**Immunophenotyping of the Blast Cells in Correlations with the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients with Acute Myeloid Leukemia: Single Center Experience**

Irina Panovska-Stavridis *University Clinic of Hematology, Skopje Republic of Macedonia* 

### **1. Introduction**

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#### **1.1 Acute leukemias**

Acute leukemias are a heterogeneous group of malignancies that result from the malignant transformation of immature hematopoietic cells followed by clonal proliferation and accumulation of the transformed cells. They are characterized by aberrant differentiation and maturation of the malignant cells, with a maturation arrest and accumulation of more than 20% of leukemic blast in the bone marrow (Lichtman et al., 2010).

The natural history of acute leukemia and the response to therapy varies according to the type of blast involved in the leukemic process. Although in many instances the lineage assignment of the different types of blast cells may be recognized by simple morphological and cytochemical stains, it is necessary to employ immunological analyses with monoclonal antibodies and cytogenetic or molecular biological techniques to identify their particular differentiation features (Haferlach et al., 2007).

Acute leukemias are primarily characterized according to their differentiation along the myeloid and lymphoid lineage and they are divided into two main groups: acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In 10% to 20% of patients, the leukemic cells have characteristics of both myeloid and lymphoid cells (Lichtman et al.,2010).

The classification of the acute leukemias underwent many changes in recent years. The French-American-British (FAB) classification of AML and ALL was based on cytomorphological and cytohemistry details only. Since then, the diagnostic of acute leukemias had undergone a complete change and the routine diagnostic work-up incorporated immunophenotyping by multiparameter flow cytometry, classical cytogenetics, molecular cytogenetics (comprising diverse fluorescence in situ hybridization techniques and comparative genomic hybridization) and molecular genetics (mostly polymerase chain reaction (PCR)-based techniques and sequencing) (Bennet et al., 1997; First MIC Cooperative Study Group, 1985; Haferlach et al., 2007).

Immunophenotyping of the Blast Cells in Correlations with

subset (Schhlenk et al, 2008; Vardiman et al.,2008)

entities (Döhner et al., 2010).

**1.3 Diagnostic procedures** 

2008).

more myeloid lineages are dysplastic (Swerdlow et al., 2008).

(Vardiman et al., 2008).

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 479

t(9;11)(p22;q23); MLLT3/MLL", and now is a unique entity. Three new cytogenetically defined entities also are incorporated: "AML with t(6;9)(p23;q34);DEK-NUP214", "AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1"; and "AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1," a rare leukemia most commonly occurring in infants

Moreover, two new provisional entities defined by the presence of gene mutations between the group of cytogenetically normal AML (CN-AML) were added, "AML with mutated NPM1 (nucleophosmin)," and "AML with mutated CEBPA [CCAAT/enhancer binding protein(C/EBP), alpha]." There is growing evidence that these two gene mutations represent primary genetic lesions (so-called class II mutations), that impair hematopoietic differentiation, and when present alone in AML have favorable prognostic meaning. Mutations in the FMS-related tyrosine kinase 3 (FLT3) gene are found in many AML subtypes and are considered as class I mutations conferring a proliferation and/or survival advantage. AML with FLT3 mutations are not considered as a distinct entity, although determining the presence of those mutations is recommended by WHO because they have prognostic significance. The former subgroup termed "AML with multilineage dysplasia" is now designated "AML with myelodysplasia-related changes." Dysplasia in 50% or more of cells, in 2 or more hematopoietic cell lineages, was the diagnostic criterion for the former

However, the clinical significance of this morphologic feature has been questioned. AMLs are now categorized as "AML with myelodysplasia-related changes" if (1) they have a previous history of myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN) and evolve to AML with a marrow or blood blast count of 20% or more; (2) they have a myelodysplasia-related cytogenetic abnormality; or (3) if 50% or more of cells in 2 or

"Therapy-related myeloid neoplasms" has remained a distinct entity; however, since most patients have received treatment using both alkylating agents and drugs that target topoisomerase II for prior malignancy, a division according to the type of previous therapy is not often feasible. Therefore, therapy-related myeloid neoplasms are no longer subcategorized. Myeloid proliferations related to Down syndrome are now listed as distinct

A previous FAB classification is recognized in WHO classification as the entity AML, not otherwise specified. In this subgroup one can find the morphologic separation of AML according to the immaturity of leukemic cells as well as according to the hematopoietic lineage involved. This subtype of AML is reserved for the patients without the known cytogenetic or molecular genetic abnormalities. In some of them, there are markers

The rare forms of AML and acute leukemia of ambiguous lineage recognized in WHO classification are also associated with poor prognosis (Döhner et al., 2010; Vardiman et al.,

The diagnosis of the diverse subtypes of AML is a major challenge for modern hematology. Modern therapeutic concepts of AML are based on individual risk stratification in diagnosis and during follow-up. In the 1970s cytomorphology and cytochemistry represented the only available diagnostic tools. Nowadays, the routine diagnostic setting is completely changed

associated with prognostic significance (Swerdlow et al., 2008; Vardiman et al, 2008).

According to the proceedings in diagnostic methods and the improved understanding of the diversity of acute leukemia subtypes, the latest World Health Organization (WHO) classification of acute leukemias incorporates and interrelates morphology, cytogenetics, molecular genetics and immunologic markers and pays major attention on the importance of genetic events in the classification, prognosis and therapy of the AMLs. Its prognostic relevance is most clearly demonstrated in the AMLs characterized by recurrent chromosome translocation: t(15,17), t(8,21) and inv(16)/t(16,16) which generally have a favorable prognosis when treated with appropriate therapeutic agents. All other genetic events identified among AMLs had strong prognostic meaning but did not influence on the therapeutic decision (Swerdlow et al., 2008).

In the WHO classification of precursor B-cell and T-cell neoplasms, immunophenotying of the malignant cells plays a decisive role in the diagnosis, prognosis and clinical stratification of patients (Swerdlow et al., 2008).

Correct diagnosis of the diverse subtypes of AML and ALL play a central role for individual clinical risk stratification and therapeutic decisions.

#### **1.2 Acute myeloid leukemia**

Acute myeloid leukemia (AML) is one of the most common types of leukemia in adults. It is characterized by limited myeloid differentiation of the malignant cells. The malignant cells characteristically undergo maturation arrest at the level of the early myloblast or promyelocyte, although varying proportions of mature hematopoietic cells are leukemia derived. The cells that display myeloid markers include morphology, Auer rods (aberrant primary granules), cytochemistry (Sudan black, myeloperoxidase, or nonspecific esterase), and cell surface antigens (Lichtman et al., 2010).

AML encompasses a family of hematologic malignancies that can be categorized according to their cytogenetic and associated genetic abnormalities, which have major prognostic importance. During recent years, considerable progress has been made in deciphering the molecular genetics and epigenetic basis of AML and in defining new diagnostic and prognostic markers. A growing number of recurring genetic changes have been recognized in the new WHO classification of AML. Furthermore, novel therapies are now being developed that target some of the genetic lesions and the treatment increasingly is being individualized by prognostic groups, with a goal of developing treatment tailored to the molecular basis of the patient's malignancy (Swerdlow et al., 2008).

#### **1.2.1 WHO classification of AML**

The current WHO classification of AML reflects the fact that an increasing number of new clinico-pathologenetic entities of AML are categorized based upon their underlying cytogenetic or molecular genetic abnormalities (Swerdlow et al., 2008). A number of recurrent genetic abnormalities are adequately defined and are recognized as entities of AML. The subgroup "AML with recurrent genetic abnormalities" is comprised of entities that are defined with seven recurrent balanced translocations and inversions, and their variants. Two entities from this group: "AML with t(8;21)(q22;q22); AML1/ETO" and "AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBF/MYH11" are considered as AML regardless of bone marrow blast counts. In "APL with t(15;17)(q22;q12); PML/RAR," RAR translocations with other partner genes are recognized separately. The former category "AML with 11q23 (MLL) abnormalities" is redefined in "AML with

According to the proceedings in diagnostic methods and the improved understanding of the diversity of acute leukemia subtypes, the latest World Health Organization (WHO) classification of acute leukemias incorporates and interrelates morphology, cytogenetics, molecular genetics and immunologic markers and pays major attention on the importance of genetic events in the classification, prognosis and therapy of the AMLs. Its prognostic relevance is most clearly demonstrated in the AMLs characterized by recurrent chromosome translocation: t(15,17), t(8,21) and inv(16)/t(16,16) which generally have a favorable prognosis when treated with appropriate therapeutic agents. All other genetic events identified among AMLs had strong prognostic meaning but did not influence on the

In the WHO classification of precursor B-cell and T-cell neoplasms, immunophenotying of the malignant cells plays a decisive role in the diagnosis, prognosis and clinical stratification

Correct diagnosis of the diverse subtypes of AML and ALL play a central role for individual

Acute myeloid leukemia (AML) is one of the most common types of leukemia in adults. It is characterized by limited myeloid differentiation of the malignant cells. The malignant cells characteristically undergo maturation arrest at the level of the early myloblast or promyelocyte, although varying proportions of mature hematopoietic cells are leukemia derived. The cells that display myeloid markers include morphology, Auer rods (aberrant primary granules), cytochemistry (Sudan black, myeloperoxidase, or nonspecific esterase),

AML encompasses a family of hematologic malignancies that can be categorized according to their cytogenetic and associated genetic abnormalities, which have major prognostic importance. During recent years, considerable progress has been made in deciphering the molecular genetics and epigenetic basis of AML and in defining new diagnostic and prognostic markers. A growing number of recurring genetic changes have been recognized in the new WHO classification of AML. Furthermore, novel therapies are now being developed that target some of the genetic lesions and the treatment increasingly is being individualized by prognostic groups, with a goal of developing treatment tailored to the

The current WHO classification of AML reflects the fact that an increasing number of new clinico-pathologenetic entities of AML are categorized based upon their underlying cytogenetic or molecular genetic abnormalities (Swerdlow et al., 2008). A number of recurrent genetic abnormalities are adequately defined and are recognized as entities of AML. The subgroup "AML with recurrent genetic abnormalities" is comprised of entities that are defined with seven recurrent balanced translocations and inversions, and their variants. Two entities from this group: "AML with t(8;21)(q22;q22); AML1/ETO" and "AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBF/MYH11" are considered as AML regardless of bone marrow blast counts. In "APL with t(15;17)(q22;q12); PML/RAR," RAR translocations with other partner genes are recognized separately. The former category "AML with 11q23 (MLL) abnormalities" is redefined in "AML with

therapeutic decision (Swerdlow et al., 2008).

clinical risk stratification and therapeutic decisions.

and cell surface antigens (Lichtman et al., 2010).

molecular basis of the patient's malignancy (Swerdlow et al., 2008).

of patients (Swerdlow et al., 2008).

**1.2 Acute myeloid leukemia** 

**1.2.1 WHO classification of AML** 

t(9;11)(p22;q23); MLLT3/MLL", and now is a unique entity. Three new cytogenetically defined entities also are incorporated: "AML with t(6;9)(p23;q34);DEK-NUP214", "AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1"; and "AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1," a rare leukemia most commonly occurring in infants (Vardiman et al., 2008).

Moreover, two new provisional entities defined by the presence of gene mutations between the group of cytogenetically normal AML (CN-AML) were added, "AML with mutated NPM1 (nucleophosmin)," and "AML with mutated CEBPA [CCAAT/enhancer binding protein(C/EBP), alpha]." There is growing evidence that these two gene mutations represent primary genetic lesions (so-called class II mutations), that impair hematopoietic differentiation, and when present alone in AML have favorable prognostic meaning. Mutations in the FMS-related tyrosine kinase 3 (FLT3) gene are found in many AML subtypes and are considered as class I mutations conferring a proliferation and/or survival advantage. AML with FLT3 mutations are not considered as a distinct entity, although determining the presence of those mutations is recommended by WHO because they have prognostic significance. The former subgroup termed "AML with multilineage dysplasia" is now designated "AML with myelodysplasia-related changes." Dysplasia in 50% or more of cells, in 2 or more hematopoietic cell lineages, was the diagnostic criterion for the former subset (Schhlenk et al, 2008; Vardiman et al.,2008)

However, the clinical significance of this morphologic feature has been questioned. AMLs are now categorized as "AML with myelodysplasia-related changes" if (1) they have a previous history of myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN) and evolve to AML with a marrow or blood blast count of 20% or more; (2) they have a myelodysplasia-related cytogenetic abnormality; or (3) if 50% or more of cells in 2 or more myeloid lineages are dysplastic (Swerdlow et al., 2008).

"Therapy-related myeloid neoplasms" has remained a distinct entity; however, since most patients have received treatment using both alkylating agents and drugs that target topoisomerase II for prior malignancy, a division according to the type of previous therapy is not often feasible. Therefore, therapy-related myeloid neoplasms are no longer subcategorized. Myeloid proliferations related to Down syndrome are now listed as distinct entities (Döhner et al., 2010).

A previous FAB classification is recognized in WHO classification as the entity AML, not otherwise specified. In this subgroup one can find the morphologic separation of AML according to the immaturity of leukemic cells as well as according to the hematopoietic lineage involved. This subtype of AML is reserved for the patients without the known cytogenetic or molecular genetic abnormalities. In some of them, there are markers associated with prognostic significance (Swerdlow et al., 2008; Vardiman et al, 2008).

The rare forms of AML and acute leukemia of ambiguous lineage recognized in WHO classification are also associated with poor prognosis (Döhner et al., 2010; Vardiman et al., 2008).

#### **1.3 Diagnostic procedures**

The diagnosis of the diverse subtypes of AML is a major challenge for modern hematology. Modern therapeutic concepts of AML are based on individual risk stratification in diagnosis and during follow-up. In the 1970s cytomorphology and cytochemistry represented the only available diagnostic tools. Nowadays, the routine diagnostic setting is completely changed

Immunophenotyping of the Blast Cells in Correlations with

**1.3.5 Molecular genetics** 

**1.4 Prognostic factors** 

least, conventional therapy.

**1.4.1 Patient-related factors** 

**1.4.2 AML-related factors** 

**1.4.2.1 Cytogenetics** 

the Molecular Genetics Analyses for Diagnostic and Clinical Stratification of Patients… 481

like acute promyelocytic leukemia (APL). Also, it allows the distinction of the disease with widely different prognosis. For example AML t(8;21)(q22;q22) with favorable risk versus

Numerous genetic abnormalities that escape cytogenetic detection like gene mutations and gene expression abnormalities are more recently discovered among CN-AML. Molecular diagnosis by reverse transcriptase- polymerase chain reaction (RT-PCR) for the frequent gene fusions, such as AML1/ETO, CBFMYH11, MLLT3/MLL, DEK/NUP214, can also be useful in certain circumstances. RT-PCR, for which standardized protocols are already published, is also an excellent option to detect recurrent cytogenetic rearrangements, if chromosome morphology is of poor quality, or if there is typical marrow morphology but the suspected

Prognostic factors of an AML case may be subdivided into those related to patient characteristics and general health condition and those related to characteristics particular to the AML clone. The former subset usually predicts treatment-related mortality (TRM) and becomes more important as patient age increases while the latter predicts resistance to, at

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).

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,

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

and cytogenetic and molecular abnormalities in leukemic cells (Döhner et al., 2010).

AML abn 3q26 with adverse risk (Döhner et al., 2010; Grimwade D.,2001).

cytogenetic abnormality is not present. (Gabert et al., 2003; Beillard et al, 2003).

and it consists of classic cytogenetics, molecular cytogenetics, molecular genetics and immunophenotyping by multi-parameter flow cytometry (Döhner et al., 2010 )**.** 

#### **1.3.1 Cytomorphology**

First steps in the diagnostic work-up of a patient with suspected AML is a morphological evaluation of a classical bone marrow aspirate and a peripheral blood smear by using a May-Grunwald-Giemsa or a Wright-Giemsa stain. It is recommended that at least 200 leukocytes on blood smears and 500 nucleated cells on marrow smears to be counted. For a diagnosis of AML, a marrow or blood blast count of 20% or more is required, except for AML with t(15;17), t(8;21), inv(16) or t(16;16), and some cases of erythroleukemia. Myeloblasts, monoblasts, and megakaryoblasts are included in the blast count. Erythroblasts are not counted as blasts except in the rare instance of pure erythroid leukemia (Bennet et al,1997; Döhner et al., 2010; Panovska-Stavridis et al., 2008).

#### **1.3.2 Cytochemistry**

Lineage involvement could be identified with cytochemistry by using myeloperoxidase (MPO) or Sudan black B (SBB) and nonspecific esterase (NSE) stains. Detection of MPO (if present in > 3% of blasts) indicates myeloid differentiation, but its absence does not exclude a myeloid lineage because early myeloblasts and monoblasts may lack MPO. SBB staining parallels MPO but is less specific. NSE stains show diffuse cytoplasmic activity in monoblasts (usually 80% are positive) and monocytes (usually 20% positive). In acute erythroid leukemia, a periodic acid-Schiff (PAS) stain may show large globules of PAS positivity (Bennet et al,1997; Döhner et al., 2010, Panovska-Stavridis et al., 2008).

#### **1.3.3 Immunophenotyping**

Application of immunophenotyping together with the cytomorpohology and cytohemistry has a crucial role in the initial diagnosis of all cases with a suspected or proven diagnosis of acute leukemias. Immunophenotyping allows the discrimination of different cell population on the basis of their size, granularity, and antigen expression patterns. Flow cytometry is a powerful technology for characterization and analysis of cells. It simultaneously measures and analyzes multiple physical characteristics of single particles, usually cells, as they move in a fluid stream through a beam of light through an optical and/or electronic detection apparatus. Flow cytometry uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate specific multi-parameter data from particles and cells in the size range of 0.5nm to 40nm diameter (Panovska-Stavridis et al., 2008).

The applied methodology detects cell surface antigens in a suspension of viable cells and cytoplasmic and nuclear antigens in previously fixed and stabilized cell suspension with the application of monoclonal antibodies conjugated with different fluorochromes. It permits simultaneous detection (multiparameter analyzes) of more than two membrane and nuclear or cytoplasmic antigens by means of double or multiple immunostaining (Bain et al., 2002; Bene et al., 1995; Döhner et al., 2010; Panovska-Stavridis et al., 2008).

#### **1.3.4 Cytogenetics**

Chromosome abnormalities are detected in approximately 55% of adult AML. Conventional cytogenetic analyses are part of the standard diagnostic approach of a patient suspected with AML. This allows the identification of genetics entities that deserve targeted treatments like acute promyelocytic leukemia (APL). Also, it allows the distinction of the disease with widely different prognosis. For example AML t(8;21)(q22;q22) with favorable risk versus AML abn 3q26 with adverse risk (Döhner et al., 2010; Grimwade D.,2001).
