Ultrastructural study of liver sinusoids of mice during invasion by leukemic myelocytes.

Results of an ultrastructural study of liver of RF mice during invasion by leukemic myelocytes were reported. In early stages of infiltration, leukemia cells adhered to the endothelial cells of the sinusoidal wall; gaps 1-4 mu in diameter then developed in the endothelium, and leukemia cells passed through the gaps to enter the extravascular space. Other sinusoids became occluded by leukemic myelocytes, the endotheilium disintegrated, and the occluding cells thus became extravascular. In late stages of infiltration, when leukemia cells migrated back into the sinusoids, the endothelium was continuous and leukemia cells passed through temporary pores located within endothelial cells.

Ultrastructural features of basophil and mast cell granulopoiesis in blastic phase Philadelphia chromosome-positive leukemia.

Ultrastructural and ultracytochemical studies were performed on blood and bone marrow specimens from 18 patients with Philadelphia chromosome-positive blastic leukemia; 7 patients were in blast transformation following a typical history of chronic myelogenous leukemia and 11 patients presented with "acute leukemia." The patients were divided into 2 morphologic groups on the basis of light microscopic and cytochemical observations. In group I, which consisted of 11 patients, the proliferating cells were "lymphoid" in appearance and demonstrated many cytochemical, biochemical, and immunologic features similar to those of the lymphoblasts of non-T, non-B acute lymphoblastic leukemia. In group II, which consisted of 7 patients, the proliferating cells were myeloid in appearance. On the basis of ultrastructural observations, the 11 group I patients were divided into 2 subgroups, A and B. Subgroup IA, consisting of 5 patients, was characterized by blasts that demonstrated no differentiating features. In subgroup IB, consisting of 6 patients, 20-30% of the leukemic cells contained inclusions that resembled leukemic mast cell or basophil granules. The leukemic cells in the 7 group II patients manifested myeloid characteristics by light microscopy and prominent basophil and mast cell granulopoiesis by electron microscopy. Abnormalities of other myeloid cell lines were also observed in both the lymphoid and myeloid groups of patients.

Immunologic, biochemical, and ultrastructural characterization of the leukemia cell in F344 rats.

Immunologic, biochemical, and morphologic characteristics of the mononuclear cell from the leukemia of F344 rats were determined. The cells were morphologically similar to large granular lymphocytes (LGL). Surface marker analysis revealed Fc gamma receptors, no Fc gamma receptor or complement receptor activity, and an inability to spontaneously rosette guinea pig erythrocytes. Leukemia cells also had a surface immunoglobulin that hemagglutinated normal rat erythrocytes. The surface immunoglobulin and Fc gamma receptors dissociated from the cell after 2 hours of in vitro incubation, but Fc gamma receptor activity was reexpressed after 6 hours of in vitro incubation. Cells were capable of adherence to glass surfaces but had a low capacity for phagocytosis of latex beads. Cytochemical analysis revealed a consistent, strongly positive reaction for esterase that was sensitive to NaF. The cytochemical profile of the leukemia cell was similar to that described for LGL.

Expression of a cell surface glycoprotein (p180) related to cell-substratum adhesion during differentiation of mouse myeloid leukemia cells.

Mouse myeloid leukemia M1 cells were induced to differentiate in vitro into macrophages and granulocytes by various inducers including ascitic fluid. Differentiated M1 cells induced with ascitic fluid expressed a differentiation-associated cell surface glycoprotein with a molecular weight of 180,000 (p180), which can be labeled by lactoperoxidase-catalyzed radioiodination or metabolic labeling with L-[14C]fucose. p180 was also induced by treatment with conditioned medium of hamster embryo cells, dexamethasone, dibutyryl cyclic adenosine 3':5'-monophosphate, and prostaglandin E1. Ascitic fluid, conditioned medium of hamster embryo cells, and dexamethasone induced all the differentiation-associated properties tested, whereas dibutyryl cyclic adenosine 3':5'-monophosphate and prostaglandin E1 induced lysozyme activity and adhesiveness to the substratum but not phagocytosis, locomotive activity, Fc receptors, or morphological changes. The adherent cells induced by dibutyryl cyclic adenosine 3':5'-monophosphate produced a large amount of p180, while the floating cells produced very little, but no difference was detected in the lysozyme activities of the two cell types. These results suggest that p180 is associated with cell-substratum adhesion of differentiated M1 cells.

Growth of human malignant micromegakaryocytes in vitro.

Mononuclear cells from the peripheral blood of a patient with megakaryoblastic transformation of Philadelphia chromosome-positive chronic myelogenous leukemia were cultured. Morphological and cytochemical studies and cell ploidy determinations were done daily for 4 days. PAS staining of the cells increased progressively during culture. Ultrastructural study of circulating and cultured cells revealed demarcation membranes and alpha granules indicating the cells were micromegakaryocytes. Deoxyribonucleic acid synthesis, determined by 3H-thymidine uptake, peaked at 72 hours. The DNA content of cultured cells was diploid at all times. All 15 metaphases analyzed at 72 hours were Ph1-positive. Malignant (Ph1-positive) megakaryoblasts and micromegakaryocytes grown successfully were capable of partial cytoplasmic maturation as demonstrated by glycogen deposition and increase in subcellular organelles, while endoreduplication was impaired. Malignant megakaryoblasts and micromegakaryocytes can be grown successfully in short term liquid culture and have more complete maturation in vitro than observed in vivo.

Granulocytic sarcoma of the brain after renal transplantation.

A case of granulocytic sarcoma of the brain in a renal transplant recipient treated with immunosuppressive agents is presented. While the increased risk of malignant lymphoma, particularly large-cell lymphoma (reticulum cell sarcoma, histiocytic lymphoma, immunoblastic sarcoma) in these patients is well known, this appears to be the first report of a granulocytic sarcoma. Granulocytic sarcoma, a rare tumor composed of immature granulocytic elements, almost never involves the parenchyma of the brain. Histochemical examination may be necessary to distinguish this lesion from other poorly-differentiated neoplasms.

The acute monocytic leukemias: multidisciplinary studies in 45 patients.

The clinical and laboratory features of 37 patients with variants of acute monocytic leukemia are described. Three of these 37 patients who had extensive extramedullary leukemic tissue infiltration are examples of true histiocytic "lymphomas." Three additional patients with undifferentiated leukemias, one patient with refractory anemia with excess of blasts, one patient with chronic myelomonocytic leukemia, one patient with B-lymphocyte diffuse "histiocytic" lymphoma and one patient with "null" cell, terminal deoxynucleotidyl transferase-positive lymphoblastic lymphoma had bone marrow cells with monocytic features. Another patient had dual populations of lymphoid and monocytoid leukemic cells. The true monocytic leukemias, acute monocytic leukemia (AMOL) and acute myelomonocytic leukemia (AMMOL), are closely related to acute myelocytic leukemia (AML) morphologically and by their response to chemotherapy. like AML, the leukemic cells from the AMMOL and AMOL patients form leukemic clusters in semisolid media. Cytochemical staining of leukemic cells for nonspecific esterases, presence of Fc receptor on the cell surface, phagocytic ability, low TdT activity, presence of surface "ruffles" and "ridges" on scanning EM, elevations of serum lysozyme, and clinical manifestations of leukemic tissue infiltration are features which accompanied monocytic differentiation in these cases.

Ultrastructural cytochemical localizations by back-scattered electron imaging of white blood cells.

White blood cells have been studied in the back-scattered electron imaging (BEI) mode of scanning electron microscopy (SEM) with cytochemical methods for endogenous peroxidase, acid phosphatase, and a silver-staining method for nuclei. Peroxidase-positive granules were seen with good contrast and resolution in myeloid precursor cells and acid phosphatase activity was easily detected in macrophages and monoblasts. Silver staining permitted recognition of the shapes and location of the nuclei. In spite of the cytochemical procedures, cell surface structures were reasonably well-preserved in all methods, making direct correlation of BEI and secondary electron imaging (SEI) images an attractive feature in cell research with the scanning electron microscope.

Blastic transformation of a well-differentiated monocytic leukemia: changes in cytochemical and cell surface markers.

The clinical course of a patient with a well-differentiated monocytic leukemia which later underwent blastic transformation is described. Cytochemical, ultrastructural and cell surface analysis data were obtained at periods throughout her illness and correlated with the blastic transformation. Although surface markers characteristic of monocytic leukemia persisted, a deficiency of peroxidase in the granules of this patient's monocytes was observed as well as loss of alpha-naphthyl butyrate esterase staining during transformation.

Studies on chronic myeloid leukemia cell populations with colony-forming abilities in PHA-leukocyte feeder and Robinson assays.

Investigation of leukemic colony-forming cells (CFC) in PHA-supplemented cultures requires removal of T lymphocyte precursors prior to culture. Using a method of discontinuous density gradient centrifugation with concurrent depletion of E-rosette forming cells, T lymphocytes were effectively separated from light density CML bone marrow and blood cell fractions. Consequently, in light density fractions (1.056 and 1.059 g/ml) pure leukemic colony growth was obtained in the PHA-leukocyte feeder (PHA-l.f.) assay. Fraction 1.062 g/ml also yielded pure leukemic colonies in most experiments. Comparison of the density distributions of leukemic PHA-l.f. CFC and Robinson CFC revealed that both CFC populations had congruent density profiles in most patients. In others PHA-l.f. CFC were found to be of somewhat higher density than Robinson CFC. The most striking divergence was apparent in a patient in blast crisis. The findings suggest that different subsets of precursor cells within the CML population proliferate in PHA-l.f. and Robinson colony methods. Both colony techniques are thus potentially useful for discriminating subpopulations of colony-forming cells in chronic myeloid leukemia.

"Undifferentiated" large cell malignancies: an ultrastructural and immunocytochemical study.

One of the most difficult areas of surgical pathology is the classification of "undifferentiated" or "anaplastic" large cell malignant tumors. The differential diagnosis of these tumors includes poorly differentiated carcinomas, large cell malignant lymphomas, and amelanotic malignant melanomas. In this study of 56 such cases, the application of electron microscopy and, in selected instances, of indirect immunofluorescence and immunoperoxidase staining methods have helped us reach a precise histopathologic diagnosis in the majority of neoplasms that were considered to be "undifferentiated" by light microscopy.

Apparent discharge of cell fragments from blasts cells in acute leukemia. An ultrastructural study.

In the blast cells of three patients who had leukemia, formation of small surface blebs and the apparent discharge of small cytoplasmic fragments were observed by light and electron microscopy. An ultrastructural study showed that they consisted of either double membrane-lined vacuoles or solid cytoplasmic fragments. The exact cause of this phenomenon is not known, but may be related to the complex alterations in the surfaces of malignant cells.

Megakaryoblastic transformation of Ph positive chronic granulocytic leukemia.

A case of well-documented and illustrated megakaryoblastic transformation in a patient with chronic granulocytic leukemia is presented. The salient features of this case were the presence of megakaryoblasts in the peripheral blood and bone marrow and characteristic cytochemical and electron microscopic findings. In addition, the authors observed an unusual, previously unreported, similarity of the abnormal platelets with those described in the Gray platelet syndrome. A literature review of the 13 previously described cases is included.

Cytochemical diagnosis of acute myelomonocytic leukemia.

Blood and bone-marrow smears from adult patients with acute leukemias were stained for esterase reaction, consecutively with naphthol AS D-chloracetate (chloracetate esterase) followed by alpha naphthyl butyrate (nonspecific esterase). The two substrates were, respectively, granulocyte- and monocyte-specific. By this method three subgroups of acute nonlymphocytic leukemias could be distinguished. Leukemic cells may be positive for either chloracetate esterase or nonspecific esterases, and the authors believe these two subgroups represent "true" granulocytic and "pure" monocytic leukemias. In a third group, leukemic cells contained both esterases in the same cell, and it is believed this group may represent "true" myelomonocytic leukemias. In the majority of patients in this group, leukemia evolved from a preleukemic phase. When only Romanowsky-stained smears are used, the monocytoid feature and absolute elevated monocyte counts in acute granulocytic leukemia may lead to an erroneous diagnosis of the leukemia as acute myelomonocytic leukemia. This happened in five of the 13 cases in the study. The presence of granulocyte- and monocyte-specific esterases in a single cell supports the concept of a common origin of granulocytes and monocytes. The authors conclude that the combined esterase reaction can distinguish among acute granulocytic leukemia, acute monocytic leukemia, and acute myelomonocytic leukemia.

Megakaryoblastic transformation of chronic granulocytic leukaemia. An electron microscopy and cytochemial study.

Morphological, cytochemical, and ultrastructural electron microscopic (EM) studies were performed on blood and bone-marrow cells of case of Ph1-positive chronic ganulocytic leukaemia in megakaryocytic acute transformation. The entire leukaemic cell population was found to consist and of megakaryoblasts and megakaryocytes. Intermediate stages of maturation between blasts and micromegakaryocytes were observed at EM level.

Acute myelogenous leukaemia with bilateral mammary gland involvement.

A 34-year-old woman developed acute myelogenous leukaemia in the course of pregnancy and, after delivery of a normal baby, developed multiple bilateral breast masses composed of myelogenous tissue.

A comparative study of chromosome G-banding using trypsin, papain, and pretreatment with emulphogene.

G-banding of chromosome metaphase preparations derived from haemic cells of healthy individuals and from patients with acute myeloid leukaemia was performed with the aid of trypsin, papain, and pretreatment of the chromosome spreads with emulphogene before proteolytic digestion. Papain digestion revealed more distinguishable bands than did trypsin digestion. Pretreatment of the chromosome spreads with emulphogene greatly enhanced the number of distinguishable bands for both enzymes. The combination of pretreatment with emulphogene and digestion with papain revealed optimal numbers of bands for individual chromosomes essentially identical with those agreed at the Paris Conference 1971. The use of the emulphogene-papain technique appears also to offer an advantage in the banding of chromosomes from leukaemic cells.

Significance of Phi bodies in acute leukaemia.

Material from 39 patients with acute leukaemia was investigated with the peroxidase cytochemical reaction using 3,3'diaminobenzidine (DAB) and other substrates in order to test their sensitivity in detecting myeloid differentiation. The proportion of positive blasts and of cases with Auer rods in acute myeloid leukaemia (AML) was significantly greater with DAB than with benzidine. In addition, Phi bodies were demonstrated in AML blasts only when DAB was used; Phi bodies were also observed in two out of seven cases of chronic granulocytic leukaemia in "myeloid" blast crisis but were not seen in any case of acute lymphoblastic leukaemia. Phi bodies were more numerous when the reaction was carried out at pH 9.7, and their number was significantly reduced in the presence of 3-amino 1,2,4-triazole. Both findings suggest that the Phi bodies derive from catalase-containing granules (microperoxisomes) and are distinct from Auer rods, which derive from peroxidase-containing (primary) granules. Like Auer rods, Phi bodies appear to be characteristics of immature myeloid cells in leukaemia but are seen with a higher frequency than Auer rods in acute myeloid leukemia.

Variations in the activity of nucleolar organizers in different tissues, demonstrated by silver staining of human normal and leukemic cells.

A simple silver-staining technique that demonstrates those nucleolar organizing regions of metaphase chromosomes which are transcriptionally active during the preceding interphase (AgNORs) has been applied to cells obtained from the bone marrow and mitogen-stimulated peripheral blood lymphocyte cultures of hematologically normal individuals and patients with various forms of leukemia. In the majority of bone marrow cells from the normal controls and many of the patients, the number of cells with detectable AgNORs, and the staining intensities in those cells which were Ag+, were markedly reduced compared with the levels found in blood lymphocytes. The numbers of cells having satellite associations and the numbers of chromosomes participating in these associations also generally reflected the proportions of AgNORs present. When patterns of bone marrow silver staining were compared between patients with leukemia, distinct differences were found which could be correlated with cytology. It is suggested that different cell types have characteristic AgNOR staining profiles, reflecting specific regulation of ribosomal RNA synthesis in particular cell lineages. AgNOR staining may indicate, therefore, the predominant cell types that divide in the bone marrows of patients with different forms of leukemia.

3. The value of chromosome studies in the classification and management of the leukemias.

Chromosome identification techniques have shown the non-random nature of cytogenetic changes in leukemia. In addition, they have identified structural chromosome abnormalities occurring in specific types of acute leukemia as classified by the FAB criteria. Such cytogenetic sub groups are associated with differing prognoses. In acute lymphocytic leukemia, even the ploidy of the leukemic cells appears important in predicting prognosis. Identification of the Philadelphia (Ph1) chromosome has diagnostic significance in chronic granulocytic leukemia. This makes possible the classification of patients into Ph1 + and Ph1 - varieties which have different responses to therapy. Similar variations are found between the 2 groups of patients who do or do not develop additional abnormalities with blastic transformation. The implication of these findings in both acute and chronic leukemia may influence choice of therapy in the future.