Blast Cells with Monoclonal Surface Immunoglobulin in Two Cases of Acute Blast Crisis Supervening on Chronic Lymphocytic Leukaemia

Acute blast crisis occurred in two patients with previously well-confirmed chronic lymphocytic leukaemia. The finding by direct immunofluorescence of membrane-bound monoclonal immunoglobulins on the surface of the blast cells showed that they were related to B cells in the same way as the proliferating lymphocytes in most patients with chronic lymphocytic leukaemia. In one patient the surface monoclonal IgM detected on both the lymphocytes and the blast cells had the same rheumatoid antibody activity, supporting the concept that the leukaemic cells found during the acute and chronic phases of the disease originated from the same clone.     

Determination of the number and relative position of tryptophan residues in various albumins.

Trace metals were measured by neutron-activation analyses in purified nucleic acids and histone(s) of lymphocytes from patients with acute lymphocytic leukaemia or infectious mononucleosis and from normal donor DNA isolated from lymphocytes of a patient with infectious mononucleosis and a normal donor showed a high a high content of Cr2+, Sb2+, Fe2+, and Zn2+, whereas DNA of lymphoblasts from a patient with acute lymphocytic leukaemia had a lower content of these trace metals, but the Co2+ content was 20-fold higher than in DNA or normal donor lymphocytic cells. Total histones from leukaemic cells had higher contents of most of the trace metals except for Zn2+, which was present in lesser concentration than in histones from normal donor lymphocytic cells. Lysine-rich (F1) histones showed lower contents of Cr2+, Sb2+ and Co2+, whereas arginine-rich (F3) histones had significantly higher contents of these trace metals. These observations may be of interest in that F3 histones more effectively inhibit RNA synthesis in human lymphocytic cells than do other species of histones.     

Epigenetic inactivation of the hsa-miR-203 in haematological malignancies

miR-203 is a tumour suppressor microRNA (miRNA). We studied the methylation of hsa-miR-203 in 150 samples including acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL) and non-Hodgkin's lymphoma (NHL) by methylation-specific PCR, and miRNA expression by stem-loop RT-qPCR. hsa-miR-203 promoter was unmethylated in normal controls but homozygously methylated in two AML and four lymphoma cell lines, in which 5-Aza-2'-deoxycytidine treatment led to promoter demethylation and miR-203 re-expression. Restoration of miR-203 expression in lymphoma cells inhibited cellular proliferation and increased cell death, suggesting an inherent tumour suppressor activity. In primary samples, hsa-miR-203 methylation was absent in CML but detected in 5.0% ALL, 10.0% AML, 42.0% CLL and 38.8% of NHL (including six [60.0%] natural killer-cell, nine [40.9%] B-cell and four [23.5%] T cell NHL). Moreover, hsa-miR-203 methylation was associated with hypermethylation of hsa-miR-34a, -124a and -196b in NHL but not CLL. In CLL, hsa-miR-203 methylation was associated with a higher presenting Hb level (P = 0.033). The projected 10 year overall survival of the CLL patients was 58.2%, which was impacted by Rai stage and high-risk karyotypes but not hsa-miR-203 methylation. hsa-miR-203 was more frequently methylated in lymphoid than myeloid malignancies (P = 0.002). In conclusion, miR-203, a tumour suppressor gene, was hypermethylated in a tumour-specific manner with gene silencing. hsa-miR-203 was more frequently hypermethylated in lymphoid than myeloid malignancies. In NHL, hsa-miR-203 methylation was associated with concomitant methylation of other tumour suppressor miRNAs. The frequent hsa-miR-203 methylation in lymphoid malignancies suggested a pathogenetic role of hsa-miR-203 methylation.     

EXCLUSION OF SERUM FACTORS IN THE IN-VITRO DEPRESSION OF LYMPHOCYTES' RESPONSE TO MITOGENS IN CHRONIC LYMPHOCYTIC LEUKAEMIA

To find out whether serum factors are involved in the depression of the mitogenic response of lymphocytes of patients with chronic lymphocytic leukaemia, sera from these patients were used in the culture medium for normal lymphocytes. the results were considered on the basis of the dynamics of responses of these normal lymphocytes, comparing a standard human group AB serum with twenty sera from patients with CLL. No heat-stable inhibitory factors peculiar to CLL were found. In an experiment in which fresh CLL and normal sera were compared, no heat-labile inhibitory factors were apparent.     

Application of the pMHC Array to Characterise Tumour Antigen Specific T Cell Populations in Leukaemia Patients at Disease Diagnosis

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8⁺ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms’ Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8–1.4 x 10⁶). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1_126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1_950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.     

Flow cytometric characterization of chronic lymphocyte leukaemias using orthogonal light scattering and quantitative immunofluorescence

Light scattering properties and antigen distribution of lymphocytes labeled with the monoclonal antibodies CD 5 and CD 20 were determined for 19 patients with a chronic B-cell derived leukaemia. The density of the antigen detected by the monoclonal antibody CD 5 appeared to be considerably lower on malignant B-lymphocytes of the patients as compared with T lymphocytes. A large variation was observed in the amount of receptors for the monoclonal antibodies CD 5 and CD 20 on the malignant cells of the different patients. B-cell chronic lymphocytic leukaemia (B-CLL) patients were clearly distinguishable from leukaemic follicular non Hodgkin lymphoma patients (LF-NHL, formerly lymphosarcoma cell leukaemia) and from a patient with a prolymphocytoid transformation (PLT) of the B-CLL according to the amount of the antigens for CD 5 and CD 20. Within the B-CLL patient population, no relation of progression of the disease with distribution of these antigens could be observed. In one patient the extraordinary phenotype CD 20+, CD 11+, leu 8+, CD 5- of the malignant lymphocytes was observed. An experimentally simple method to differentiate between the various chronic lymphocytic leukaemias (CLL) appeared to be the determination of orthogonal light scattering properties of lymphocytes. In healthy donors one can always distinguish two populations of lymphocytes in the orthogonal light scatter histograms. Lymphocytes of B-CLL patients show one uniform population with a relatively small orthogonal light scattering signal, lymphocytes of our patients with PLT of B-CLL or with LF-NHL show one uniform population with a relatively large orthogonal light scattering signal.     

Generation of a human IgM monoclonal antibody directed against HLA class II molecules: a potential agent in the treatment of haematological malignancies

Major histocompatibility complex (MHC) class II molecules have been considered as a good target molecule for use in immunotherapy, because of the high expression in some lymphoma and leukaemia cells and, also, because of their restricted expression on human cells (monocytes, dendritic, B lymphocytes, thymic epithelial cells, and some cytokine-activated cells, such as T lymphocytes). We have obtained a human IgM monoclonal antibody directed against human leukocyte antigen (HLA) class II molecules, using transgenic mice carrying human Ig genes. The antibody BH1 (IgM/κ isotype) recognises HLA-class II on the surface of tumour cells from patients suffering from haematological malignancies, such as chronic and acute lymphocytic leukaemias, non-Hodgkin lymphomas and myeloid leukaemias. Interestingly, functional studies revealed that BH1 mAb recognises and kills very efficiently tumour cells from several leukaemia patients in the presence of human serum as a source of complement. These results suggest that this human IgM monoclonal antibody against HLA-class II could be considered as a potential agent in the treatment of several malignancies. Belén Díaz, Irene Sanjuan and Susana Magadán share authorship; Francisco Gambón and áfrica González–Fernández share leadership.     

Lysosomal acid phosphatase: difference between normal and chronic lymphocytic leukaemia T and B lymphocytes.

Lysosomal acid phosphatase was assayed in homogenates of isolated normal and B cell type chronic lymphocytic leukaemia (B-CLL) T and B lymphocytes by biochemical means. Unlike the results of cytochemical studies reported in the literature enzyme activity was considerably higher in normal B lymphocytes than in corresponding T cells. This finding offers the possibility to use acid phosphatase as a marker for normal B lymphocytes. The diminution of acid phosphatase in unseparated B-CLL lymphocytes depends predominantly upon a loss of enzyme activity in the B cell fraction indicating an intrinsic abnormality of these neoplastic lymphocytes.     

Generation of cytotoxic T lymphocytes from peripheral blood of leukaemic patients

Peripheral blood mononuclear cells from 13 patients with acute leukaemia were used to establish long-term interleukin-2-dependent cytotoxic T lymphocytes. Cells were grown in RPMI medium containing interleukin-2 (IL-2, 100 U/ml) and 2.5% conditioned medium prepared by activating normal lymphocytes with phytohaemagglutinin. Proliferation of IL-2-dependent CD3-positive lymphocytes was seen in 1 of 2 acute lymphocytic leukaemia cases (ALL), 1 of 4 acute myelogeneous leukaemia cases (AML) (M1) and 8 of 8 more differentiated AML. In 2 cases with detectable leukaemic cell markers (1 ALL and 1 AML) passageable cells were developed, that expressed normal T cell phenotypes (namely CD3, CD4, and CD8) at the expense of leukaemic cells. In 1 of 2 cases, long-term IL-2-cultured cells showed specific cytotoxic activity against autologous leukemic cells. The percentage killing against autologous and two allogeneic target cell lines at a 50/1 effector/target (E/T) ratio was 42%, 9% and 19% respectively. Similarly the cytotoxic activity of IL-2 activated from 4 different individuals against conventional tumour targets K562 and Daudi at a ratio of 50/1 was 29%–68% (median=55%) and 34%–78% (median=61%) respectively. It was also found that this killing potential of the activated cells was maintained for as long as culture was continued (median 23 days, range 17–75 days). The mechanism(s) of T cell proliferation at the expense of leukaemic blast cells in the case of a minority of leukaemic patients and the possible clinical therapeutic potential of these cells following in vitro IL-2 activation deserve further investigation.     

Antitumor activity in vitro in chronic myelogenous leukaemia revealed after treating peripheral cells with cytosine arabinoside

Summary Cytosine arabinoside (Ara-C) treatment of peripheral blood mononuclear cells from 12/12 chronicphase chronic myelogenous leukaemia (CML) patients revealed a proliferative response stimulated by their untreated leukaemic cells. Specific recognition of tumour cells by patients' normal lymphocytes was suggested by the finding that cells of siblings genotypically identical for human leukocyte antigen caused no stimulation. Lymphocytes thus stimulated by tumour cells from one of these patients were cloned by limiting dilution and tested for antileukaemic effects in cytotoxicity and proliferation assays. Cytotoxic lines were isolated that killed autologous CML targets but only a limited number of allogeneic fresh leukaemias or cell lines. These results show that anti-leukaemia effectors can be isolated from chronic-phase CML patients and suggest their potential application in adoptive immunotherapy.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 120) Abbreviations used: ANLL, acute non-lymphocytic leukaemia; Ara-C, cytosine arabinoside; CML, chronic myelogenous leukaemia; IL, interleukin; LAK, lymphokine-activated killer; NK, natural killer; PBMC, peripheral blood mononuclear cells; HLA, human leukocyte antigen     

A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes

Summary We have previously described the derivation of a monoclonal antibody, S2C6, to a novel 50 Kdalton antigen associated with human urinary bladder carcinoma. No reactions were obtained with carcinomas of unrelated origin or with normal urothelial cells. However, the antibody also reacted with a similar antigen on some cell lines of B lymphocyte origin. Using large panels of target cells we have now shown that this reactivity was entirely restricted to cells of the B lineage within the haematopoietic system. As opposed to its apparent restriction to malignant cells of the urothelium, the S2C6 antigen was expressed by normal B lymphocytes as well as by many malignant B cells (chronic lymphocytic leukaemia, hairy cell leukaemia and immunocytoma). Pre-B cells derived from acute lymphocytic leukaemia and plasma cells from multiple myeloma lacked the antigen. Expression was significantly enhanced on cultured B cells from Burkitt lymphomas and on Epstein-Barr virus-transformed lymphoblastoid cell lines including those of the pre-B phenotype derived from fetal bone marrow. As judged from the molecular size and the distribution pattern displayed by the S2C6 antigen it appears to be distinct from other B cell antigens previously described. A possible relation of the S2C6 antigen to a receptor for B cell growth factors is discussed.     

Lectin binding ability of B-chronic lymphocytic leukaemia cells.

The binding of five fluorescein-labelled lectins: peanut agglutinin (PNA), lentil agglutinin (LEN), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and asparagus pea agglutinin (ASP) to human B-cell chronic lymphocytic leukaemia (B-CLL) and B lymphocytes of normal donors was studied. The specificity of the fluorescence was demonstrated by inhibition with appropriate saccharides. The proportion of B cells was estimated using anti-B cell monoclonal antibody. Both leukaemic and normal B cells showed the binding ability of all except of one (ASP) studied lectins. We have found the differences in surface carbohydrate patterns between B-CLL and normal B lymphocytes. B-CLL cells showed the considerably lower ability to bind SBA and slightly higher expression of PNA and LEN receptors in comparison to normal B cells. The analysis of WGA binding allowed for recognizing two groups of CLL patients: one with high and the second one with low WGA receptor expression. The double marker studies revealed that B cells could simultaneously react with anti-B cell monoclonal antibody and fluorochrome labelled lectins.     

Actin and tubulin of normal and leukaemic lymphocytes.

Double labelling and the isolation of actin- and tubulin-derived peptides were used to determine the amounts of these proteins in peripheral lymphocytes from normal donors and from patients with chronic lymphocytic leukaemia. As a precaution against proteolysis, samples were boiled before assay. The actin content of chronic-lymphocytic-leukaemia (CLL) lymphocytes was 8.1 +/- 2.1% of total protein, which was lower (P less than 0.05) than the amount (12.8 +/- 3.0%) of actin found in normal lymphocytes. The tubulin content of CLL lymphocytes was 4.4 +/- 1.5% of total protein, which was also significantly less (P less than 0.05) than that of normal lymphocytes, which was found to be 6.1 +/- 1.1%.     

The lymphocytic basophilia after incubation of blood smears in distilled water

The incubation of peripheral blood smears in distilled water frequently used for the control of the digestion with ribonuclease produced distinct changes of basophilic ribonucleic acid containing structures in peripheral lymphocytes. The alteration of these structures was apparently produced by the activity of the endogenous ribonuclease since it was reduced or prevented by the lowering of the temperature or addition of the ribonuclease inhibitor. In addition, marked differences were found between peripheral blood lymphocytes of healthy persons and patients suffering from chronic lymphocytic leukaemia with respect to the sensitivity of lymphocytic basophilia to the incubation in distilled water.     

Different maturation capabilities of chronic lymphocytic leukaemia lymphocytes in vitro

Peripheral blood lymphocytes from five patients with B-derived chronic lymphocytic leukaemia were stimulated by Staphylococcus aureus strain Cowan together with T cell mitogen phytohaemagglutinin in 5-9 days suspension cultures. The responses of B lymphocytes were studied on a T cell depleted subpopulation, obtained from harvested lymphocyte cultures using the sheep red blood cell rosette technique. Proliferation tests were performed using a 3H-TdR blast cell index. The maturation process of B-lymphocytes was examined with cytoplasmic Ig studied by FITC-conjugated antisera. Results analysed indicate various degrees of maturation of B cells in different patients.     

Differences among the polyadenylated RNA sequences of human leucocyte populations: an approach to the objective classification of human leukaemias

We have constructed a complementary DNA (cDNA) library representing expressed sequences of the white blood cells from a patient with chronic granulocytic leukaemia. The library was screened by colony hybridization of 32P-labelled cDNAs synthesized from the polyadenylated RNAs of the white blood cells from patients with chronic granulocytic or chronic lymphocytic leukaemia. The autoradiographic patterns were compared and 70 recombinants were selected to comprise a panel which distinguished between these two types of leukaemia. Hybridization of this panel with complementary DNAs transcribed from the polyadenylated RNAs of a variety of normal and neoplastic leucocyte populations showed that the RNA sequences in high abundance in leucocytes from chronic granulocytic leukaemias differ quite radically from those in other leucocytes. The patterns of hybridization seen when this panel was challenged with cDNAs representing the RNAs of normal and leukaemic leucocyte populations were sufficiently different to distinguish clearly the peripheral blood leucocytes of chronic granulocytic leukaemias from other populations of white blood cells, both normal and leukaemic. We suggest that this approach might provide additional markers useful in the classification of the acute leukaemias, especially the undifferentiated leukaemias whose identification by conventional methods is uncertain.     

Development of chronic lymphocytic leukaemia after posttraumatic splenectomy

In the course of a post-splenectomy follow-up study, 2 out of 102 patients who had been splenectomized after an abdominal trauma were found to have developed chronic lymphocytic leukaemia 5 and 31 years after splenectomy, respectively. The possible association between splenectomy and secondary leukaemia is discussed.     

Detection of a soluble form of the receptor for interleukin 2 in the serum of patients with hairy cell leukaemia

The sera of patients with hairy cell leukaemia (HCL) contain a factor which inhibits the binding of anti-IL-2R antibody to its target (activated T lymphocytes). The presence of this factor, which probably corresponds to the soluble form of IL-2R (sIL-2R), can be easily detected using a simple immunocytochemical inhibition assay. In a series of patients with chronic lymphoproliferative diseases the presence of sIL-2R appeared to be specific for HCL, using the sensitivity of our test, since it could not be detected in sera from normal subjects and patients with B-cell lymphocytic leukaemia or Hodgkin's disease. Thus it might be used as an additional tool for characterizing HCL patients.     

Cholesterol esters as growth regulators of lymphocytic leukaemia cells

Objective: Alterations in plasma lipid profile and in intracellular cholesterol homoeostasis have been described in various malignancies; however, significance of these alterations, if any, in cancer biology is not clear. The aim of the present study was to investigate a possible correlation between alterations in cholesterol metabolism and expansion of leukaemia cell numbers. Materials and methods: Lipid profiles in plasma and in primary leukaemia cells isolated from patients with acute or chronic lymphocytic leukaemia (ALL and CLL) were studied. Results and conclusions: Decreased levels of HDL‐C were observed in plasma of leukaemic patients, levels of total cholesterol, LDL‐C, triglycerides and phospholipids were unchanged or only slightly increased. As compared to normal lymphocytes, freshly isolated leukaemic cells showed increased levels of cholesterol esters and reduction in free cholesterol. Growth stimulation of ALL and CLL cells with phytohemagglutinin led to further increase in levels of cholesterol esters. Conversely, treatment with an inhibitor of cell proliferation such as the mTOR inhibitor, RAD, caused decline in population growth rate of leukaemia cells, which was preceded by sharp reduction in rate of cholesterol esterification. On the other hand, exposure of leukaemic cells to two inhibitors of cholesterol esterification, progesterone and SaH 58‐035, caused 60% reduction in their proliferation rate. In addition to demonstrating tight correlation between cell number expansion and cholesterol esterification in leukaemic cells, these results suggest that pathways that control cholesterol esterification might represent a promising targets for novel anticancer strategies.     

RNA synthesis in nuclei of rat liver and of human lymphocytes.

Physico-chemical properties and RNA synthesis in the rat liver and human lymphocytes have been compared in a nuclear system in vitro. Human lymphocytes were isolated from blood of healthy donors and of chronic lymphocytic leukaemia patients. The isolated nuclei served as the source of polymerase and template DNA. 3H-CTP was incorporated into the acid insoluble fraction linearly for 60 min. The nuclei of lymphocytes contained small amounts of RNA and protein, and the isolation procedure was complicated. Rat liver nuclei seem to be less prone to clumping at high pH values and may incorporate much more 3H-CTP. The nuclear synthesis was compared with incorporation of 3H-rU and 32P-orthophosphate into nuclear RNA of intact lymphocytes. Normal cells easily incorporated 32-P, and in contrast leukaemic cells incorporated 3H-rU to a greater extent.