**5. Discussion**

Modern diagnostic approach for acute leukemias combines cytomorphology, cytochemistry, multiparameter flow cytometry, chromosome banding analysis, accompanied by diverse fluorescence in situ hybridization techniques, and molecular analyses. The correct diagnosis is essential for classification of this heterogeneous complex of disorders and plays a central role for individual risk stratification and therapeutic decisions (Haferlach et al., 2007; Lichtman et al., 2010)

Fig. 6. Distribution of patients according to the grades of the EKOG performance status

#### **5.1 Immunophenotyping in modern diagnosis of AML**

494 Advances in Cancer Therapy

In 5 of the AML case PML/RAR fusion transcript was detected. Four of those patients had morphology and immunophenotype that correlate with AML-M3 diagnosis. The five patients were first diagnosed as AML-M2 and only after the positive result for PML/RAR the diagnosis was revised as AML-M3. In all those five patients target therapy with ATRA

RT-PCR analysis also detected molecular abnormalities in the Core binding factor (CBF) in 10 AML patients; the presence of the AML/ETO1 fusion gene was confirmed in 3 patient

Molecular analyses enabled 23.7% of the cases from our study to be classified in the adequate genetic entities of AML with different prognosis requiring different therapeutic

We evaluate the distributions of the patients from our study group in the different grades of the Eastern Cooperative Oncology Group (EKOG) performance status scale (Oken et al. 1982)(Figure 6). All the patients with EKOG performance status higher than grade 2 and

Furthermore, all the obtained results were correlated and consecutively effective treatment

Modern diagnostic approach for acute leukemias combines cytomorphology, cytochemistry, multiparameter flow cytometry, chromosome banding analysis, accompanied by diverse fluorescence in situ hybridization techniques, and molecular analyses. The correct diagnosis is essential for classification of this heterogeneous complex of disorders and plays a central role for individual risk stratification and therapeutic decisions (Haferlach et al., 2007;

Fig. 6. Distribution of patients according to the grades of the EKOG performance status

**4.4 Results from the analyses of the clinical data of the patients** 

strategy for each single acute leukemia patient was selected.

patients with serious co morbidities were not suitable candidates for alloSCT.

was initiated.

approach.

**5. Discussion** 

Lichtman et al., 2010)

and CBF/MYH11 in 7 patients.

Assignment of lineage is critical in the diagnostic evaluation of acute leukemia, as treatment for AML and ALL markedly differs. Myeloid and lymphoid lineage may be distinguished based on cellular morphology, cytochemical staining, and expression of lineage-specific antigens (Döhner et al., 2010). Analyses of diagnostic evaluation of acute leukemia in our study showed that immunophenotyping was necessary for lineage assignment in 4 (8.9%) cases that were morphologically and cytochemically undifferentiated, and also for correction of the lineage that was assigned based on morphology and cytochemistry in 3(3.8%) additional cases.

Flow cytometric immunophenotyping is a powerful technological tool that aids in the diagnosis, classification and monitoring of hematological malignancies. It is essential in the diagnosis of AML, as it demonstrates a particular lineage involvement and has a prognostic significance in the majority of AML cases. We used the panel based on the recommendation of the EGIL group and BTSH. Our analyses comprised of a two step process with the first panel of markers being applied to all cases of acute leukemia and the second only in patients with AML that did not demonstrate a clear myeloid commitment. We also evaluated further lymphoid antigen positive AML cases by using the second panel of McAb (Stelzer & Goodpasture, 2000).

The second panel of McAb was aimed at identifying uncommon types of AML, such as those with megakaryocytic or elytroid differentiation and the exclusion or conformation of diagnosis of non-hematological malignancy. In our study, we applied the second AML panel in 27 cases; 4 which did not demonstrate clear myeloid commitment and 23 cases with lymphoid antigen positive AML cases. With our primary McAb we were able to differentiate AML form ALL in 96.8% of cases. Only in one patient (AML-M6-1.5%) lineage differentiation was assigned after staining with the secondary McAB panel and one case of non-hematological malignancy was confirmed.

Immunophenotyping was crucial in all cases of poorly differentiated myeloid leukemia (AML-M0), megakaryoblatstic leukemia (AML-M7) and in some case of monoblastic leukemia (AML-M5) and those with primitive erythroid cells as predominant leukemic cells (AML-M6). This is also important for recognizing an AML case that co-expresses lymphoidassociated antigens. In addition, immunophenotyping enables recognition of unusual forms of acute leukemia: designated acute biphenotypic or acute mixed lineage leukemia. The leukemia associated immunophenotype (LAIP) of the blast cells is a useful tool for detection of minimal residual disease in AML cases (Döhner et al., 2010; Stelzer & Goodpasture, 2000; Swerdlow et al., 2008).

In order to classify AML cases in the different AML entities we correlated the immunological data from immunophenotyping with the FAB morphological and cytochemical classification. One of the difficulties in knotting the flow cytometric data with traditional morphology is the lack of routine flow cytometric data analyses that would ensure correlation of the immunophenotyping data to abnormal morphologic counterpart. Our approach was based on the fact that complete eight-part differential of the myeloid lineage in the normal bone marrow could be done with correlations of the expression of CD45 expression versus light side scatter (SSC) characteristics of the cells. Extension of this technique to the analyses of leukemias allows abnormal cell to be recognized as independent clusters in CD45/SSC histograms with pattern of CD45 and SSC expression that correlate to the same pattern of the morphologically similar cells in normal bone marrow. Flow-cytometric analyses by using CD45 gating strategy reveled that leukemic

Immunophenotyping of the Blast Cells in Correlations with

R1

0

marker CD15

Marcucci et al., 2000).

64

128

SS Lin

192

256

100 101 102 103 104 CD45-PE-Cy5 Log Comp

**5.2 Molecular analyses in the modern diagnosis of AML** 

AML subtypes (Marcucci et al., 2000; Perea et al.2006; Wang et al., 2008)

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 497

A B Fig. 7. Histogram analyses of M5-myloblasts; A: Blast cells are biger and more granular and have higher CD45,B: Blasts cells express CD117 and the myeloid expression maturation

Results from the immunophenotying guided us to perform exact molecular analyses and further define some specific genetic entities of AML. We tested our AML patients for the presence of PML/RAR, AML1/ETO and CBF/MYH11 fusion transcripts, by using RT-PCR method. Results from those analyses additionally improved the exact classification of the AML subtypes and made target and specific therapy available for some of the specific

Nonrandom chromosomal abnormalities are identified at the cytogenetic level in approximately 55% of all adult primary or de novo AML patients and have long been recognized as important independent prognostic indicators for achievement of complete remission (CR), duration of first CR, and survival after intensive chemotherapy treatment. Two of the most prevalent cytogenetic subtypes of adult primary or de novo AML are t (8; 21) (q22; q22) and inv (16) (p13q22). These abnormalities result in the disruption of genes encoding subunits of the CBF, which is a heterodimeric transcriptional factor involved in the regulation of normal hematopoiesis and are collectively referred as CBF-AML. At the molecular level, t(8;21)(q22;q22) and inv(l6)(p13q22) result in the creation of novel fusion genes, AML1/ETO and CBF/MYH11.Detection of t(8;21)(q22;q22) or inv(16)(p13q22) in adult patients with primary AML is a favorable independent prognostic indicator for achievement of cure after intensive chemotherapy or bone marrow transplantation, and may serve as a as a paradigm of the risk-adapted treatment in AML (Döhner et al. 2010&

In almost all studies of adult primary AML, the highest CR rate (approximately 90%) and the longest disease free survival (DFS) at *5* years (approximately 50%) have been associated with CBF-AML cases. It has been suggested that the superior outcome of AML patients with CBF gene rearrangements compared with other AML patients may be attributed to an increased sensitivity of the leukemic blasts to cytarabine that in combination with anthracyclines represents the backbone of AML treatment. The collective analysis of all data regarding the treatment of CBF-AML indicates that incorporation of multiple courses of high dose of cytarabine as consolidation therapy should be considered as the therapeutic

standard for primary adult CBF-AML (Marcucci et al., 2001; Perea et al.2006).

myeloblasts demonstrate very similar CD45/SSC characteristics to normal myeloblasts, especially the early myeloblasts. In fact, leukemic cells generally demonstrate CD45/SSC characteristics that closely resemble their nearest normal morphological counterparts in the bone marrow. In the histograms the leukemic cells can be easily recognized as an abnormal cluster of cells in CD45/SSC "space" which is usually occupied by normal myeloid blasts. Using this method we define in our study group each category of AML as defined by FAB system (Stelzer & Goodpasture, 2000). Early myeloblasts are presented at Figure 6. Flow cytometric analyses show that those leukemic cells demonstrate low light forward and side scatter (FSC&SSC), which means that those cells are small and don't contain any granules. They also have low CD45 expression.

The predominant immunophenotype characteristics of early myeloblasts are an expression of all pan-myeloid antigens: CD13, CD33, CD117 and HLA-DR and lack expression of more mature myeloid antigens such as CD15 and CD14. Also, high proportion of the leukemic cells expresses CD34. With the maturation process of the blast cells, CD34 expression becomes more heterogeneous and weaker. The densest is at AML-M0 blasts, which typically express only one myeloid-associated antigen plus. Myeloid blasts that arise as a result of the genetic change in more mature myeloid progenitor lose the characteristics of the early myeloblasts. For example, AML-M3 blasts usually lack the expression of CD34 and HLA-DR.

At Figure 7 are presents more mature myeloblasts, M5 blast. It is obvious that they are bigger when compared with the AML-M1 blasts presented on Figure 6, and contain some more granules (have higher FSC&SSC). Immunophenotypically, they are characterized with expression of some more mature myeloid markers as CD14 and CD15, and in most of the case with lost CD34 expression.

The results from our study showed that routine immunophenotyping improved the diagnosis in 12 (15, 5%) cases with acute leukemia which was essential for more appropriate individual clinical stratification of the patient with acute leukemia. Our data demonstrate that flow cytometry in correlation with the morphological classification criteria for each subtype of AML can be used for initial classification of each FAB AML entities and and justify routine implementation of flow cytometry analyses in the diagnostic evaluation of AML cases.

Fig. 6. Histogram analyses of early M1 myeloblasts ; A: Blast cells demonostrate low FSC&SSC, B:Blast cells have low CD45 expression

myeloblasts demonstrate very similar CD45/SSC characteristics to normal myeloblasts, especially the early myeloblasts. In fact, leukemic cells generally demonstrate CD45/SSC characteristics that closely resemble their nearest normal morphological counterparts in the bone marrow. In the histograms the leukemic cells can be easily recognized as an abnormal cluster of cells in CD45/SSC "space" which is usually occupied by normal myeloid blasts. Using this method we define in our study group each category of AML as defined by FAB system (Stelzer & Goodpasture, 2000). Early myeloblasts are presented at Figure 6. Flow cytometric analyses show that those leukemic cells demonstrate low light forward and side scatter (FSC&SSC), which means that those cells are small and don't contain any granules.

The predominant immunophenotype characteristics of early myeloblasts are an expression of all pan-myeloid antigens: CD13, CD33, CD117 and HLA-DR and lack expression of more mature myeloid antigens such as CD15 and CD14. Also, high proportion of the leukemic cells expresses CD34. With the maturation process of the blast cells, CD34 expression becomes more heterogeneous and weaker. The densest is at AML-M0 blasts, which typically express only one myeloid-associated antigen plus. Myeloid blasts that arise as a result of the genetic change in more mature myeloid progenitor lose the characteristics of the early myeloblasts. For example, AML-M3 blasts usually lack the expression of CD34 and HLA-

At Figure 7 are presents more mature myeloblasts, M5 blast. It is obvious that they are bigger when compared with the AML-M1 blasts presented on Figure 6, and contain some more granules (have higher FSC&SSC). Immunophenotypically, they are characterized with expression of some more mature myeloid markers as CD14 and CD15, and in most of the

The results from our study showed that routine immunophenotyping improved the diagnosis in 12 (15, 5%) cases with acute leukemia which was essential for more appropriate individual clinical stratification of the patient with acute leukemia. Our data demonstrate that flow cytometry in correlation with the morphological classification criteria for each subtype of AML can be used for initial classification of each FAB AML entities and and justify routine

A B

Fig. 6. Histogram analyses of early M1 myeloblasts ; A: Blast cells demonostrate low

implementation of flow cytometry analyses in the diagnostic evaluation of AML cases.

They also have low CD45 expression.

case with lost CD34 expression.

R1

0

64

128

SS Lin

192

256

<sup>0</sup> <sup>64</sup> <sup>128</sup> <sup>192</sup> <sup>256</sup> FS Lin

FSC&SSC, B:Blast cells have low CD45 expression

DR.

Fig. 7. Histogram analyses of M5-myloblasts; A: Blast cells are biger and more granular and have higher CD45,B: Blasts cells express CD117 and the myeloid expression maturation marker CD15

#### **5.2 Molecular analyses in the modern diagnosis of AML**

Results from the immunophenotying guided us to perform exact molecular analyses and further define some specific genetic entities of AML. We tested our AML patients for the presence of PML/RAR, AML1/ETO and CBF/MYH11 fusion transcripts, by using RT-PCR method. Results from those analyses additionally improved the exact classification of the AML subtypes and made target and specific therapy available for some of the specific AML subtypes (Marcucci et al., 2000; Perea et al.2006; Wang et al., 2008)

Nonrandom chromosomal abnormalities are identified at the cytogenetic level in approximately 55% of all adult primary or de novo AML patients and have long been recognized as important independent prognostic indicators for achievement of complete remission (CR), duration of first CR, and survival after intensive chemotherapy treatment. Two of the most prevalent cytogenetic subtypes of adult primary or de novo AML are t (8; 21) (q22; q22) and inv (16) (p13q22). These abnormalities result in the disruption of genes encoding subunits of the CBF, which is a heterodimeric transcriptional factor involved in the regulation of normal hematopoiesis and are collectively referred as CBF-AML. At the molecular level, t(8;21)(q22;q22) and inv(l6)(p13q22) result in the creation of novel fusion genes, AML1/ETO and CBF/MYH11.Detection of t(8;21)(q22;q22) or inv(16)(p13q22) in adult patients with primary AML is a favorable independent prognostic indicator for achievement of cure after intensive chemotherapy or bone marrow transplantation, and may serve as a as a paradigm of the risk-adapted treatment in AML (Döhner et al. 2010& Marcucci et al., 2000).

In almost all studies of adult primary AML, the highest CR rate (approximately 90%) and the longest disease free survival (DFS) at *5* years (approximately 50%) have been associated with CBF-AML cases. It has been suggested that the superior outcome of AML patients with CBF gene rearrangements compared with other AML patients may be attributed to an increased sensitivity of the leukemic blasts to cytarabine that in combination with anthracyclines represents the backbone of AML treatment. The collective analysis of all data regarding the treatment of CBF-AML indicates that incorporation of multiple courses of high dose of cytarabine as consolidation therapy should be considered as the therapeutic standard for primary adult CBF-AML (Marcucci et al., 2001; Perea et al.2006).

Immunophenotyping of the Blast Cells in Correlations with

treatment groups (Marcucci et al., 2000).

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 499

RT-PCR methods for detecting the PML/RARfusion transcript also provide the "gold standard" approach for confirming a diagnosis of APL. In addition to its high specificity and sensitivity, it is essential for defining PML breakpoint location thereby establishing the

Standard cytogenetic analysis is currently the most common method for identifying the most common recurrent cytogenetic abnormalities t(8;2l)(q22;q22) and inv( 16)(pl3q22) or t(16;16)(p13;q22) in AML patients. This technique also allows detection of secondary chromosomal abnormalities such as del (9q),-X and -Y which are frequently associated with t(8;21)(q22;q22), or +8 and +22 more often found with inv(16)(p13q22). Still, despite the recent improvements in the cytogenetic methodology and the use of complementary techniques such as fluorescence in situ hybridization and comparative genomic hybridization to increase the rate of successful karyotyping, the possibility that subtle structural chromosomal aberrations are missed remains (Marcucci et al., 2000). In t(8;21) (q22;q22) or inv(16) (p13q22), failure to detect submicroscopic (cryptic) rearrangements of the involved genes leads to false-negative results that may ultimately impact the correct stratification of this patient population in prognostic and risk-adapted therapy groups. Sensitive molecular methodologies such as RT-PCR have been successfully used to detect cryptic CBF abnormalities in diagnostic samples of AML patients with karyotypes that are otherwise negative for t(8;21)(q22;q22) or inv(16)(p13q22). Andrieu et al.,1997 reported detection of AML1/ETO fusion transcripts by RT-PCR in cases with unsuccessful cytogenetic studies, normal karyotypes, or karyotypes other than t(8:2 l)(q22;q22) such as del(8q), i(8)(q10) or del(9q). Langabeer et al., 1997 used a two-step RT-PCR to screen for the AMLI/ETO and CBF/MYHll fusion transcripts in AML patients entered into the U.K. MRC AML 10, 11, and 12 trials and compared the results with conventional cytogenetics. All patients with cytogenetic evidence of t(8;21)(q22;q22) or inv(16)(pl3q22) were positive by RT-PCR for the corresponding fusion transcripts. RT-PCR was also positive in 31 cases (19 cases of AML1/ETO and 12 cases of CBF/MYH11) without CBF cytogenetic abnormalities, increasing the overall detection rate of "t(8;21)(q22;q22)" and "inv(16)(p13q22)" from 8.1% to 12.9% and from 6.5% to 10.3%, respectively. The authors of these studies concluded that all primary AML should be routinely tested for the presence of the CBF fusion genes by molecular screening to improve genomic stratification of AML patients in risk-related

Although patients with CBF-AML have a relatively good prognosis, a substantial number of them relapse and eventually die of their disease. Because relapse after intensive treatment is likely to occur as the result of failure to completely eradicate the leukemic blasts, evaluation of MRD by a sensitive molecular technique such as PCR based techniques has been proposed to detect persistence of malignant clones and predict disease relapse in AML patients in CR. This strategy has been successful in CML (Radich et al.,1995) and acute promyelocytic leukemia (Grimwade et al., 2002; Wang et al., 2008), but its clinical

RQ-PCR provides the most sensitive parameters for AML with reciprocal gene fusions PML/RAR, CBF–MYH11, AML1–ETO (Haferlach et al., 2007). The score of gene expression of the respective fusion transcripts after consolidation therapy in relation to gene expression at diagnosis correlates significantly with prognosis [Haferlach et al., 2007]. The

applicability to other subgroups of acute leukemia remains controversial.

**5.2.1 Why we used RT-PCR method for detection of the genetic abnormalities?** 

target for reliable monitoring of the minimal residual disease (MRD).

The prognostic impact of the CBF gene rearrangements appears equally significant in the setting of BMT of AML patients in first CR. However, the iatrogenic morbidity and mortality of BMT suggest that patients with CBF-AML should not receive this therapeutic modality as initial treatment. The collective analysis of all data regarding the implementation of all SCT in AML treatment suggest that although allo SCT and auto SCT may have a potential role in the initial management of AML other than CBF-AML, the treatment-related morbidity and mortality of SCT represent a therapeutic limitation for treatment of CBF-AML patients. Considering the high probability of cure that these patients can achieve with intensive chemotherapy, it is reasonable to spare them the toxicity of SCT as consolidation in first complete remission (Löwenberg et al., 2008a; Koreth et al, 2009; Marcucci et al., 2000; Marcucci et al., 2001 & Perea et al.2006).

APL or AML-M3 is a distinct subtype of AML which is characterized by a t(15;17) translocation leading to a *PML-RARA* fusion gene. Historically, recognition of this form of AML as a separate entity was important for the clinicians, not because the chemotherapy used as treatment differed substantially from that used for the other subtypes of AML, but because the relatively common occurrence of life-threatening coagulopathy mandated special supportive maneuvers, including the use of low-dose heparin and aggressive blood product support. In the past, induction mortality was often significant, with some older series reporting an incidence approaching 50%. APL was typified with the worst features associated with leukemia: a fulminant disorder that struck primarily young people, had devastating effects on an individual's life, and resulted in death for a large number of patients during the initial phases of treatment. The last two decades have seen a fundamental shift from this paradigm, with APL now recognized as one of the most curable forms of acute leukemia. Introduction of the differentiation therapy with ATRA into the treatment of APL completely revolutionized the management and outcome of this disease, and presents the first model of targeted therapy for cancer. This agent represents one of the most spectacular advances in the treatment of human cancer, providing the first paradigm of molecularly targeted treatment. Treatment of APL with ATRA combined with anthracycline-based chemotherapy yields a CR rate of approximately 90% for newly diagnosed APLs. The relapse rate is approximately 20%, and with the development of new molecular target therapies such as arsenic trioxide, a cure can now be expected even for relapsed patients. After the advent of ATRA, the introduction of arsenic trioxide (ATO), probably the most biologically active single drug in APL has provided a valuable addition to the armamentarium and may have contributed to further improvements in the clinical outcome of this disease. Several treatment strategies using these agents, usually in combination with chemotherapy, have provided excellent therapeutic results with survival rates exceeding 70% in multicenter clinical trials. Cure of patients with APL depends not only on the effective use of combination therapy involving differentiating and classical cytotoxic agents, but also, critically, upon supportive care measures that take into particular account the biology of the disease and the complications associated with molecularly targeted therapies. Moreover, it is important to consider diagnostic suspicion of APL as a medical emergency (uncommon in AML) that requires several specific and simultaneous actions, including immediate commencement of ATRA therapy, prompt genetic diagnosis, and measures to counteract the coagulopathy (Grimwade et al., 2002; Wang et al., 2008).

The prognostic impact of the CBF gene rearrangements appears equally significant in the setting of BMT of AML patients in first CR. However, the iatrogenic morbidity and mortality of BMT suggest that patients with CBF-AML should not receive this therapeutic modality as initial treatment. The collective analysis of all data regarding the implementation of all SCT in AML treatment suggest that although allo SCT and auto SCT may have a potential role in the initial management of AML other than CBF-AML, the treatment-related morbidity and mortality of SCT represent a therapeutic limitation for treatment of CBF-AML patients. Considering the high probability of cure that these patients can achieve with intensive chemotherapy, it is reasonable to spare them the toxicity of SCT as consolidation in first complete remission (Löwenberg et al., 2008a; Koreth et al, 2009;

APL or AML-M3 is a distinct subtype of AML which is characterized by a t(15;17) translocation leading to a *PML-RARA* fusion gene. Historically, recognition of this form of AML as a separate entity was important for the clinicians, not because the chemotherapy used as treatment differed substantially from that used for the other subtypes of AML, but because the relatively common occurrence of life-threatening coagulopathy mandated special supportive maneuvers, including the use of low-dose heparin and aggressive blood product support. In the past, induction mortality was often significant, with some older series reporting an incidence approaching 50%. APL was typified with the worst features associated with leukemia: a fulminant disorder that struck primarily young people, had devastating effects on an individual's life, and resulted in death for a large number of patients during the initial phases of treatment. The last two decades have seen a fundamental shift from this paradigm, with APL now recognized as one of the most curable forms of acute leukemia. Introduction of the differentiation therapy with ATRA into the treatment of APL completely revolutionized the management and outcome of this disease, and presents the first model of targeted therapy for cancer. This agent represents one of the most spectacular advances in the treatment of human cancer, providing the first paradigm of molecularly targeted treatment. Treatment of APL with ATRA combined with anthracycline-based chemotherapy yields a CR rate of approximately 90% for newly diagnosed APLs. The relapse rate is approximately 20%, and with the development of new molecular target therapies such as arsenic trioxide, a cure can now be expected even for relapsed patients. After the advent of ATRA, the introduction of arsenic trioxide (ATO), probably the most biologically active single drug in APL has provided a valuable addition to the armamentarium and may have contributed to further improvements in the clinical outcome of this disease. Several treatment strategies using these agents, usually in combination with chemotherapy, have provided excellent therapeutic results with survival rates exceeding 70% in multicenter clinical trials. Cure of patients with APL depends not only on the effective use of combination therapy involving differentiating and classical cytotoxic agents, but also, critically, upon supportive care measures that take into particular account the biology of the disease and the complications associated with molecularly targeted therapies. Moreover, it is important to consider diagnostic suspicion of APL as a medical emergency (uncommon in AML) that requires several specific and simultaneous actions, including immediate commencement of ATRA therapy, prompt genetic diagnosis, and measures to counteract the coagulopathy (Grimwade et al., 2002;

Marcucci et al., 2000; Marcucci et al., 2001 & Perea et al.2006).

Wang et al., 2008).

### **5.2.1 Why we used RT-PCR method for detection of the genetic abnormalities?**

RT-PCR methods for detecting the PML/RARfusion transcript also provide the "gold standard" approach for confirming a diagnosis of APL. In addition to its high specificity and sensitivity, it is essential for defining PML breakpoint location thereby establishing the target for reliable monitoring of the minimal residual disease (MRD).

Standard cytogenetic analysis is currently the most common method for identifying the most common recurrent cytogenetic abnormalities t(8;2l)(q22;q22) and inv( 16)(pl3q22) or t(16;16)(p13;q22) in AML patients. This technique also allows detection of secondary chromosomal abnormalities such as del (9q),-X and -Y which are frequently associated with t(8;21)(q22;q22), or +8 and +22 more often found with inv(16)(p13q22). Still, despite the recent improvements in the cytogenetic methodology and the use of complementary techniques such as fluorescence in situ hybridization and comparative genomic hybridization to increase the rate of successful karyotyping, the possibility that subtle structural chromosomal aberrations are missed remains (Marcucci et al., 2000). In t(8;21) (q22;q22) or inv(16) (p13q22), failure to detect submicroscopic (cryptic) rearrangements of the involved genes leads to false-negative results that may ultimately impact the correct stratification of this patient population in prognostic and risk-adapted therapy groups. Sensitive molecular methodologies such as RT-PCR have been successfully used to detect cryptic CBF abnormalities in diagnostic samples of AML patients with karyotypes that are otherwise negative for t(8;21)(q22;q22) or inv(16)(p13q22). Andrieu et al.,1997 reported detection of AML1/ETO fusion transcripts by RT-PCR in cases with unsuccessful cytogenetic studies, normal karyotypes, or karyotypes other than t(8:2 l)(q22;q22) such as del(8q), i(8)(q10) or del(9q). Langabeer et al., 1997 used a two-step RT-PCR to screen for the AMLI/ETO and CBF/MYHll fusion transcripts in AML patients entered into the U.K. MRC AML 10, 11, and 12 trials and compared the results with conventional cytogenetics. All patients with cytogenetic evidence of t(8;21)(q22;q22) or inv(16)(pl3q22) were positive by RT-PCR for the corresponding fusion transcripts. RT-PCR was also positive in 31 cases (19 cases of AML1/ETO and 12 cases of CBF/MYH11) without CBF cytogenetic abnormalities, increasing the overall detection rate of "t(8;21)(q22;q22)" and "inv(16)(p13q22)" from 8.1% to 12.9% and from 6.5% to 10.3%, respectively. The authors of these studies concluded that all primary AML should be routinely tested for the presence of the CBF fusion genes by molecular screening to improve genomic stratification of AML patients in risk-related treatment groups (Marcucci et al., 2000).

Although patients with CBF-AML have a relatively good prognosis, a substantial number of them relapse and eventually die of their disease. Because relapse after intensive treatment is likely to occur as the result of failure to completely eradicate the leukemic blasts, evaluation of MRD by a sensitive molecular technique such as PCR based techniques has been proposed to detect persistence of malignant clones and predict disease relapse in AML patients in CR. This strategy has been successful in CML (Radich et al.,1995) and acute promyelocytic leukemia (Grimwade et al., 2002; Wang et al., 2008), but its clinical applicability to other subgroups of acute leukemia remains controversial.

RQ-PCR provides the most sensitive parameters for AML with reciprocal gene fusions PML/RAR, CBF–MYH11, AML1–ETO (Haferlach et al., 2007). The score of gene expression of the respective fusion transcripts after consolidation therapy in relation to gene expression at diagnosis correlates significantly with prognosis [Haferlach et al., 2007]. The

Immunophenotyping of the Blast Cells in Correlations with

**5.4 Approach to AML diagnostic algorithms** 

prognostic information (Lowenberg 2008).

risk stratification and therapeutic decisions.

optimize the individual risk stratification for each AML patient.

older than 60years.

(Haferlach et al., 2007).

promptly applied.

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 501

chronic cardiac, pulmonary, hepatic or renal disorders or diabetes suffer greater acute toxicity from chemotherapy. Older patients may also have decreased bone marrow regenerative capacity, even after successful leukemia cytoreduction. Inability to tolerate long periods of pancytopenia and malnutrition or the nephrotoxicity of drugs such as amino glycosides or amphotericin remains a major barrier to successful treatment (Piccirillo et al.2004; Sorror et al, 2005). Six 6(9,3%) patients from our study had seriouos comorbidities which limited the aplication of intensive chemotherapy in their treatment. Five of them were

Modern diagnostic approaches in the AML should be created as integral and basic parts of optimized treatment concepts for the benefit of the each individual patient. The ultimate test of any disease diagnostic algorithm approach is its usefulness in guiding the selection of effective treatment strategies. 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 determinants (e.g., secondary AML, white blood cell count at diagnosis)

A comprehensive approach in diagnosis, classification, and treatment follow-up in patients with acute leukemias can, therefore, be suggested by diagnostic algorithms, which also show the relationship and the hierarchy between single methods. These standard guidelines for AML mostly result from a combination of different methods and intend to add

Our diagnostic algorithm for AML (as shown in Fig. 8) starts with cytomorphology and cytochemistry. These methods should be performed in combination: cytomorphology and cytochemistry allow rapid classification of the acute leukemias and further enables the choice of the antibody panel for flow cytometric analyses. In case cytomorphology gives indices for characteristic aberrations—in the FAB subtypes M3/M3v for t(15;17)/PML/ RAR, in FAB M4eo for inv(16)/CBF/MYH11, or in M1/2 with the characteristic long Auer rods for the t(8;21)/AML1/ETO, PCR analyses for these rearrangements should be

Especially in case of suspicion for APL due to clinical symptoms or due to the morphological findings, RT-PCR for PML/RARshould be initiated as soon as possible (Schoch et al., 2002). We think that, regardless of the morphological, cytochemical and immunological futures of the blast cells, RT-PCR for the fusion oncogene PML/RARare recommendable in all AML cases. The RT- PCR method is the most sensitive and rapid technique for detection of this oncogene and provides an optimal basis for MRD analyses. Further we suggest all AML cases which are PML/RARnegative to be tested for the presence of the reciprocal fusions genes that describe CBF-AML, AML1/ETO and CBF/MYH11.Those analyses provides the basis for sub-classification of the AML cases in prognostically relevant subclasses. This is the prerequisite for adequate individual clinical

Moreover, we correlated the obtained result for the applied multimodal diagnostic approach with the patient age, EKOG performance status and comorbidities, which allowed us to

prognostic value of quantitative PCR in MRD diagnostics in these subgroups was demonstrated in several studies. Patients with an MRD level<1% after induction chemotherapy in relation to initial diagnosis had a relapse rate of 8% in contrast to 91% in the patients with MRD levels of ≥1% in the investigation on the CBF-leukemias by Krauter et al.,2003. Marcucci et al, 2000, were able to define a distinct transcript copy number in inv(16)/CBFB/ MYH11 below which relapse was unlikely and above which relapse occurred with high probability. A Gruppo Italiano Malattie Ematologiche Maligne dell' Adulto (GIMEMA) trial showed that molecular switch from CR to PML/RAR positivity was followed by hematologic relapse after a median time of 3 months in 95% of all APL cases (Diverio et al. 1998). Data from different studies indicate that of all AML subtypes, quantitative PCR monitoring could be best established for the reciprocal gene fusions.

Molecular analyses enabled 23.7% of the cases of our study to be classified in the adequate genetic entities of AML with different prognosis requiring different therapeutically approach.

#### **5.3 Analyses of the clinical characteristics of the patients in the era of modern diagnosis of AML**

#### **5.3.1 Analyses of the EKOG performance status**

Several studies support the use of the ECOG performance status as a measure of physical functioning and prognosis in patients with AML (Oken et al. 1982). A retrospective study of data from five Southwestern Oncology Group (SWOG) trials that included 968 patients with AML found that the mortality rate within 30 days of initiation of induction therapy is dependent upon both the patient's age and ECOG performance status at diagnosis. A second retrospective analysis of 998 patients age 65 or greater (range 65 to 89; median 71 years) who underwent intensive induction chemotherapy reported eight-week mortality rates of 23, 40, and 72 percent for patients with ECOG PS of zero to 1, 2, and 3 to 4, respectively. One-year overall survival rates for the same groups were 35, 25, and 7 percent, respectively. A third retrospective study of 2767 patients with non-APL AML from the Swedish acute leukemia registry also reported that older patients with an ECOG PS of zero to 1 had 30 day death rates after intensive chemotherapy of less than 15 percent, while patients with a PS of 3 or 4 had higher early death rates regardless of patient age ranging from 26 to 36 percent. Seventy percent of patients up to age 80 years had a PS of zero to 2. A prospective trial of induction chemotherapy with cytarabine plus daunorubicin in 811 older adults (median age 67 years, range 60 to 83 years) with ECOG PS of zero to 2 reported a 30 day mortality rate of 11 percent (Appelbaum et al, 2006). We used the EKOG performance status score in initial randomization of our AML patients for different induction and consolidation approaches, in order to avoid intensive induction and consolidation treatment for patient with EKOG performance status higher than 2. Only 8 (12.5%) patients from our study group have EKOG performance status higher than 2, but only 3 of those patients were younger than 60 years of age.

#### **5.3.2 Analyses of the comorbidities**

Comorbidity is an also a distinct additional clinical entity that exists or may occur during the clinical course of patient with a primary disease (i.e. AML). Comorbid conditions are poor prognostic factors especially in older patients with AML. Patients with age-related chronic cardiac, pulmonary, hepatic or renal disorders or diabetes suffer greater acute toxicity from chemotherapy. Older patients may also have decreased bone marrow regenerative capacity, even after successful leukemia cytoreduction. Inability to tolerate long periods of pancytopenia and malnutrition or the nephrotoxicity of drugs such as amino glycosides or amphotericin remains a major barrier to successful treatment (Piccirillo et al.2004; Sorror et al, 2005). Six 6(9,3%) patients from our study had seriouos comorbidities which limited the aplication of intensive chemotherapy in their treatment. Five of them were older than 60years.
