Diagnosis of acute basophilic leukemia

De novo acute basophilic leukemia (ABL) is a rare form of acute leukemia. Most frequently, the blast cells are morphologically undifferentiated, and the recognition of the presence of coarse basophilic granules may be the first step in diagnosis of this rare disorder. These granules are metachromatic and MPO negative. Immunophenotyping shows myeloid markers and some more specifically associated antigens such as CD9 or CD25 which are strongly expressed. Lymphoid, erythroid or megakaryocytic markers are not significantly expressed. In the absence of basophilic granules, some cases are classified as AML M0 if they express myeloid markers, or undifferentiated leukemia if no markers are present. If specific immature basophilic or theta granules are present, only an electron microscopic study will enable the diagnosis of a basophilic lineage assignment. Some cases may be misdiagnosed if all these steps are not followed. After all these investigations, two types of ABL may be defined: 1-A pure ABL, monophenotypic with basophilic lineage involvement alone, which should be classified as AML M8 . Genetic studies in these cases are very important for understanding the leukemic process and in a few cases, we can suspect c-MYB oncogene involvement but further investigations are still necessary. 2- More frequently, acute leukemia can be a mixture of blasts from different lineages with an important but variable participation of mature or immature basophilic cells. These cases must be classified as AML/Baso or multiphenotypic acute leukemias and often present Phl -chromosomal abnormality.     

Expression of platelet glycoproteins by erythroid blasts in four cases of trisomy 21

In four patients with trisomy 21 (three constitutional, one acquired) with a morphological undifferentiated leukemia, diagnosis of erythroid leukemia was established by both immunophenotyping and ultrastructural studies. Indeed, a majority of blasts from three patients expressed several erythroid markers such as carbonic anhydrase 1, spectrin beta chain, and glycophorin A. In addition, band 3 and hemoglobin were immunologically detected in a fraction of the blast cells from two cases. At ultrastructural level, a majority or all blast cells exhibited erythroid differentiation features such as theta granules and ferritin molecules. However, platelet glycoproteins GP Ib, GP IIb, and GP IIIa were also immunologically detected in a fraction (from 14-82%) of the blasts. Since the ultrastructural study indicated that some promegakaryoblasts were also present in three patients, double labeling between erythroid markers (glycophorin A or carbonic anhydrase I) and platelet glycoprotein (Ib or IIIa) was performed and showed a clear overlap between the two kinds of markers. A similar approach was performed at ultrastructural level and indicated that blast cells with ultrastructural erythroid features of differentiation may have three distinct phenotypes, i.e., presence of glycophorin A without platelet glycoproteins or, conversely, the presence of platelet glycoproteins without glycophorin A and coexpression of glycophorin A and platelet glycoproteins. Expression of glycophorin A correlated directly with the differentiation level of the erythroid blasts, whereas platelet glycoproteins were essentially expressed in the more primitive leukemic erythroid cells. The GP Ib synthesized by these blasts was subsequently studied. The GP Ib alpha mRNA analyzed by Northern blot from these erythroid cells was identical in size with that from megakaryocytic cells as was the molecular weight of the GP Ib molecule from both after immunoprecipitation by a monoclonal antibody. Therefore, "in vivo" erythroid leukemic cells may express the main platelet glycoproteins including GP Ib.     

Early Erythroblastic Leukemia Presenting as De Novo Acute Leukemia

Early erythroblastic leukemia is a newly defined type of leukemia in which the blasts have the same characteristics as erythroid precursors at the level of CFU-E. The blasts are characterized by the presence of carbonic anhydrase I, CD 36 antigen, platelet peroxidase (PPO)-like activity, and ferritin-containing granules. Early erythroblastic leukemia appears to have characteristic clinical features; in the original report of nine cases, only one patient had typical de novo acute leukemia.

We report here a case of early erythroblastic leukemia that presented! as de novo acute leukemia. The blasts from this patient had almost the same ultrastructural and phenotypical characteristics as those of the originally reported cases, even though our case was not examined for anti-carbonic anhydrase I antibodies.

As a single marker, PPOl activity can no longer be considered specific for the megakaryocyte-platelet lineage, even though the significance of transient expression of PPO-like activity in immature erythroblastic cells at the level of CFU-E still remains to be clarified.

When leukemic blasts show positivity for CD36 and negativity for megakaryocytic or monocytic markers, the diagnosis of early erythroblastic leukemia should be suspected and electron microscopical characteristics should be studied.     

Acute transformation of chronic large granular lymphocyte leukemia associated with additional chromosome abnormality

A patient with large granular lymphocyte (LGL) leukemia that transformed into an acute or aggressive form after 20 months of the chronic phase is reported. The patient's leukemic cells were mature, medium-sized lymphocytes with sparse azurophil granules and the surface phenotypes of the cells were CD2+, CD3-, CD11+, and CD16+. Molecular analysis showed a germ line configuration in both T-cell receptor beta-chain genes and T-cell receptor tau-chain genes. A clonal anomaly of chromosome (trisomy 8) was demonstrated in peripheral blood cells. LGL after acute transformation of the disease displayed large blastic morphology with prominent nucleoli, intense basophilic cytoplasm, and numerous granules. Karyotypic analysis demonstrated a mosaic of trisomy 8 and trisomy 8 with an additional marker chromosome. Thus, transformation of chronic LGL leukemia into an acute or aggressive form in this patient was associated with morphologic and karyotypic changes of the leukemic cells. Patients with a stable form of chronic LGL leukemia should be examined carefully for the possible acute crisis associated with a clonal evolution.     

Ultrastructural and ultracytochemical differences between megakaryoblastic leukemia in children and adults. Analysis of 49 patients.

BACKGROUND. Acute megakaryoblastic leukemia (AMKL) has two peaks in distribution of incidence (in adults and children 1 to 2 years of age) and is frequently seen in children with Down syndrome. The current study was undertaken to disclose whether there were any differences between these groups. METHODS. Electron microscopic and ultrastructural cytochemical features of 49 children and adults with a AMKL or chronic myelogenous leukemia (CML) in megakaryoblastic crisis were compared. RESULTS. Blast cells from children with AMKL, including those with and without Down syndrome, had immature features lacking typical alpha granules and a demarcation membrane system (DMS). However, blast cells from patients with AMKL with Down syndrome had more theta, electron-lucent, and basophil-like granules, suggesting that the blast cells had more potential to differentiate into other cell lines than megakaryocytes. The AMKL blast cells of adult patients showed a higher percentage of platelet peroxidase (PPO) positivity than other subgroups, and they occasionally contained typical alpha granules and DMS. This indicated that the blast cells of adults with AMKL were more mature than those of children and CML in megakaryoblastic crisis. CONCLUSIONS. By electron microscopic analysis, leukemic megakaryoblasts differed between children with AMKL with and without Down syndrome, adults with AMKL, and patients with CML in megakaryoblastic crisis.     

Immunological characterization of the leukemic megakaryocytic line at light and electron microscopic levels

Twenty cases of leukemia involving platelet precursors have been identified by a panel of monoclonal and polyclonal antiplatelet antibodies and by the ultrastructural demonstration of platelet peroxidase (PPO). The two techniques were in close agreement both for identification and for the quantitation of the blast cells except in three cases where PPO was present in the absence of the immunological markers. The immunological appearance of the leukemic megakaryocytic precursors was identical to that of their normal counterparts; the cells were positive with J 15 (anti GP IIb-IIIa complex), C 17 (anti GP IIIa), J 2 (anti GP 26,000) AN 51 (anti GP Ib). A diffuse cytoplasmic labelling was observed with anti factor VIII vwF and anti platelet factor 4 (PF 4). In addition, the leukemic maturation was quite similar to normal megakaryocyte differentiation since in micromegakaryocytes the expression of Gp Ib was strong and an intense granular pattern of labelling with anti factor VIII vwF and anti PF 4 was observed. In no case was the leukemic megakaryocytic series labelled by anti-erythroid antibodies, anti myeloid antibodies or J 5, B 1, OKT 11 antibodies. Using ultrastructural immunoferritin with J 15 it was possible to demonstrate that labelling with this antibody only occured on PPO-positive cells. Immunogold or peroxidase labelling with AN 51 at the EM level in cases of mixed leukemia showed that Gp Ib was absent from proerythroblasts and myeloblasts. Therefore, in no case were specific platelet markers expressed in the leukemias of other cell lineages.     

Intracellular immunoglobulin in lymphocytes from patients with chronic lymphocytic leukemia: An immunoelectron microscopic study

The presence of ultrastructural distribution of intracellular immunoglobulin (Ig) in leukemic cells from patients with chronic lymphocytic leukemia (CLL) and in normal B cells were investigated by an immunoelectron microscopic technique. Most normal B cells had no intracellular Ig in spite of the presence of surface Ig. However, leukemic cells from 10 to 11 patients with CLL contained intracellular Ig and showed various staining patterns. In eight patients, Ig was present in the perinuclear space (PN), the endoplasmic reticulum (ER) and, if identified, the Golgi complexes. In four patients, most cells had diffuse staining of the cytoplasm. In five patients, Ig was detected in the ER-associated structures or the vesicles, in addition to the PN and ER. These findings suggest that CLL cells have a greater capacity to produce Ig than those of normal B cells and include various clones with distinct staining patterns of intracellular Ig.     

Hodgkin's disease and myelomonocytic leukemia: an ultrastructural and immunocytochemical study.

The ultrastructual and immunologic features of the initial Reed-Sternberg and Hodgkin cells are compared with the ultimate leukemic cell type in a child with Hodgkin's disease who subsequently developed acute myelomonocytic leukemia (AMML) following 29 months of chemotherapy. Hodgkin tumor cells contained cytoplasmic IgG and ultrastructurally resembled large immunoblasts, containing one or two round nuclei with large bizarre nucleoli, many polyribosomes, sparase endoplasmic reticulum, underdeveloped Golgi lamellae, and few cytoplasmic granules. The Hodgkin tumor cells displayed no evidence of phagocytosis. The leukemic monocytic cells did not contain cytoplasmic IgG and, ultrastrucally, exhibited and indented and irregular nuclear profile with less prominent nucleoli, numerous pleomorphic granules, a moderate number of free ribosomes, short segments of endoplasmic reticulum, and stacked Golgi lamellae. The cell surface was irregular and occasionally appeared involved in endocytic activity. These results indicate that the Hodgkin tumor cells originated from B lymphocytes rather than tissue macrophages, whereas the leukemic monocytes arose from the bone marrow-derived monocyte-macrophage series. The findings suggest further that AMML developing after Hodgkin's disease consitutes a second neoplasm rather than a leukemic transformation of Hodgkin tumor cells.     

T-cell lineage of a Friend virus-induced lymphatic leukemia in athymic rats

Athymic (rnu/rnu) and euthymic rats inoculated with the Friend virus-associated lymphatic leukemia virus developed lymphocytic leukemia. Neoplastic cells from these animals were evaluated by means of indirect immunofluorescence and flow cytofluorometry with monoclonal antibodies Ox-1, Ox-7, and W3/25, which react with surface antigens present on normal rat lymphoid cell populations. Lymphoid cells from leukemic animals revealed characteristic alterations in cell surface fluorescence profiles when compared to normal, healthy controls. Athymic and euthymic leukemic rats were similar in that many cells from both the spleen and bone marrow had markers on the cell surface normally found on thymocytes but not on mature peripheral lymphocytes. These studies provided evidence supporting the presence of T-lineage lymphocytes in the athymic rat. Further, this population of early or "pre"-T-lymphocytes included the predominant leukemia cell type induced by the Friend virus-associated lymphatic leukemia virus.     

Phenotypic and ultrastructural profile of M5 leukemia cells in peripheral blood and skin infiltrate

The leukemic cells circulating in the peripheral blood and invading the skin of a patient with type M5 myelomonocytic leukemia were compared using ultrastructural, cytochemical and immunological criteria. Neoplastic cells exhibited more differentiated morphologic features in the skin than in peripheral blood, resembling tissue macrophages. The cytochemical pattern did not show any appreciable difference, whereas the surface antigenic profile was dissimilar. Most circulating leukemic cells were Leu M1+ and Leu M3+, and the percentage of OKM1+ and OKIa-1+ cells varied in two different blood samples examined. Conversely, OKIa-1 monoclonal antibody stained virtually all the leukemic cells infiltrating the skin in the absence of any appreciable reactivity with the other monoclonal antibodies. The phenotype of the malignant cells in the skin did not vary during the clinical course of the disease. These observations suggest that the cutaneous microenvironment is able to induce leukemic cells to mutate their phenotypic features towards a more mature state, or that only relatively differentiated circulating leukemic cells are able to leave the bloodstream and colonize the skin.     

Ultrastructure and cytokinetics of leukemic myeloblasts containing giant granules.

Leukemic myeloblasts containing abnormal granules were studied with ultrastructural, cytochemical, and thymidine labeling techniques to evaluate defects in granulogenesis and proliferation. Giant granules (1 to 3 micron in diameter) and Auer rods were observed in leukemic cells from two patients, and only rarely were both abnormal granule types observed in the same cell. The lysosomal origin of these abnormal granules was demonstrated by their content of peroxidase, esterase, and anionic glycoconjugates. Fusion of small dense granules (less than 0.2 micron in diameter) appeared to be increased in cells containing Auer rods and/or giant granules, but fusion of intact primary granules (0.2 to 0.4 micron in diameter) and sequestration of cytoplasmic contents were observed only in giant granules and not in Auer rods. Although the small granules that fused to form giant granules and Auer rods appeared similar, there was no evidence for transformation of giant granules into Auer rods. In one patient, cells with abnormal granules could easily be distinguished from the larger population of cells that lacked abnormal granules. The perturbation of these two distinct populations by chemotherapy was evaluated with thymidine labeling experiments. A high percentage (2- or 3-fold greater) of the abnormally granulated myeloblasts incorporated tritiated thymidine when compared to myeloblasts without abnormal granules in the same specimen. This difference could have resulted from an underlying metabolic defect which affected both granulogenesis and cell division. These results demonstrate that the formation of giant granules in leukemic cells is morphologically similar to that observed in the Chediak-Higashi syndrome and that leukemic cells with abnormal granules may differ cytokinetically from uninvolved leukemic cells.     

Establishment and characterization of four human monocytoid leukemia cell lines (JOSK-I, -S, -M and -K) with capabilities of monocyte-macrophage lineage differentiation and constitutive production of interleukin 1

Four monocytoid cell lines, JOSK-I, -S, -M, and -K, were newly established successfully from peripheral blood of two cases of acute monocytic leukemia and one case each of acute myelomonocytic leukemia and chronic myelogenous leukemia in myelomonocytic blast crisis. In order to establish permanent cell lines, cultures of leukemic blasts were initiated in 96-well microtiter plates. Each cell line grew in a suspension culture with a doubling time of 24-32 h and has been serially maintained for over 20 mo. Each line had immature monocytic properties as judged from the results of cytological, immunochemical, and functional analyses. The cells showed a positive reaction for alpha-naphthyl butyrate esterase which was completely inhibited by sodium fluoride and exhibited immature monocytic features on electron microscopic observation. They also had surface markers specific for the monocyte-macrophage lineage. Chromosome analyses showed that each line had a variety of marker chromosomes; furthermore, these established lines exhibited high potentialities involving morphological and functional differentiation into more mature monocytic cells when induced by several chemical inducers. We also found that two of the established cell lines produced much interleukin 1 activity without any stimuli. These new lines might be valuable for studying the regulation of monocyte-macrophage differentiation and host defense mechanisms.     

A T chronic lymphocytic leukemia with large granular lymphocytes. Phenotype and functions of leukemic cells under in vitro treatment by differentiation inducers

We reported the morphologic, phenotypic and functional characteristics of leukemic cells with natural killer (NK) properties in a case of T chronic lymphocytic leukemia with large granular lymphocytes. These cells were cytologically and cytochemically characterized as phosphatase acid positive large granular lymphocytes (LGL), and presented parallel tubular arrays at the ultra structural level. They displayed a CD2, CD3, CD8, CD11, Leu 7, and Leu 11 positive phenotype while they lacked B cell markers including surface immunoglobulins. In addition, they expressed human leukocyte antigens (HLA) Class I, but no Class II antigens. These phenotypic studies were also performed after cells were cultured in vitro with 12-0-tetradecanoyl phorbol 13-acetate, gamma interferon, 5-azacytidine, sodium butyrate, phytohemagglutinin, and interleukin 2 (IL2). The cell surface markers underwent several significant changes. Among them we noted a higher percentage of labeled cells with anti-CD6 and CD7 monoclonal antibodies (moAbs), and a positivity with an anti-CD19 (B4) moAb. The leukemic LGL spontaneously developed a NK activity on K 562 tumor cells, which was not affected under the various T and B cells growth factors because they became more sensitive to IL2; but they were also stimulated by a 50-kilodalton (KD) B cell growth factor (BCGF) factor devoid of any T cell proliferation activity. Together these results gave a better characterization of azurophilic granules containing T chronic lymphocytic leukemia, and enabled the documentation of the differentiation of LGL with NK activity.     

Correlation between Intracellular and Extracellular Lysozyme in Acute (Myelo)Monocytic Leukemia

The localization of myeloperoxidase (MPO) or lysozyme was studied in leukemic cells by electron microscopy in an attempt to investigate why the level of lysozyme activity increases in the serum of patients with acute (myelo)monocytic leukemia. The specimens were obtained from 3 patients with acute monocytic leukemia and 4 with acute myelomonocytic leukemia with extremely elevated serum lysozyme levels (more than 100 μg/ml, normal: ∼10µg/ml). The granules containing MPO or lysozyme, surrounded by microfilaments, were observed in the periphery of the cytoplasm. MPO positive granules were considered tot contain lysozyme. Some granules appeared to be degranulation from the cell-membrane of viable leukemic cells. This observation supports the concept that there is a mechanism by which lysozyme-positive granules degranulation from viable leukemic cells and as a result the level of lysozyme activity becomes elevated in the serum. It also appears that MPO is discharged into the serum at the same time as lysozyme: is released.     

髓系/自然杀伤细胞祖细胞急性白血病(附1例报道并文献复习)

目的：提高对髓系／自然杀伤细胞祖细胞(myeloid／Datural killer cell precursor,M／NKP)急性白血病的认识。方法：报告1例M／NKP急性白血病并复习相关文献。结果：M／NKP急性白血病起源于髓系和NK细胞共同的祖细胞。白血病细胞形态学特征类似急性淋巴细胞白血病L型．胞体大小不等,核圆或不规则,核仁明显,胞质苍白,无嗜天青颗粒,髓过氧化物酶(MPO)染色阴性,免疫表型特征为表达CD7,CD33,CD34,CD56和HLA-DR,其他NK细胞及T、B细胞分化抗原阴性。临床特征为髓外浸润较常见,急性髓系白血病化疗方案疗效相对较好,但预后差。结论：M／NKP急性白血病具有独特的临床和实验室特征,是有别于其他NK细胞及髓系细胞恶性疾病的一类独立疾病。     

Immunofluorescence and histologic studies of virus-induced murine lymphocytic leukemias.

Mice were inoculated with either the lymphatic leukemia virus associated with Friend and Rauscher viruses or with the Graffi or BALB/Tennant leukemia viruses. A total of 48 leukemic mice were examined histologically and by immunofluorescence. All four viruses induced a histologically similar disease that particularly involved tha thymus-dependent lymphoid regions. Lymphocytes from the spleens and thymuses of leukemic animals were examined for Thy-1 antigen and immunoglobulins on the cell surface; all the leukemias were composed of T- cells and/or nonreactive cells lacking both the Thy-1 antigen and immunoglobulins. By immunofluorescence, the spleen and thymus from the same leukemic animal frequently showed different cell-surface markers, though no morphologic differences were seen in leukemias involving the different classes of lymphocytes.     

Heterogeneity in TPA-induced differentiation of B-chronic lymphocytic leukemia cells: development of hairy cell or plasmacytoid features are time and dose dependent

Cells from 11 chronic lymphocytic leukemic (CLL) patients were induced to differentiate with various doses of tetradecanoyl phorbol-13-acetate (TPA) and the degree of induction was followed up to six days by measuring the expression of two surface membrane markers (SmIg and GP-70), Ig secretion, tartrate-resistant acid phosphatase (TRAP), and ultrastructural changes. The results indicate dose and time dependency of the TPA effect and a great heterogeneity in the response to TPA among cells from different CLL patients. Furthermore, the two main TPA-induced features, the "plasmacytoid" or "hairy cell" features depended on the dose and duration of treatment with the phorbolester. The plasmacytoid features were more frequently encountered at low doses (1 ng/ml) of TPA and were evident after short exposures to TPA (1-2 days). The hairy cell features were more obvious after incubation with higher doses of TPA (10-100 ng/ml) or at Day 6 with lower doses of TPA. The differentiation features measured, including cell morphology, surface membrane markers, Ig secretion, and TRAP staining, appeared to be independent of each other suggesting an autonomous pathway of differentiation for some of these features.     

Megakaryoblastic leukemia in an infant. Establishment of a megakaryocytic tumor cell line in athymic nude mice

A 17-month-old infant with clinical and pathologic features of acute megakaryoblastic leukemia and myelofibrosis developed soft tissue metastases. Tissue from an orbital metastasis was biopsied and transplanted into a nude mouse. Histologically the tumor was composed of pleomorphic cells with single convoluted nuclei or multilobed nuclei and prominent granular cytoplasm and had an alveolar histologic pattern in some areas. The ultrastructural features of the tumor cells include multilobed nuclei with prominent nucleoli and cytoplasmic granules with the characteristics of alpha-granules. The tumor has been successfully passaged over a 1-year interval and appears to be a stable megakaryoblastic tumor cell line (CHRF-288). Cells from the tumor line are reactive for factor VIII related antigen and also have GpIIb IIIa complex antigen on the plasma membrane surface. This tumor line may be a useful system for investigation of megakaryocytic functions including the production and regulation of the various factors they produce.     

The Surface Phenotype of Lymphatic Leukemia, Cells: An Immunoscanning Electron Microscopy Study

Bone-marrow cells from 11 cases of T- and 18 cases of B-lymphatic leukemia, at different maturation stages, were examined by scanning electron microscope (SEMI. All cases were extensively studied for the expression of surface markers by immunofluorescence. In addition six cases of T- and 10 cases of B-cell leukemia were labeled with a panel of monoclon;tl antibodies (including CD3, CD5, CD7, CD10, CD4, CD8, CD19, CD20 and CD22) and, after incubation with a colloidal gold conjugate, observed with SEM in the back-scattered electron imaging mode. Early stages of leukemic lymphoid B- and T-cell differentiation are characterized by prevalently smooth cell surfaces. Short stub-like microvilli constantly appear on more mature T cells, while complex surface features like small ruffles and pleomorphic microvilli are present in well-differentiated B-cell proliferations. Surface microvilli can be interpreted als structural features of lymphoid cells, progressively expressed with maturation and differentiation of leukemic as well as normal cells.     

Monocytic involvement by monosomy 7 preceded acute myelomonocytic leukemia in a patient with myelodysplastic syndrome

Novel techniques were used to detect which cell lineages were affected by monosomy 7 in a patient who had myelodysplastic syndrome and later developed acute leukemia. The patient had had paroxysmal nocturnal hemoglobinuria for 20 years before developing refractory anemia with excess of blasts. Cytogenetic analysis at the myelodysplastic stage disclosed monosomy 7 in bone marrow mitoses. Restriction fragment length polymorphism analysis of fractionated white blood cells with the chromosome 7-specific probes MetH and MetD revealed that blood monocytes and most bone marrow erythroblasts but not blood granulocytes or lymphocytes were affected by monosomy 7. The patient later developed acute myelomonocytic leukemia with blast cells positive for markers of the myelomonocytic lineage but negative for granulocytic markers in a standard surface marker analysis. The leukemic blast cells had monosomy 7 as determined by direct cytogenetic investigation. Thus, the monocytes were found to be affected by monosomy 7 in this patient 8 months before her myelodysplastic syndrome progressed to acute myelomonocytic leukemia, and the affected cells had the same biologic markers at both stages of the disease.