Establishment of a Ph1-positive human cell line (BV173).

A new cell line (BV173) derived from a patient with Philadelphia chromosome (Ph1)-positive acute leukemia was compared with the Ph1-positive K562 and NALM-1 lines, which display the phenotypic characteristics of erythroid and pre-B cells, respectively. BV173 cells retained the Ph1 chromosome and had the morphologic and cytochemical features of undifferentiated blast cells. They lacked the membrane characteristics of mature B- or T-lymphocytes and did not react with monoclonal antibodies to the myelomonocytic cell lineage. Although they reacted with anti-glycophorin A antiserum, they failed to produce hemoglobin after butyric acid treatment. This line was similar to NALM-1 in that it bore common acute lymphoblastic leukemia antigen and la-like antigen, reacted with monoclonal antibodies directed against early stages of hematopoietic cell differentiation, and presented the nuclear enzyme terminal deoxynucleotidyl transferase. However, it differed from NALM-1 because it did not express cytoplasmic IgM, a marker of pre-B-cells. The new line can be considered a clonal expansion of leukemia cells blocked at an earlier differentiation stage than that for the other Ph1-positive cell lines.

Evidence that B-lymphocytes carry the nuclear pocket abnormality associated with bovine leukemia virus infection.

Peripheral blood lymphocytes from 3 cows with persistent lymphocytosis were separated on nylon wool columns into nylon wool-adherent and nonadherent populations. The percentage of cells coated with surface immunoglobulin (B-cells) and the frequency of lymphocytic nuclear pockets in each sub-population were then determined. In each case the adherent population consisted predominantly of B-cells with an increased nuclear pocket frequency, whereas the nonadherent cells were 98.99% negative for surface immunoglobulin (non-B-cells) and contained essentially no nuclear pockets. These findings provided additional evidence that the B-subpopulation of cells was highly involved in bovine leukemia oncogenesis.

Use of multiparameter studies and scanning electron microscopy in the interpretation and attempted correlation of surface morphology with cell type in 135 cases of human leukemias.

Multiparameter studies and scanning electron microscopy (SEM) were performed on cells obtained from 135 cases of leukemia in an attempt to clarify whether there was a reliable correlation between surface morphology and cell type as defined by cytochemistry, membrane markers, and transmission electron microscopy. These studies also attempted to determine whether SEM could be used to distinguish lymphoid and nonlymphoid leukemias, to recognize different types of lymphoid leukemia, and to define the cell type involved in cases of unclassified leukemia. The results of this study suggest that there is a good correlation between surface morphology as seen by SEM and cell type identified by multiparameter techniques. In most cases, nonlymphoid leukemic cells could be distinguished from lymphoid leukemic cells on the basis of their surface morphology. SEM did not appear to contribute to the diagnosis of unclassified leukemia, but more cases of this nature must be studied. Despite the fact that acute lymphoblastic leukemia cells frequently showed fewer microvilli than did other lymphoid leukemias, overlap of surface features in about one-third of the cases did not enable SEM to be used as a reliable means of distinction. The above conclusions appear to be supported by preliminary scanning immunoelectron microscopic observations on leukemic cells. It is concluded that SEM is a useful aid to other modes of microscopy in leukemia but should not be used on its own to establish diagnosis.

Strong, specific anti-human leukemia antisera prepared with the use of purified cell membrane antigen.

Two rabbits immunized with 15 micrograms of a purified human thymus leukemia-associated antigen preparation and boosted once with the same amount of the antigen preparation yielded antisera that showed strong specificity for human leukemic T-cells without any prior absorptions. These antisera from the two rabbits showed a 50% killing of cells at antiserum dilutions of 5700- and 1600-fold, respectively, against JM, a leukemic T-cell line, and slightly weaker activity against MOLT-4, another leukemia T-cell line. These antisera, without any absorption, showed no or minimal reaction against two nonmalignant B-cell lines (RPMI 1788 and RPMI 8057), a leukemic non-T, non-B-cell line (NALM-16), a leukemic pre-B-cell line (NALM-1), normal peripheral blood lymphocytes, and T-cells isolated from peripheral blood lymphocytes. Antiserum 7557, which showed the higher antibody activity, was further studied by an absorption test using various human cell lines. The antiserum showed strong activity against all three leukemic T-cell lines tested, i.e., CCRF-CEM, RPMI 8402, and CCRF-HSB-2, whereas it showed no significant activity against other cell lines which included two leukemic non-T, non-B-cell lines (KM-3 and NALM-6), NALM-1 and RPMI 1788. These are the first anti-human leukemia antisera, except for monoclonal hybridoma antibodies, that showed good specificity for leukemia cells without prior absorption. The present procedure of immunizing animals with a small amount of human thymus leukemia-associated antigen preparation isolated from cell membrane will also be useful for obtaining strong, specific antisera of other cell membrane antigens.

Association of specific chromosome abnormalities with type of acute leukemia and with patient age.

Complete data regarding age, sex, karyotype, and French-American-British Cooperative Group classification were available for 239 unselected patients with acute nonlymphocytic leukemia. Of these, 128 were classified as having acute myeloblastic leukemia (M1 or M2); within the acute myeloblastic leukemia group, 83 (65%) of the patients were chromosomally abnormal. Except for 16 patients with a t(8;21), the percentage of patients with an abnormal karyotype increased with age, particularly above the age of 50 years. Besides the patients with t(8;21), there were 29 patients with loss of part or all of chromosomes 5 and/or 7, 11 patients with +8, and 27 patients with other abnormalities. Of 70 patients with acute myelomonocytic leukemia (M4), on the other hand, 28 (40%) were chromosomally abnormal, only three had loss of chromosomes 5 or 7, one was -7, +8, and our were +8, whereas 20 had other abnormalities. This difference may reflect different etiological factors in these two types of leukemia.

Role of cytoplasmic lipids in altering diphenylhexatriene fluorescence polarization in malignant cells.

Prior studies of fluorescence anisotropy (polarization) with diphenylhexatriene in normal and malignant cell populations have shown differences which have been attributed to an altered membrane lipid composition in cancer. We studied fresh tumor cells from patients with diverse lymphoid neoplasms and found a discrete range of whole-cell fluorescence polarization values (P values) for each type of neoplasm. Following cell fractionation, however, the P values of isolated plasma membranes from malignant cells did not differ significantly from the values obtained with normal donor lymphocytes. Therefore, the altered whole-cell fluorescence polarization measurements in malignant cells are not likely to be due to gross lipid changes in the plasma membrane. Histochemical staining and cell fractionation revealed the presence of cytoplasmic lipid accumulations, and these had extremely low P values, which could account for the low P values of malignant cells. Complementary studies of lymphoid cell lines showed whole-cell fluorescence polarization measurements to be extremely sensitive to exogenous lipid supplements, but membrane values remained stable. We conclude that alterations in membrane lipid fluidity, as measured by the diphenylhexatriene probe, are not consistently found in lymphoid neoplasms and hence cannot presently be invoked to account for the malignant behavior of these cells. However, intracellular neutral lipid accumulation appears to be a common feature of the lymphoid neoplasm. The lipid alterations described could be characteristic of cell immaturity or proliferation rather than malignancy; nevertheless, they may convey unappreciated biological consequences.

Large granular lymphocyte (LGL) lymphoproliferative diseases: naturally cytotoxic tumors in man and experimental animals.

It has recently become clear that in the spleen and blood of both rodents and man that a unique subpopulation of lymphocyte is the mediator of virtually all of the inherent natural killer (NK) and antibody-dependent cell-mediated cytotoxic (ADCC) activity. Because of their large size, eccentric kidney-shaped nucleus and prominent cytoplasmic granules, these cytotoxic cells, termed large granular lymphocytes (LGL), can be readily identified in Geimsa stained cytocentrafuge preparations. Unfortunately, the relatively low numbers of these cells in normal lymphoid tissues has made the detailed analysis of LGL quite difficult. Recently however, a number of investigators have reported both rodent and human leukemias or leukocytosis in which there was an abnormally high number of circulating lymphocytes with either the appearance and/or function of LGL. The present manuscript reviews this literature with an emphasis on the biological and clinical characteristics of this lymphoproliferative disease. Emphasis is also placed on the usefulness of these cells for the detailed analysis of LGL morphology and function.

Ultrastructure of meiotic chromosomes in boys undergoing chemotherapy for leukemia.

Meiotic chromosomes from four boys treated with combination chemotherapy for acute lymphoblastic leukemia were analysed ultrastructurally by serial sectioning and reconstruction of spermatocyte chromosomes. Analysis of the 1012 chromosomes in 22 zygotene and pachytene spermatocyte nuclei showed that in most of the cells that reached the pachytene stage, one or more of the following abnormalities were found in the intranuclear structures: decreased condensation of euchromatin, centromeric heterochromatin, diffuse heterochromatin, or abnormal nucleolar morphology. The total number of recombination nodules and -bars was within normal limits, but in some nuclei the ratio between the two types of structure was inappropriate for the stage, indicating developmental asynchrony. With the exception of one nucleus, there was no increase in abnormalities of pairing and synaptonemal complex formation. The number of structural aberrations in autosomal and sex chromosomes was similar to that found in the reference material. The repair system responsible for resolving chromosome interlockings in zygotene was functioning. It is not possible to predict which of the short term effects found will become permanent and thus pose a risk of genetically abnormal conceptions. New means of classifying meiotic chromosomes, of evaluating the synchrony of development, and of analysing the telomere attachment pattern are described.

Adult T-cell leukemia: a report on two white patients.

Two white European males are reported with adult T-cell leukemia (ATL), a disease first described in Japan, but recently also in the U.K. and U.S.A. Both patients presented with lymphadenopathy, but without a mediastinal mass. In addition, one patient had skin infiltrates and the other had hepatosplenomegaly. Morphologic and ultrastructural examination of the blasts in bone marrow and lymph node biopsy revealed a predominance of polymorphic lymphoid cells with pronounced nuclear irregularities and a semi-mature chromatine pattern. Histopathology of the lymph nodes showed a diffuse infiltration with medium-sized lymphoblasts with irregular nuclei. The blasts in the bone marrow formed E rosettes with sheep erythrocytes, lacked terminal deoxynucleotidyl transferase (Tdt) activity but expressed the Ia-like antigen; although the majority of the cells reacted with a polyclonal anti-T-cell serum, they were negative for OKT3. In one patient a helper/inducer phenotype (OKT4+) was found in the lymphoblasts of bone marrow and lymph node, while in the other only in the lymph node. The difference between bone marrow and lymph node phenotype is discussed. To our knowledge, these are the first two European patients reported with ATL, a disease clearly different from convoluted T-cell acute lymphocytic leukemia.

Detection of antibodies to adult T-cell leukemia (ATL)-associated virus in ATL patients' sera by immunoelectron microscopy.

In an attempt to demonstrate the presence of antibodies to ATL-associated type-C virus particles (ATLV), an indirect immunoferritin method of immunoelectron microscopy was performed on an ATLV-producing human cord T-cell line (MT-2) and short-term cultures of ATL cells. All five sera from ATL patients seropositive to ATL-associated antigens (ATLA) but not three sera from healthy adults were positive for the ferritin labeling of ATLV and plasma membranes in both MT-2 and short-term cultured ATL cells. Sera absorbed with sheep red blood cells or human T-cell acute lymphatic leukemia cells labeled ATLV much more intensely with ferritin than with plasma membranes. These results demonstrated at the ultrastructural level that anti-ATLA-positive sera contained antibodies to surface glycoproteins and/or structural proteins of ATLV and that they differed from anti-Forssman or anti-T-cell antibodies. It was also demonstrated that anti-ATLA-positive sera showed the same reactivity with ATLV from both MT-2 and short-term cultured ATL-cells by the immunoferritin method.

Niagara sky blue 6B--a new stain for granulocytic cells.

Using an acidified solution of Niagara sky blue 6B, granules in mature granulocytic cells from peripheral blood and bone marrow stained bright blue to blue-green. Granules in immature granulocytes like myelocytes and promyelocytes stained purple. In leukemic myeloblasts and leukemic monocytes, granular-appearing structures stained purple. In leukemic lymphoblasts, purple granular structures were not visualized. As such, Niagara sky blue 6B can be used to identify granulocytic cells at all maturational stages.

Acute leukemia of adults. Ultrastructural, cytochemical and histologic observations in 100 cases.

In order to establish guidelines for categorization of acute leukemia, marrow histology, special stains, and electron microscopy were performed in 100 adult leukemia cases. Differential counts for each stain were performed, and the results were combined with those obtained by electron microscopy for final classification. Myeloid (non-lymphoid) leukemia was most common (83 cases), and there were 13 lymphoid cases, two cases of erythroleukemia, and two undifferentiated leukemias. Histologic studies of marrow particles revealed significant admixtures of lymphocytes, plasma cells, and abnormal erythroid elements, particularly in acute myelomonocytic leukemia. Interpretations of cytochemical results were confirmed by ultrastructural studies in 90% of the cases. Clinically significant discrepancies between cytochemical and ultrastructural interpretations were rarely found. Only two myeloid and five lymphoid cases did not mark typically with any cytochemical stain. The single most reliable special stain was the periodic acid-Schiff stain, whereas the stain most difficult to interpret was the alpha-naphthyl acetate esterase stain. Sudan black stain was particularly helpful in demonstrating Auer rods. Five of six cases unclassified by cytochemistry were categorized by electron microscopy. Although patterns of cytochemical staining were delineated for each type of leukemia, significant intragroup variation in cytochemical reactions was recognized. This study indicates that ultrastructural examination should be routinely used for categorization of cases with equivocal cytochemical findings and for analysis of unclassified cases.

Morphologic and cytochemical observations on the overt leukemic phase of therapy-related leukemia.

Comparison of morphologic and cytochemical features of the leukemic cells of patients with overt acute nonlymphocytic leukemia following therapy for primary malignancies (2 degrees-ANLL) to those of patients with ANLL de novo revealed several differences. No Auer rods were seen in cells of patients with 2 degrees-ANLL, but they were seen in 54 (42%) of 129 cases of ANLL de novo. Only one (4.7%) of 21 patients with 2 degrees-ANLL was classified as M-4 by FAB criteria, compared to 46 (36%) of 129 patients with ANLL de novo classified as M-4. Only one (10%) of 10 patients with 2 degrees-ANLL had more than 10% peroxidase-positive leukemic cells, whereas 19 (95%) of 20 of a control group of patients with ANLL de novo of similar FAB subtypes had more than 10% positive cells. Two of 11 (18%) 2 degrees-ANLL patients had 10% or more naphthol-AS-D chloroacetate esterase-positive cells, in contrast to nine (47%) of 19 patients with ANLL de novo. Nonspecific esterase activity was present in 10% or more of cells in only one (10%) of 10 patients with 2 degrees-ANLL studied, as compared to seven (35%) of 20 of the control group. Previous reports indicate that patients with 2 degrees-ANLL have cytogenetic abnormalities and therapeutic responses that differ from those in ANLL de novo; our findings indicate that there are morphologic and cytochemical differences as well.

The leukemic phase of histiocytic lymphoma. Histologic, cytologic, cytochemical, ultrastructural, immunologic and cytogenetic observations in a case.

A case of histiocytic lymphoma that progressed to a leukemic phase was studied by various methods. Although the cells were found to bear a superficial morphologic resemblance to histiocytes, they more closely resembled transformed lymphocytes. Immunologic markers strongly supported a B-lymphoid origin in this case, while cytogenetic analysis indicated a large number of consistent chromosomal rearrangements, including a translocation involving chromosomes Nos. 8 and 14 which has been previously reported to occur primarily in Burkitt's lymphoma.

Secondary leukemia following treatment of Hodgkin's disease: ultrastructural and cytogenetic data in two cases with a review of the literature.

Two cases of secondary acute nonlymphocytic leukemia developing after combined chemo-radiotherapy for Hodgkin's disease (HD) are reported. The first case was a 28-year-old woman with PSIIIsA HD, treated with total lymphoid irradiation followed by combination chemotherapy that was almost entirely ABVD (Adriamycin, bleomycin, vinblastine, dacarbazine), who developed acute monoblastic leukemia three years after the diagnosis of Hodgkin's disease. We believe this to be the first reported case of secondary leukemia associated with the combination of radiotherapy and ABVD chemotherapy. The second case was a 37-year-old man with Stage IVB Hodgkin's disease, treated with radiotherapy and MOPP (nitrogen mustard, vincristine, procarbazine, prednisone) who developed acute myeloblastic leukemia five years after the diagnosis of Hodgkin's disease. Both cases showed typical changes of panmyelosis demonstrated by cytochemical and ultrastructural studies. In both cases, bone marrow cells had a dominant clone with a markedly abnormal karyotype. The nature of therapy-related secondary leukemia after Hodgkin's disease and its relationship to current modes of treatment are discussed.

Langerhans cells increase in the dermal lesions of adult T cell leukaemia in Japan.

In cases of adult T cell leukaemia neoplastic T cell infiltration in the skin was accompanied by an increase in Langerhans cells. This is in keeping with the view that Langerhans cells may induce antigen-specific and allogenic T cell activation.

Analysis and rearrangement of human karyotypes by computer.

A system useful for the collection and analysis of a large number of abnormal karyotypes and for studies on the relationship between cytogenetic abnormalities and clinical features of diseases is reported. The program is based on the disintegration and rearrangement of abnormal karyotypes recorded according to the 1978 International System for Human Cytogenetic Nomenclature (ISCN).

Surface morphology of lymphoreticular cells: review of data obtained from scanning electron microscopy.

This illustrated review summarizes the current status of knowledge relating to the surface morphology of normal and malignant lymphocytes and other leukocytes, as seen by SEM, with particular emphasis on lymphoreticular disorders. Problems of interpretation of the SEM data and their significance are discussed.

Characterizing "difficult" acute leukemias. A combined electron microscopic and immunological marker study.

The techniques of transmission electron microscopy (TEM), including ultrastructural myeloperoxidase cytochemistry (MPO), and immunological marker analysis, have been used to classify 58 "difficult" cases of acute leukemia where a precise diagnosis could not be made on the basis of conventional light microscopy and cytochemistry. TEM with MPO proved most valuable in characterizing 15 cases of acute myeloid leukemia and its variants, as well as defining complex cellular subpopulations in 11 cases of chronic myeloid leukemia in blast crisis. Immunological marker studies provided conclusive evidence of lymphoid differentiation in 18 cases of acute lymphoblastic leukemia and related disorders. In addition, the combined techniques were used to document 14 cases of terminal transferase-positive acute myeloid leukemia. This study demonstrates that these 2 techniques provide overlapping and complementary information for accurate diagnosis and classification of morphologically difficult hematological malignancies.

4. Influence of classification systems on the treatment of leukemia.

In this paper the influence of the newer aids to the classification of leukemia is considered in relation to the treatment of the disease.