Diagnosis of acute promyelocytic leukemia – M3 according to the FAB classification

Acute promyelocytic leukemia and its atypical variant (M3 and M3v according to the FAB classification):
• accounts for 5–8% of all acute non-lymphoblastic leukemias (ONLL);
• part of the blasts has nuclei of characteristic lobed two-lobed shape;

• blasts, as a rule, contain abundant bright-colored grit and tufts of Auer rods, with an atypical microgranular variant (M3v), small grit is detected only by ultrastructural examination;
• blasts contain peroxidase, lipids, granulocyte esterase in the maximum amount, moderate amount of non-specific esterase resistant to sodium fluoride, PAS-positive substance in diffuse form;

• Immunophenotype is represented by expression of the myeloid antigen CD33, less often CD13 and CD15. Coexpression of CD2 and CD9 is sometimes observed. HLA-DR and CD34 are generally absent;
• in 95% of cases translocation is detected (15; 17) (q22; ql2); (PML / RARa). In addition to this translocation, t (ll; 17) (q23; q21), t (5; 17) (q32; 12), t (ll; 17) (ql3; q21) can also be observed. Acute promyelocytic leukemia in accordance with the WHO classification is distinguished into an independent category of acute non-lymphoblastic leukemia (ONLL) with repeated cytogenetic abnormalities.

Acute myeloblastic leukemia with maturation (M2 according to FAB classification) – diagnostics

• is 30-45% of all ONL;
• in the bone marrow more than 10% of neutrophilic cells at different stages of maturation and less than 20% of monocytes;
• blasts with extensive cytoplasm and, as a rule, with grit, sometimes with large granules of the type of Chediak-Higashi, with Auer sticks;
• blasts contain significant amounts of myeloperoxidase, lipids, ASD-chloroacetate esterase, as well as non-specific esterase and PAS-positive substance in diffuse form;
• blasts express myeloid antigens CD33, CD11, CD13, CD15, react with MCA to peroxidase and lysozyme, in cases with t (8; 21) coexpress lymphoid marker CD19, sometimes CD56 antigen;
• a dysplasia of cells of the neutrophilic series is determined: impaired segmentation of nuclei such as the pseudo-Selger anomaly; hypogranulation of cytoplasm, sometimes Auer’s sticks in mature forms;
• the number of eosinophils can be increased, there are no signs of dysplasia in them;
• translocation (8; 21) is detected in approximately 40% of cases. In accordance with the WHO classification, these observations are categorized into an independent category of acute non-lymphoblastic leukemia (ONLL) with recurring anomalies.

Acute basophilic cell leukemia (M2baz according to the FAB classification) – diagnosis

Acute basophilic cell leukemia (M2baz according to the FAB classification):
• is about 0, 5% of all acute non-lymphoblastic leukemia (ONLL);
• blasts of medium size, have oval or two-lobed nuclei;
• blasts contain coarse polymorphic granularity, positive with metachromatic coloration;
• blasts do not contain peroxidases, lipids and granulocyte esterase, stained with alcian blue;
• electron microscopic examination determines the structures characteristic of basophilic precursors and mast cells: electron-dense particles separated inside and crystalline material;
• translocation can be detected (6; 9) or 12p- deletion, in rare cases t (9; 22) (q34; qll).

Modern diagnosis of acute leukemia

The basis for the modern diagnosis of acute leukemia was the FAB classification proposed in 1976 by a group of French, American and British hematologists. This classification is based on the criteria obtained in the morphological and cytochemical study of normal and leukemic cells. The morphocytochemical approach made it possible to diagnose most cases of AML: acute myeloblastic, promyelocytic, monoblastic leukemia, and erythromyelosis.

A comparative analysis of the morphological features of leukemic blasts allowed us to establish the signs of myeloid differentiation: graininess in myeloblasts, Auer sticks in the form of beams in leukemic promyelocytes, monocytoid form of nuclei in monoblasts. With the help of cytochemical methods in myeloblasts, enzymes specific for cells of the granulocyte series are detected: myeloperoxidase (MPO) and ASD-chloroacetate esterase;

For myeloid cells, including blasts, the presence of a PAS-positive substance in a diffuse form is characteristic. At the same time, in lymphoblasts, as in all lymphoid cells, the PAS-positive substance is deposited in the form of granules. The absence of signs of myeloid differentiation and the presence of PAS-positive substance in the form of granules, characteristic of lymphocytes, give reason to isolate acute lymphoblastic leukemia.

All variants of ONLL (acute non-lymphoblastic leukemia) were designated by the abbreviation M and the corresponding number (MO — M7), all ALL (acute lymphoblastic leukemia) – by the letter L (Л1 — ЛЗ). The proposed criteria are recognized by most hematologists, including in our country, however, in some cases, the variant of acute leukemia remained undifferentiable.

The use of the achievements of ultrastructural studies, immunology and molecular biology further allowed us to supplement and clarify the classification of FAB. Using electron microscopic studies, a special subtype of leukemic promyelocytes with fine grit not detected by light microscopy, as well as leukemic megakaryoblasts containing specific platelet peroxidase were characterized.

Since the 1980s, the immunophenotypic method has been used to diagnose acute leukemia. The use of monoclonal antibodies (ICA) Cases of acute leukemia with an unclear line of differentiation (undifferentiated, bilinear or biphenotypic) have been proposed to be divided into an independent category.

Diagnosis of acute leukemia – classification

Diagnosis of acute leukemia includes the study of peripheral blood, bone marrow, in some cases – trepanobiopsy. All leukemias are divided into two large groups: acute non-lymphoblastic (ONLL), or myeloid (AML), and acute lymphoblastic (ALL) leukemia.

Anemia, thrombocytopenia and neutropenia are characteristic signs that are found in the majority of patients at diagnosis. T

rombocytopenia is determined in the vast majority of patients. The lowest rates of platelets (10-15 • 109 / l and below) are characteristic of acute promyelocytic leukemia. In 1-2% of cases of ONLL, thrombocytosis is noted in peripheral blood (more than 400 × 109 / L), sometimes combined with an increase in the number and dysplasia of megakaryocytes. A similar pattern is specific for patients with ONLL with chromosome 3 abnormality.

The number of leukocytes in the peripheral blood of patients with acute leukemia can vary within very wide limits – from 0.8 • 109 / l to 200 • 109 / l. In the hemogram, in most cases, blast cells are detected, their number is very variable (1-95%). It should be borne in mind that in some cases at the time of diagnosis, blasts in the blood may not be detected. As a rule, when calculating a blood formula, pronounced neutropenia draws attention.

Punctate of the bone marrow usually contains a sufficient number of myelokaryocytes (30-400 x 10 9 / L), low cellularity is noted in rare cases. Hypocellular bone marrow in combination with leukopenia is observed more often with promyelocytic leukemia.

A characteristic symptom of acute leukemia is blast metaplasia: the number of blast cells exceeds 20%. Normal hemopoiesis sprouts in the bone marrow are narrowed. In some cases, an increase in the number of pathological forms of normoblasts (erythromyelosis) or megakaryocytes (with anomalies of chromosome 3) is observed.

Acute lymphoblastic leukemia in children: diagnosis

Acute lymphocytic leukemia accounts for up to 80% of leukemias in children. Most of the remaining nosologies include acute myeloid / acute non-lymphocytic leukemia (AML / ONLL). Chronic myeloid leukemia and other myeloproliferative disorders are rare.

The clinical picture of leukemia in children. Clinical signs and symptoms result from infiltration of the bone marrow or other organs with leukemic blast cells.

In most children, the onset of the disease goes without expressed complaints for several weeks with the following symptoms:

•  malaise;
• infections;
• pallor of the skin and mucous membranes;
• abnormal hematomas;
• hepatosplenomegaly;
• enlarged lymph nodes;
• pain in the bones.

In some children, the disease progresses very quickly.

In most cases, but not in all children, a blood test is abnormal (low Hb and thrombocytopenia), as well as evidence of the spread of blast cells. A bone marrow examination is necessary to confirm the diagnosis and identify the immunological and cytogenetic characteristics that provide useful prognostic information.

Both acute lymphoblastic leukemia and AML are classified based on morphology. Immunological phenotyping further classifies ALL into subclasses. B-cell (75%) and T-cell (15%) subtypes are the most common subclasses.

The prognosis and some aspects of the clinical picture change with other subtypes, and accordingly treatment is selected for this. The prognosis of acute lymphoblastic leukemia depends on age, tumor load (determined by the number of leukocytes), the speed of response to the initial chemotherapy and the presence or absence of certain cytogenetic / molecular genetic abnormalities in the tumor cells.

High leukocyte counts (> 50×109 / l), under 1 year old or over 10 years of age with bone marrow blasts and submicroscopic leukemia levels (minimal residual disease) at the end of the first phase (induction) of treatment are important variables in determining the intensity of treatment.

Cytogenetic studies of the bone marrow in diagnosis are important for identifying certain prognostic factors that can lead to a correction in the intensity of therapy.

Cytogenetic studies in acute leukemia – karyotypes

Frequent detection of changes in the karyotype in leukemic cells was a prerequisite for further progress in understanding the pathogenesis of acute leukemia, the development of diagnostic and prognostic models for various variants of acute leukemia, evaluating the effectiveness of therapy and detecting early recurrence.

The definition of karyotype has become the “gold standard” in diagnosing and predicting the course of acute leukemia.

In acute lymphoblastic leukemia, the presence of t (9; 22), determined in 30% of adult patients, t (4; ll), -7, or +8 (rarely observed) indicates an unfavorable prognosis. An altered karyotype is observed in approximately 80% of patients with acute myeloblastic leukemia. Detection in AML t (15; 17), t (8; 21) and inv (16) is prognostically favorable, while +8, -5, del (5q), -7, del (7q), -20, +11, +13, inv (3) and the involvement of llq23 are unfavorable signs.

Certain clinical variants of acute leukemia, such as M3, are associated with t (15; 17); myelocytic with maturation – with t (8; 21); acute myeloblastic leukemia with pathological bone marrow eosinophilia, with t (8; 14); t (2; 8); t (8; 22) with inv (16) or del 16p; megakaryocytic – with t (l; 22). Use for the treatment of podophyllotoxins or anthracyclines can lead to the appearance of translocations within 1–3 years after completion of chemotherapy. After alkylating drugs, the occurrence of -5, -7 and complex chromosomal abnormalities are most typical within 2-9 years after completion of therapy.

Genetic features (cytogenetic and molecular biological), along with other conditions (prior treatment, history of myelodysplastic syndrome (MDS)), significantly affect the course of acute leukemia, but are not always associated with the distinguished FAB categories. The drawbacks of the FAB classification led to the creation of a new classification that combines genetic and clinical aspects with morphology, cytochemistry and the immunophenotype of the hematopoietic system tumors. The work on its creation was completed in 1997 by a group of experts of the European Association of Hematopathology and the Society of Hematopathology.

The adopted WHO-classification is more rational: each selected nosological form has clinical significance and can be diagnosed by a pathologist.

Some specific cytogenetic disorders in acute myeloblastic leukemia (AML) are associated with typical morphology and affect the course of the disease. With the exception of the MoH with t (15; 17), they do not always correlate with the FAB category. This allowed us to identify four separate nosological units under the heading “acute myeloblastic leukemia (AML) with characteristic cytogenetic translocations.”

Cases with these cytogenetic disorders, but low blastosis, previously referred to as MDS, are classified as AML. It was decided to include in the WHO classification of AML with multilinear dysplasia, AML with a history of MDS, and AML associated with previous treatment.

In 2001, in accordance with the decision of the WHO commission, the preservation of FAB terminology and the separation of acute lymphoblastic leukemia (ALL) into forms L1, 2 and 3 was considered inexpedient, since the predictive value of morphological variants L1 and L2, as well as their correlation with the immunophenotype and cytogenetic markers are not obvious, and the form of L3 ALL is equivalent to Burkitt’s lymphoma in the leukemia phase.

Tumors from progenitor cells, regardless of whether they affect the lymph node or involve the bone marrow in the tumor process, are biologically the same. This led to the conclusion that ALL and lymphoblastic lymphoma are one disease with a different clinical picture. Due to the fact that in most cases, tumors from progenitor cells proceed with bone marrow damage, it was decided to abandon the term “acute lymphoblastic leukemia.”

Immunophenotyping of acute leukemia – goals, objectives

The introduction of monoclonal antibodies into practice allowed us to identify specific receptors, antigens and other molecules (markers) on the membrane and / or in the cytoplasm of blast cells. Currently, more than 300 antigenic groups have been identified, which are called differentiation clusters (CD).

The objectives of immunophenotyping in acute leukemia:

1) confirmation of the diagnosis established on the basis of studying the morphology and cytochemistry of the blasts;
2) establishment of a diagnosis with questionable morphological and cytochemical results;
3) diagnosis of biphenotypic acute leukemia;
4) detection of aberrant antigen expression for the detection of a neoplastic clone with signs of complete remission (minimal residual disease);
5) division of acute leukemia into prognostic groups.

Cells of acute lymphoblastic leukemia of T-cell origin are characterized by the expression of T-cell markers CD2, CD5, CD7 (less commonly CD1); sometimes B-cell markers CD10 and CD21 can also be ex-compressed. In cases of B-ALL, CD19, CD10, CD22 and, depending on the stage of maturation, CD20 and surface immunoglobulin are expressed.

In acute myeloid leukemia, CD13, CD15, CD33, and CD14 are expressed (with the monocytoid component). Expression of Tdt (terminal deoxynucleotide-diltransferase) is noted in most lymphoid blasts and in 20% of cases of acute myeloblastic leukemia. CD34 can be expressed on blast cells of all lines, especially poorly differentiated.

HLA-DR is determined on B-ALL blasts, most AML (except for MV) and in rare cases with T-ALL.

In most cases of biphenotypic leukemias (a combination of phenotypes of two and even three lines), blasts show coexpression of markers of several lines; in more rare cases, the same patient simultaneously has blasts of different origin (myeloid and lymphoid) – bilinear acute leukemia.

Classification of acute leukemia

In 1976, a group of experts from France, the USA and the UK (FAB) proposed a classification of acute leukemia, based on the morphological and cytochemical features of blast cells.

Currently, you should also use the results of immunophenotyping and cytogenetic research necessary to determine the individual prognosis and the choice of adequate therapy (intensification, myelot transplantation or standard treatment, which often includes long-term maintenance chemotherapy).

In accordance with the FAB classification, on the basis of morphological examination of blood and bone marrow and cytochemical reactions, 8 types of acute myeloblastic leukemia (AML) (M0-M7) and 3 types of acute lymphoblastic leukemia (L1-L3) are distinguished. Currently, the FAB classification of ALL is not used; in AML, immunophenotyping and cytogenetic study of blast cells are almost always used additionally.

The most important for differential diagnosis is the allocation of lymphoid or myeloid lesions of acute leukemia, since there are significant differences in the treatment of patients with AML and ALL. Due to the fact that modern therapy programs for all morphological types of AML, with the exception of ALI, are usually similar, and the prognosis is usually unfavorable, the use of only the FAB classification is impractical.

It should also be borne in mind that in some patients, morphological and cytochemical studies conducted by qualified laboratory physicians either do not allow verification of the variant of RL, or give an erroneous diagnosis in some patients (in the study of peripheral blood and bone marrow preparations by various experts, the diagnosis is confirmed no more than than in 80-90% of cases).

In these cases, immunophenotyping of blast cells is indicated. Finally, at present, cytostatic therapy programs are differentiated depending on the immunological variant (first of all in ALL) and the nature of cytogenetic changes. For all these reasons, morphological and cytochemical examination of blood and bone marrow in most cases should be complemented by immunophenotyping and cytogenetic studies.

Differential diagnosis of acute leukemia

Differential diagnosis of acute leukemia is carried out with other diseases of the hematopoietic system (non-Hodgkin’s lymphomas, myelodysplastic syndromes, blast crisis of chronic myeloid leukemia, multiple myeloma, aplastic or megaloblastic anemia), leukemoid reactions (most often associated with lymphoma cytosarcidoproteinoproteinoproteinoproteinoproteinoprostocrostostostostosidocaptophaloprooptosinopoproteinza anemia, anesthesia-anemia-anemia-anemia-anemia-anaineathrotostostostosidrotherapy syndrome, blast crisis, chronic myeloid leukemia, blast crisis). pronounced monocytosis in tuberculosis and other infectious diseases), “exit” from agranulocytosis, metastasis of solid tumors in the bone marrow.

The study of the bone marrow almost always allows to exclude non-hematological pathology, since in acute leukemia there is an increase in the content of blast cells. Detection of at least 20% of typical blast cells in a smear of peripheral blood and / or bone marrow eliminates most hematological diseases that have similar clinical and laboratory manifestations.

The exceptions are:

1) one of the variants of myelodysplastic syndrome (MDS) (refractory anemia with an excess of blasts – RAIB), which usually differs from acute leukemia only by the percentage of blast cells in the bone marrow;

2) blast crisis of chronic myeloblastic leukemia.

In 30% of patients over 60, the development of acute myeloblastic leukemia is preceded by one of the variants of myelodysplastic syndrome (MDS) (most often RIBS). In both cases, the results of treatment and the prognosis are usually unfavorable, therefore the differences between refractory anemia with an excess of blasts (RAIB) and secondary acute myeloblastic leukemia (AML) developed from myelodysplastic syndrome (MDS) do not have significant clinical significance.

For differential diagnosis with blast crisis of chronic myeloblastic leukemia (CML), it is necessary to consider the history of the disease, clinical data, the results of cytogenetic research and immunophenotyping. When formulating a diagnosis, it is necessary to isolate secondary acute leukemias (almost always acute myeloblastic leukemias (AML)) that developed after radiotherapy and / or chemotherapy for tumor and non-tumor diseases.

Myelogram with acute leukemia.

Trepanobiopsy is not a mandatory study for acute leukemia, but it is necessary to conduct it at low cellularity of the bone marrow or “dry” punctate to exclude aplastic anemia and subleukemic myelosis.

Cytological examination of the cerebrospinal fluid in acute leukemia is performed in all patients with acute leukemia before treatment. In the absence of pathology in acute lymphoblastic leukemia, myelomonoblastic and monoblastic acute leukemia, further prevention of neuroleukemia is carried out. With the development of neuroleukemia, it is treated, the results of which are evaluated on the basis of an analysis of the cellular composition of the cerebrospinal fluid.

Biochemical studies in acute leukemia. In most cases, biochemical parameters are within normal values, however, in certain cases of acute leukemia (ALL, monoblastic leukemia), impaired renal function (increased creatinine level) may be observed due to their infiltration with tumor cells. Specific renal infiltration and / or their increase can be documented using ultrasound or computed tomography. In some cases (with acute leukemia with hyperleukocytosis, acute lymphoblastic leukemia with organomegaly), tumor lysis syndrome is detected already in the debut of the disease.

More often, however, this syndrome occurs with the rapid lysis of cells during chemotherapy and is characterized by hypocalcemia, hyperkalemia, increased LDH levels, and hyperuricemia with the development of renal failure.

Instrumental studies for acute leukemia are not critical for acute leukemia, but their results can influence the nature of the treatment and the prognosis of the disease. Thus, radiography of the chest organs reveals an increase in mediastinal lymph nodes, pneumonia; electrocardiography – rhythm and / or conduction disturbances due to specific myocardial infiltration, anthracycline cardiomyopathy, etc.