Acute megakaryoblastic leukemia (M7 according to FAB classification) – diagnosis

Acute megakaryoblastic leukemia (M7 according to FAB classification):
• makes up 3-5% of all acute non-lymphoblastic leukemias (ONLL);
• in the bone marrow, the progenitors of the megakaryocyte series account for more than 50%;

• blasts are of two types:
1) with irregular outlines and pronounced basophilia of the cytoplasm, moderate nuclear-cytoplasmic ratio;
2) rounded with a high nuclear-cytoplasmic attitude, resembling lymphoblasts in appearance;

• in blasts, moderate activity of α-naphthylacetate esterase, resistant to sodium fluoride, and the absence of butyratesterase, is determined. In some cases, PAS-positive substance is located in the form of large clusters along the edge of the membrane against the background of diffuse staining of the cytoplasm;
• platelet peroxidase is determined by ultrastructural examination in the nuclear membrane and the endoplasmic reticulum of blasts;

• blasts express platelet antigens CD41 (glycoprotein IIb / IIIa) and / or CD61 (glycoprotein IIIa), less often CD42 (glycoprotein Ib), since the latter is expressed in more mature forms. For the diagnosis of M7 ONLL, it is necessary that the number of antigen-positive cells be more than 50%. CD13, CD33, CD36 are also detected;
• A variant of M7 leukemia in children with Down syndrome on the background of a transient myeloproliferative disease is separately highlighted. A number of patients may experience spontaneous remission.

Acute erythromyelosis and erythroid leukemia (M6a and M6b according to the FAB classification) – diagnosis

Acute erythromyelosis and erythroid leukemia (M6a and M6b according to the FAB classification): • accounts for 5-6% of all acute non-lymphoblastic leukemia (ONLL);

1) acute erythromyelosis:

• in the bone marrow, erythroid progenitors account for more than 50% of all cells, myeloblasts, more than 20% of cells of the non-erythroid population;
• cells of the red row have pronounced signs of dysplasia: megaloblastoid changes, dissociation of nuclear maturation and cytoplasm, uneven staining of the cytoplasm, the presence of Jolly bodies, multi-core forms. They increase the number of siderophilic granules, determine the positive PAS-reaction in diffuse and / or granular form;
• erythroid progenitors react with ICA to glycophorin A and hemoglobin A;
• myeloblasts contain granularity, single Auer sticks, are positive in reactions to peroxidase, ASD-chloroacetate esterase and lipids;
• blasts express myeloid lineage antigens CD13, CD33, CD117, and also sometimes CD34 and HLA-DR, react with MCA to peroxidase;

2) erythroid leukemia:

• the blast population prevails in the bone marrow (more than 80%);
• blasts of medium or large size with rounded nuclei, delicate chromatin structure and 1–2 nucleoli, cytoplasm basophilic, without grain, sometimes contains vacuoles;
• blasts do not have myeloperoxidase, lipids, contain granules of PAS-positive substance, nonspecific esterase, acid phosphatase;
• there are no myeloid immunological markers in blasts, early forms express CD36, more mature erythroid glycophorin A antigen;
• Multiple chromosomal rearrangements are detected, often involving chromosomes 5 and 7.

Acute monoblastic leukemia (M5a according to FAB classification) – diagnostics

Acute monoblastic leukemia without maturation (M5a according to FAB classification):
• accounts for 5–8% of all acute non-lymphoblastic leukemias (ONLL);
• blasts of large size, irregular outlines, with moderate or severe cytoplasmic basophilia, with rounded nuclei with 1-2 nucleols; in rare cases, erythrophagocytosis is observed;
• blasts contain a significant amount of nonspecific esterase suppressed by sodium fluoride, a small amount of peroxidase and / or lipids in individual cells;
• blasts express the myeloid antigens CD33, CD13, CD117 and monocytic – CD14, as well as CD4, CDllb, CDllc, CD64, CD36, react with MCA to lysozyme;
• translocations (9; 11), (11; 19), (4; 11) are often detected with the participation of the MLL gene localized at llq23. In accordance with the WHO classification, these observations are categorized as the acute non-lymphoblastic leukemia category with recurring cytogenetic abnormalities.

Acute monoblastic leukemia with maturation (M5b according to the FAB classification):

• makes up 3-6% of all acute non-lymphoblastic leukemias (ONLL);
• more than 20% of blasts have a characteristic monocytoid form of the nuclei, a weakly basophilic cytoplasm with a small granularity and vacuolization;
• blasts contain a significant amount of nonspecific esterase suppressed by sodium fluoride, a small amount of peroxidase and / or lipids in individual cells;
• blasts express the antigens CD33, CD13, CD14, CD15 and react with the ICA to lysozyme;
• t (9; ll), t (ll; 19), t (4; ll) are often detected with the formation of the MLL gene localized at llq23. In accordance with the WHO classification, these observations are categorized into an independent category of acute non-lymphoblastic leukemia (ONLL) with recurring anomalies.

Acute myelomonoblastic leukemia (M4 according to the FAB classification) – diagnosis

1) biclonal variant of acute myelomonoblastic leukemia:
• makes up 15-25% of all acute non-lymphoblastic leukemias (ONLL);
• blasts are represented by two types of cells – myeloblasts and monoblasts (biclonal variant);
• blasts express antigens CD33, CD13, CD14, CD15, react with MCA to peroxidase and lysozyme;
• llq23 or t (8; 21) anomaly can be detected;

2) bifenotypic variant of acute myelomonoblastic leukemia:
• makes up 1% of all acute non-lymphoblastic leukemias (ONLL);
• blasts are represented by a single cell type (bifenotypic variant);
• blasts are morphologically characterized as M2 myeloblasts;
• blasts contain peroxidase, lipids, granulocyte esterase and nonspecific esterase simultaneously suppressed by sodium fluoride in all cells;
• the immunophenotype of blasts is the same as that of cells in the biclonal variant;

3) myelomonoblastic with eosinophilia:
• M4eo accounts for about 10–12% of all acute non-lymphoblastic leukemia (ONLL);
• blasts are represented by two types of cells – myeloblasts and monoblasts;
• atypical eosinophils are present in the bone marrow, their number may exceed 6%. In eosinophilic myelocytes and metamyelocytes, large dark-colored granularity is detected;
• in blasts and eosinophils, inversion of chromosome 16 or t (l6; 16) is detected.

In accordance with the WHO classification, these observations are allocated to an independent category of acute non-lymphoblastic leukemia (ONLL) with recurring cytogenetic abnormalities.

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.”