**3. Material and methods**

484 Advances in Cancer Therapy

available body of evidence suggests that these AMLs are unlikely to profit from an alloSCT

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

 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

Correct diagnosis of the diverse subtypes of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) play a central role for individual clinical risk stratification and therapeutic decisions. Modern therapeutic concepts of AML are based on individual risk stratification at diagnosis and during follow-up. The ultimate test of any disease diagnostic algorithm approach is its usefulness in guiding the selection of effective treatment strategies. (Haferlach et al., 2007, Löwenberg et al, 2008b) As discussed above, cytogenetic as well as various genomic markers (gene mutations, gene overexpression) may provide input for algorithms for remission induction and post-remission treatment decisions. At the same time, it remains appropriate to realize that prognostic factors in fact remain a moving target and they are only relevant to therapies available at a given time. Algorithms that provide a basis for risk-adapted therapeutic choices may include immunological markers, cytogenetic factors, molecular markers as well as clinical parameters (e.g., age, attainment of an early or late complete remission) and hematological

On the other hand, the more carefully AML is studied, the clearer it becomes that there is considerable heterogeneity between cases with respect to morphology, immunological phenotype, associated cytogenetic and molecular abnormalities and, more recently, patterns of gene expression. This is reflected in the substantially different responses to treatment. Some entities are becoming so distinct that they are regarded as different diseases with

In order to improve and simplify the diagnosis and management of AML patients that are diagnosed and treated at the at the University Clinic of Hematology-Skopje we conducted a prospective study to establish and standardize a diagnostic algorithm based on minimal screening tests which will facilitate risk adapted therapy for each single AML patient. The aims of our study were: first, to establish the correct lineage assignment of the blast cells, second, to evaluate the incidence of the favorable genetic markers PML/RAR, AML1/ETO and CBF/MYH11 among the AML cases, then to correlate the obtained results with the patient age, comorbidities, and performance status and consecutively to select the effective

determinants (e.g., secondary AML, white blood cell count at diagnosis).

treatment strategy for each single acute leukemia patient.

(Schlenk et al., 2008).

future (Schlenk et al., 2008).

grafts).(Löwenberg et al, 2008a)

specific approaches to treatment.

**2. Motivation and aim of the study** 

#### **3.1 Patients and samples**

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; Beillardet al, 2003).

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

Immunophenotyping of the Blast Cells in Correlations with

Jose.CA.USA) ((Bain et al., 2002).

al., 2008)

al., 2002).

**3.5 Molecular analysis** 

and probe are shown in Table 2.

**3.6 Statistical analysis** 

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 487

For the detection of cytoplasmic and nuclear antigens we used commercially available permeabilization/fixation solutions (FACS Permeabilization solution BDBiosciencies;San

We incubated 100ml of specimens (peripheral blood or bone marrow) and appropriate McAb for 15min at room temperature, than added the permeabilization solutions and/or lysing solution and repeated the incubation procedure. After the incubation we washed the samples three times with PBS-A, than re-suspended the cell with isotones solution and acquired the data. As control we used a lysed, but unstained sample (Panovska-Stavridis et

Acquired data were analyzed with software by using CD45 gating strategy (Borowitz et al, 1993). This technique involves incubation of all samples with fluorochrome - labeled CD45 McAb and with the McAb for which reactivity needs to be established with an alternative fluorochrome). In the initial step of the analyses, gating was set up on a CD45-positive versus light side-scatter dot plot. The procedure allowed the discrimination between the blast cell population and normal cells and the exclusion of platelets and debris. Thereafter, if necessary, it was possible to perform another gating, to separate the cells positive with the McAb under study (Borowitz et al, 1993). Leukemias were first screened by the primary panel and, if necessary, further characterized by the McAb of the secondary panel (Bain et

Antigen expression was considered positive if 20% or more blast cells reacted with a particular antibody, except reactivity of blasts cells with MPO. It was considered positive if 10% or more of mononuclear cells were MPO positive (Bain et al., 2002; Döhner et al., 2010).

Mononuclear cell preparation, RNA isolation, and cDNA synthesis were performed at the Department of Molecular Biology, Immunology and Pharmacogenetics, Faculty of Pharmacy-Skopje, according to standard procedures. Aliquots of 5 μL of cDNA (100 ng RNA equivalent) were used for Real-time Quantitative polymerase chain reaction (RQ-PCR) with primers and dual-labeled probes as described by Gabert et al. Positions and nucleotide

The RQ-PCR reaction was performed in a 25-μl reaction volume using 12.5 μl (1x) Master Mix (Applied Biosystems), 300nM primers and 200nM probes on a Mx3005P(TM) QPCR System (Stratagene) under the following conditions: 95C for 10min, followed by 50 cycles of 95C for 15s, 50C for 1min. In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript was amplified in parallel to the fusion gene (FG) transcript. Since ABL (Abelson) gene transcript expression did not differ significantly between normal and leukemic samples, ABL was used as a control gene in this study (24). Positions and nucleotide sequences of the primers

Statistical analyses were performed by using the statistical analyses software SPSS 18.0 and by applying the Descriptive statistics (cross tabulation, frequencies, descriptive ratio statistics), bivariate statistics (means, t-test, correlation (bivariate, partial, distances), nonparametric tests and linear regression statistical methods. The Level of probability for obtaining the null hypothesis, in accordance with the international conventions for bio-

sequences of the primers and probes are shown in Table 2.

medical sciences was 0.05 or 0.01 (Armitage et al., 2002).
