a "one size fits all" approach. Although, the vast majority of AML patients can be individually characterized on the basis of the distinct chromosomal aberrations and molecular markers, treatment of AML is still based on quite unspecific cytotoxic therapy. For almost 40 years, the use of continuous infusion of cytarabine combined with another agent, usually an anthracycline, the "3+7" regimen, has been the mainstay of therapy (Yates et al, 1973). Response rates for induction with standard chemotherapy ranged from 70% to 80% for adults aged less than 60years which are enrolled in clinical trials and average in 50% for patients older than 60 years (Lichtman et al.,2010) .

Alternative consolidation therapies by applying additional chemotherapy, autologous stem cell transplantation (autoSCT) or alloSCT based on the initial cytogenetic and molecular studies are available (Cornelissen J.J.et al.(2007); Fernandez H.F.,2010; Koreth et al, 2009)

As more sophisticated molecular techniques have become available, it is clear that it is still possible to detect residual disease when all morphological and functional criteria for remission are met. Techniques such as "real–time"-quantitative polymerase chain reaction (RQ-PCR) are capable of detection at a level of 1 in 104 or 1 in 105 residual cells, but such markers are available for only a minority of cases in which the molecular lesion has been characterized(Freeman et al., 2008).

AlloSCT is the most effective antileukemic modality that is characterized by immune mediated graft-versus-leukemia effect of the transplanted cells that reduce the risk of relapse considerably and improve the relapse-free survival but also is associated with the increased risk of death and morbidity. Therefore, the alloSCT advantage has to be carefully balanced against the excess mortality (ranging between 10% and 40%) and morbidity due to transplantrelated complications, such as infection and graft-versus-host disease that are typically connected with alloSCT and can diminish all of the benefit of a reduced risk of relapse. (Cornelissen et al., 2007). For this reason, allogeneic stem cell transplantation is usually avoided in a type of AML that has a pattern of cytogenetics with a relatively favorable prognosis, such as AML with the chromosomal translocations t(8;21) or inv(16)/t(16;16)(Marcucci et al., 2000; Perea et al.,2006). In the latter subtypes the risk of relapse is in the order of 35% to 40% or less. By contrast, a transplant is treatment of choice for all other patient whose leukemia cells bear a cytogenetic or molecular abnormality that predicts a high or intermediate risk of relapse after chemotherapy (Löwenberg et al., 2008b; Koreth et al, 2009). Exception could be done for two additional genotypically defined subsets of AML that are categorized as low-risk within the large category of CN-AML. Each of those entities has a risk of relapse of about 35%. The first genotype is defined by the presence of 'favorable' mutations in *NPM1* and the absence of concurrent 'unfavorable' *FLT3*-internal tandem duplications (*NPM1*mut /*FLT3*-ITDneg).61 This genotype accounts for approximately 16% of all newly diagnosed patients younger than 60 years old. There is no demonstrable benefit from transplantation in patients with *NPM1*mut/*FLT3*-ITDneg AML. The second subset of AML with 'favorable' mutations in the transcription factor gene *CEBPA* (*CEBPA*mut) could not be analyzed in this way because of a lack of statistical power due to a limited number of cases. The latter low-risk subtype *CEBPA*mut accounts for 8% of all AML. Nevertheless, the

Immunophenotyping of the Blast Cells in Correlations with

Fig. 1. Average age of the patients in the study group

**3. Material and methods 3.1 Patients and samples** 

Beillardet al, 2003).

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 485

A total of 76 adult (>15 years) patients (from initially 77 tested) with acute leukemia who were consecutively admitted at the Clinic of Hematology-Skopje from January through December 2008 were enrolled in this study. The median age of the patients (41 men, 35 women) was 52 + 18.66 years (range 16-80), and most of the patients 37 (48.7%) were between 55 and 75 years old. The diagnosis was made by standard morphological examination and cytochemical analyses of bone marrow smears according to the criteria established by the FAB Cooperative Study Group (Bennet et al, 1997) and confirmed by immunophenotyping of bone marrow aspirates and/or peripheral blood samples (Bain et al., 2002; Bene et al., 1995 ) following the criteria of the European Group for the Immunological Classification of leukemias (EGIL) and the British Committee for Standards in Hematology (BCSH) (Bain et al., 2002; Bene et al., 1995). Consecutively, patients were further stratified in the adequate genetic AML entities according to the results of the molecular analyses. The samples contained more than 20% of blast cells (most of which had more than 50%). All patients were tested for the presence of the fusion transcript of the mayor recurrent cytogenteic abnormalities in AML (PML/RAR, AML1/ETO, CBF/MYH11) by RT-PCR, according to standard procedures. (Gabert et al.2003;

available body of evidence suggests that these AMLs are unlikely to profit from an alloSCT (Schlenk et al., 2008).

Thus, it could be concluded that patients with AML with t(8;21), AML with inv(16)/t(16;16), AML with *NPM1*mut/ *FLT3*-ITDneg and AML with *CEBPA* mutations should not considered for alloSCT. Nevertheless, it is important to stress that sufficient evidence so far exist only for the AML with recurrent cytogenetic abnormalities and the benefits for the two additional genetic low-risk AML entities should be validated in the larger studies in the future (Schlenk et al., 2008).

 In the near future these results will also need to be considered more specifically in the light of the extended scale of allogeneic stem cell transplantation strategies with respect to reduced-intensity conditioning regimens and in relationship to different transplant sources and donor types (matched unrelated and haploidentical donors, umbilical stem cell grafts).(Löwenberg et al, 2008a)
