Acute leukemias of unclear differentiation line

The category of such leukemias include acute leukemias, in which neither morphological, nor cytochemical, nor immunophenotypic methods allow to establish the differentiation of blasts along any line of hemopoiesis (acute undifferentiated leukemia), as well as observations when morphological and / or immunophenotypic data indicate the presence signs of maturation along two lines of hemopoiesis: myeloid and lymphoid or B- and T-lymphoid (acute bilinear and acute bifenotypic leukemia).

In acute undifferentiated leukemia, the morphological and cytochemical indicators of the blasts do not have any characteristic features. Cells react only with µa to early hematopoiesis precursors – stem-cell CD34, contain the enzyme deoxynucleotidyltransferase (TdT), and also express the antigens CD38 and histocompatibility class II HLA-DR.

The First European Group on the Immunological Classification of Leukemia (EGIL) recommended that further studies of this undifferentiated variant be conducted to prove the myeloid or lymphoid pattern of cell differentiation. It is proposed to use a wide panel of 33 immunological markers. According to the authors, the study of blast cells should be carried out in 2 stages. First, it is necessary to study the linear non-specific antigens TdT, CD34, HLA-DR and establish B, T and myeloid linear directivity. Then it is necessary to determine the stage of differentiation of leukemic cells. To clarify the diagnosis, it is proposed to investigate the rearrangement of immunoglobulin genes and T-cell receptors.

Cases of undifferentiated acute leukemia are sometimes referred to as stem-cell. It has been established that the group of patients with such leukemia is heterogeneous according to the blasts phenotype. The following immunodvariants of this acute leukemia are distinguished: 1. CD34 +, HLA-DR-, CD38-; 2. CD34 +, HLA-DR +, CD38 ~; 3. CD34 +, HLA-DR \ CD38 +.

Acute non-lymphoblastic leukemia due to therapy – diagnosis

Acute non-lymphoblastic leukemia and myelodysplastic syndrome can be caused by two types of therapy: either using alkylating agents and / or radiation therapy, or type II topoisomerase inhibitors. In the latter case, lymphoid leukemias can also be observed.

After therapy with alkylating agents and radiation, acute non-lymphoblastic leukemia occurs after 5 years or more. The risk of acute non-lymphoblastic leukemia depends on the total cumulative dose of the drugs, the age of the patient, and the combined use of the two methods of treatment. The onset of the disease is manifested in the form of myelodysplastic syndrome with cytopenia, multiline dysplasia.

Dysgranulo- and dyseritropoiesis are observed in the majority of patients, the number of basophils is increased. Often (up to 60% of cases) ring sideroblasts are found, in neutrophils, peroxidase deficiency is determined. As a rule, there is myeloblastic acute non-lymphoblastic leukemia, but there can also be myelomonoblastic, erythroblastic, megakaryoblastic and, less commonly, promyelocytic variants. The immunophenotype of blasts is characterized by heterogeneity.

They express myeloid antigens (CD13, CD33), early CD34, CD56 and / or lymphoid CD7. In the karyotype, unbalanced translocations or deletions of the 5th and / or 7th chromosome are often detected with the loss of all or part of the long arm of the chromosome. There are anomalies of other chromosomes: 1st, 12th, 14th, 18th, complex chromosomal aberrations.

Acute leukemia after therapy with topoisomerase II inhibitors occurs faster than after the use of alkylating agents. The latent period is on average 33-334 months. Among the drugs distinguish epipodophyllotoxins (etoposide and teniposide) and anthracyclines. Acute leukemias caused by these drugs, as a rule, belong to monoblastic or myelomonoblastic, although other variants of acute nonlymphoblastic leukemia, as well as acute lymphoblastic leukemia can occur. The most frequent violation of the karyotype is the presence of the llq23 abnormality as a result of translocations (9; 11), (11; 19), (6; 11).

In addition, translocations can be detected (8; 21), (3; 21), (6; 9). ALL is usually associated with t (4; ll) (q21; q23). In secondary acute leukemia, remission is less frequent and shorter than in primary.

Acute non-lymphoblastic leukemia with recurring cytogenetic abnormalities

Characteristic cytogenetic abnormalities in acute non-lymphoblastic leukemia (ONLL) are usually represented by balanced translocations. Such recurring, or non-random, cytogenetic anomalies include t (8; 21), t (15; 17), t (l6; 16), inv (16) and an anomaly llq23, resulting from the translocation of chromosome 11 with various partners (about 20) [61]. For most of these leukemias, a high frequency of complete remissions and a more favorable prognosis are typical. Some morphological features are characteristic of them.

Acute myeloid leukemia with t (8; 21) (q22; q22); (AML1 / ETO). This anomaly is detected in 5-12% of cases of acute non-lymphoblastic leukemia (ONLL), more often in young patients, occurs predominantly with myeloblastic leukemia with maturation (M2 according to FAB classification), sometimes with myeloblastic leukemia without maturation (Ml) or myelomonoblastic (M4).

Blasts express both myeloid (CD13, CD33), and often the lymphoid antigen CD 19. CD34 and CD56 are less commonly defined. For elements of the neutrophilic series, marked dysplasia, the presence of pelgheroid forms, homogeneous pink staining of the cytoplasm are characteristic. In mature neutrophils, in some cases Auer rods can be detected. The number of eosinophils may be increased, but there is no dysplasia in them. The presence of t (8; 21) gives a basis, when the number of blasts is less than 20%, to diagnose ONLL, and not MDS.

In ONLT with t (8; 21), the prognosis is good, with a high frequency of complete remissions and a long duration of survival with adequate treatment. Expression of the CD56 antigen worsens the prognosis.

Acute myeloid leukemia with inv (16) (pl3; q22) or t (16; 16) (pl3; q22); (CBFb / MYH11). In this case, leukemia (M4eo according to the FAB classification) in the bone marrow myeloblasts, monoblasts and anomalous eosinophil component are determined. Immunophenotyping on blasts reveals common myeloid antigens (CD13, CD33, MPO) and markers of monocytic differentiation (CD14, CD11b, CD11c, CD64, CD36, and also lysozyme). Promyelocytes and myelocytes of the eosinophilic series are distinguished by the presence of pathological granularity.

Determining the type of blast cells in acute leukemia

The study of α-naphthyl acetaserase plays a major role in the diagnosis of monoblasts M5a and M5b. This enzyme is found in most hematopoietic cells, but its isoform, inhibited by sodium fluoride, is characteristic only of cells of the monocyte-macrophage line. In determining non-specific esterase, various substrates are used with equal success: a-naphthyl acetate, butyrate, AS-chloroacetate, etc. Their diagnostic value is close.

The activity of the enzyme in monoblasts without maturation and with maturation, as a rule, is equally high. At the same time, using a panel of various substrates for nonspecific esterase, it is possible to carry out a presumptive differential diagnosis of monoblast and megacaryoblastic leukemias before immunophenotyping.

The result of the PAS reaction has a certain diagnostic value, since a characteristic of most myeloid blasts is the diffuse arrangement of the PAS-positive material in contrast to the lymphoblasts, which are characterized by the arrangement of the reaction product in the form of granules. The results of this study help to distinguish between MO ONLL and ALL. In some cases, in myeloblasts of type MO and erythroblasts, the PAS-positive substance may be in the form of granules.

Different types of blast cells in acute non-lymphoblastic leukemia (ONLL) differ in the antigenic structure of their membranes. The expression of antigens CD33, CD13 is characteristic of the cells of the granulocyte line, they react with ICA to peroxidase. The monocytic line blasts interact with the ICA for lysozyme. The presence of glycophorin A molecules is characteristic of erythroid cells, and CD41 and CD61 are characteristic of megakaryocytes.

Immunological data do not allow a differential diagnosis of granulocytic line blasts, however, the use of immunophenotyping data is crucial for the diagnosis of M0, M6 and M7 variants, since the morphological and cytochemical indicators of these cells do not allow to establish the type of blasts.

Mielosarcoma – morphology, diagnosis

Melosarcoma. This form is included in the WHO classification in the section of acute non-lymphoblastic leukemia (ONLL):

• extramedullary tumor from blast or maturing myeloid cells in patients with various myeloproliferative diseases or myelodysplastic syndromes;
• the presence of myelosarcoma in patients with acute non-lymphoblastic leukemia (ONLL) may be the first manifestation of the disease or a relapse of ONLL during remission. In patients with myeloproliferative diseases, it is associated with the onset of a blast crisis;
• blasts are more often represented by myeloblasts, but they can be cells of any myeloid line;
• type of blasts is determined on the basis of morphocytochemical and immunological studies;
• myeloblasts with maturation can have a translocation (8; 21), myelomonoblasts with eosinophils – inv16 or t (16; l6), monoblasts – anomaly 11q23.

The determination of the type of blast cells and, accordingly, the variant of acute non-lymphoblastic leukemia (ONLL) is based on the complex characteristics of the morphological, cytochemical and immunophenotypical features of leukemic elements.

The morphological criteria for the characteristics of blasts include cell size (ratio of macro-, meso- and microforms), the shape of nuclei (round, folded, monocytoid), the presence of grain and / or Auer rods, nuclear-cytoplasmic ratio (high, moderate or low). It is on the basis of morphological signs that leukemic myeloblasts and monoblasts are divided into cells with the presence or absence of signs of maturation.

Cytochemical markers of granulocyte blasts are peroxidase, lipids detected by Sudan black B, and ASD-chloroacetate esterase. The content of these markers in myeloblasts varies considerably, sometimes only one of them is detected. In the case of a dubious response, it is imperative to carry out two cytochemical reactions – peroxidase and lipids, in order to avoid a possible error.

The activity of ASD-chloroacetate esterase is significantly lower than peroxidase, therefore the definition of this enzyme is of lower diagnostic value. Low peroxidase activity in myeloblasts is a poor prognostic sign.

Acute myeloblastic leukemia with myelofibrosis (panmielosis with myelofibrosis according to the WHO classification) – diagnosis

Acute myeloblastic leukemia with myelofibrosis (panmielosis with myelofibrosis according to the WHO classification):
• a rare form of acute non-lymphoblastic leukemia (ONLL);
• cytopenia in the peripheral blood, poor uninformative punctate of the bone marrow;

• morphological, cytochemical and immunophenotypic studies of cells are difficult;
• blasts react with MCA to myeloid antigens CD33, CD13, CD117 and MPO. In the presence of erythroid or megakaryocyte antigens, an appropriate subtype of blasts is established;
• in trepanate – hyperplasia and dysplasia of varying severity of individual myeloid shoots, clusters of young elements, including blasts; • the degree of fibrosis varies, in most cases an increase in reticulin fibers is observed;
• the karyotype is usually altered. Complicated chromosomal abnormalities involving chromosome 5 or 7 are most frequent.

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.