Autologous responses to human leukaemic cells in mixed leucocyte culture

Summary Cryopreserved leukaemic blasts and remission non-T cells from 22 patients with acute leukaemia (15 lymphocytic, 7 non-lymphocytic) were tested as stimulators of autologous remission T cells and normal allogeneic T cells in primary and secondary MLC. In most cases the autologous response elicited by leukaemic cells was less than or equal to that elicited by remission non-T cells. However, T cells from 2 patients in long-standing first remission from ANLL displayed greater proliferation in response to leukaemic blasts than to remission non-T cells in both primary and secondary MLC. The results are suggestive of sensitization of these 2 patients to leukaemia-specific antigens, but other possible explanations are discussed. Abbreviations used: MLC, mixed leucocyte culture; ANLL, acute non-lymphocytic leukaemia; ALL, acute lymphoblastic leukaemia; AMLR, autologous mixed lymphocyte reaction; NK cells, natural killer cells; MNC, mononuclear cells     

Leucostasis, an underestimated cause of death in leukaemia

Massive sludging of leukaemic cells in blood vessels is a frequent and often lethal complication of leukaemia. In a retrospective clinicopathological study on the causes of death in 52 patients with acute myeloid leukaemia and myeloproliferative disease, pulmonary leucostasis was found in 40% of the patients. In many of these patients the vessels of the heart, brain and testes were also involved. In search for signs and symptoms specific for leucostasis, the clinical records of the 21 patients with leucostasis (the study group) were compared to those of 20 patients without leucostasis (the control group). Dysfunction of the organs most affected by leucostasis, namely lungs, heart and brain, was found more often in the study group than in the controls, but the combination of unexplained fever with cardiopulmonary and/or central nervous system failure occurred almost exclusively and in half of the patients with leucostasis. Leucostasis occurs predominantly, but not exclusively, in patients with high leucocyte counts, and especially, but again not exclusively, when the leucocyte counts rise sharply.     

FUNCTIONAL CELL COMPARTMENTS IN A RAT MODEL FOR HUMAN ACUTE MYELOCYTIC LEUKAEMIA

Functional cell compartments were studied in a rat model for human acute myelocytic leukaemia (AML). This was done by tracing the distribution of injected ⁵¹Chromium-labelled leukaemic cells in the body. It was concluded that two functional compartments can be distinguished in acute leukaemia, i.e., a rapidly exchangeable pool of cells (including the circulating blood pool, the marginal noncirculating blood pool and the rapidly exchangeable tissue pool; RETP) and a slowly exchangeable tissue pool (SETP). The sizes of these various compartments were roughly quantified at various stages of the disease by calculations based on the principle of isotope dilution and organ weight measurements. As the leukaemia progresses, the size of the SETP increases significantly relative to the size of the RETP. Simultaneously, the exchange rates of leukaemic cells between the organs and the blood decrease. The blood transit time of leukaemic cells was also significantly prolonged, as is the case in human AML.     

Functional cell compartments in a rat model for human acute myelocytic leukaemia.

Functional cell compartments were studied in a rat model for human acute myelocytic leukaemia (AML). This was done by tracing the distribution of injected 51Chromium-labelled leukaemic cells in the body. It was concluded that two functional compartments can be distinguished in acute leukaemia, i.e., a rapidly exchangeable pool of cells (including the circulating blood pool, the marginal noncirculating blood pool and the rapidly exchangeable tissue pool; RETP) and a slowly exchangeable tissue pool (SETP). The sizes of these various compartments were roughly quantified at various stages of the disease by calculations based on the principle of isotope dilution and organ weight measurements. As the leukaemia progresses, the size of the SETP increased significantly relative to the size of the RETP. Simultaneously, the exchange rates of leukaemic cells between the organs and the blood decrease. The blood transit time of leukaemic cells was also significantly prolonged, as is the case in human AML.     

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.     

Differences in electrophoretic mobility and cytochemistry of leukemic T-cells with identical immunologic phenotype (CD1 positive)

Leukaemic cells of acute T-lymphoblastic leukaemia and T-lymphoblastic lymphoma with secondary leukaemic course of disease which showed the same immunological phenotype (cortical thymocytes, thymocyte stage II), could be further differentiated according to their cytochemical pattern and electrophoretic mobility (EPM). Leukaemic cells of T-ALL usually reveal a high EPM, whereas EPM of leukaemic cells of T-lymphoblastic lymphoma was significantly lowered. In cytochemical respect acid phosphatase was positive in both cases of leukaemia. An activity of acid esterase, however, could only be demonstrated in leukaemic cells of T-lymphoblastic lymphoma. The findings are interpreted as manifestation of a different stage of maturity of both T-cell clones with the same immunological phenotype.     

Testicular disease in acute lymphoblastic leukaemia in childhood. Report on behalf of the Medical Research Council's Working Party on leukaemia in childhood.

In three trials conducted by the Medical Research Council on acute lymphoblastic leukaemia in childhood the incidence of testicular infiltration in relation to presenting features and treatment was analysed. Initial severe thrombocytopenia was associated with an increased incidence. Testicular infiltration was occasionally found during treatment in patients with high blood leucocyte counts at diagnosis but the main incidence in patients with all types of disease occurred within one year of stopping treatment. The incidence may be increased when "second-line" drugs, especially cyclophosphamide, have been used. A reappraisal of the value of additional drugs in maintenance treatment of patients with good prognosis is needed.     

Src kinase signaling in leukaemia

Role of Src kinases in acute lymphoblastic leukaemia has been recently demonstrated in leukaemia mouse model. Retained activation of Src kinases by the BCR-ABL oncoprotein in leukaemic cells following inhibition of BCR-ABL kinase activity by imatinib indicates that Src activation by BCR-ABL is independent of BCR-ABL kinase activity and provides an explanation for reduced effectiveness of the BCR-ABL kinase activity inhibitors in Philadelphia chromosome-positive acute lymphoblastic leukaemia. Simultaneous inhibition of kinase activity of both BCR-ABL and Src kinases results in long-term survival of mice with acute lymphoblastic leukaemia. Leukaemic stem cells exist in acute lymphoblastic leukaemia, and complete eradication of this group of cells would provide a curative therapy for this disease.     

Correlation of clinical and anatomical findings in childhood meningitis]

Clinical and anatomical findings of 76 prophylactically not irradiated children with acute leukaemia were compared. In the group of 17 children (22%) with clinically manifest symptoms of CNS involvement in the course of the illness leukaemic infiltrates were identified in the CNS at the autopsy and during histopathological examinations even in 70% in spite of clinical healing and normalization of the cerebrospinal fluid finding. In children who had either no clinical symptoms of CNS leukaemia (42 children) or only terminal symptoms in the last 24 hours of their life (17 children) leukaemic infiltrations were found in 25.4% of the cases. Non-myeloid types showed 44.5% of histopathological changes, myeloid types 22.7%. Clinical main symptoms and anatomical findings were correlated to the corresponding groups of patients and typical illustrations were added.     

An in vitro testing of chemoresistance in leukaemic patients

The fact that leukaemic cells are primarily or secondarily resistant to cytostatics is a serious phenomenon, which leads to the failure of chemotherapy of malignant diseases in clinical practise. Some detoxification and transporting systems are responsible for the generation of chemoresistance on the cellular level and the decrease of effectiveness in treatment. In vitro testing of chemoresistance of leukaemic cells is presently an inseparable component of “tailoring” therapy in the developing field of predictive oncology. The aim of this work was to estimate profiles of drug resistance, based on the predictive in vitro test, and to help in choosing the most effective cytostatic. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoline (MTT) assay was used, based on the direct effect of cytostatics on the viability of leukaemic cells in vitro. The number of living leukaemic cells was evaluated by a computer program, where LC₅₀ (concentration of cytostatics lethal to 50% of leukaemic cells) was established from the achieved dose-relation curves. Seventy-one samples of leukaemic cells isolated from the patients’ peripheral blood or bone marrow were examined. All samples were tested to 3 cytostatics minimally. It was found by the in vitro assay, that resistance to dexamethasone, prednisolone, etoposide and vincristine is increased in patients with acute myeloid leukaemia disease, compared to the acute lymphoblastic leukaemia patients. In patients with a relapsed disease population, leukaemic cells are highly heterogeneous in the MTT assay. It was concluded that the MTT assay can be used to study drug interactions in vitro in leukaemia samples. The type of interaction was highly different between patients, and depended on drug concentrations.     

Generation time of leukaemic blast progenitor cells

Previous studies have indicated that the generation time of human leukaemic cells is longer than that of normal haematopoietic cells. We have employed a modification of the thymidine (TdR)-suicide technique to measure directly the generation time of leukaemic progenitor cells capable of colony formation. The results obtained with two human leukaemic cell lines (KG-1 and HL-60) and with blast progenitor cells from two patients with acute myelogenous leukaemia indicate generation times ranging from 9 X 0-22 X 0 hr and S-phase durations ranging from 5 X 5-8 X 0 hr. Using the same technique, the generation time of normal bone marrow CFU-c was determined to be 9-11 hr. These findings suggest that the proliferation rate of human leukaemic blast progenitor cells is similar to that of normal haematopoietic stem cells.     

Interferon production by human leukaemic leucocytes.

The Sendai virus-induced interferon (IF) production by partially purified human leucocyte suspensions of normal donors and of leukaemic patients have been investigated in vitro. (i) An increased production was observed with leucocytes taken from patients suffering from chronic myelogenous leukaemia (CML) during exacerbation, but the production was approximately normal with cells taken during remission. (ii) IF production was not influenced by large doses of cytostatics (DBM, 5-FU, FUDR, 5-HU, 6-MP, cyclophosphamide) irrespective of whether normal or CML leucocytes were used. (iii) In contrast, production was inhibited in both systems by inhibitors of RNA or protein synthesis (actinomycin D, puromycin, cycloheximide). (iv) CML leucocytes produced IF for a prolonged period of time as compared to normal leucocytes. (v) Leucocytes from children with acute blastic leukaemia and those from adults with chronic lymphoid or acute paramyeloblastic leukaemia produce, in contrast to normal leukocytes, approximately as much IF in the absence as in the presence of serum. It is concluded that the Sendai virus-induced IF synthesis in CML leucocytes requires de novo protein synthesis.     

Serum Ferritin in Acute Leukaemia at Presentation and during Remission

In patients with acute myeloblastic leukaemia the mean serum ferritin concentration showed a twenty-five-fold increase compared with normal people, and in children with acute lymphoblastic leukaemia (A.L.L.) there was a thirteen-fold increase. The high concentration of circulating ferritin seemed to be related to increased synthesis by leukaemic cells. The return of serum concentrations to normal in A.L.L. patients after successful chemotherapy suggested that ferritin concentration may be a useful index of active disease and may help in prognosis.     

Decreasedin vitro chemosensitivity of tumour cells in patients suffering from malignant diseases with a poor prognosis

The aim of the study was to assess the predictive value of MTTin vitro assay for evaluation of tumour cell resistance/sensitivity to cytotoxic drugs. We analyzed 105 samples of malignant cells of different origin. The study included patients with a diagnosis of acute and chronic lymphatic leukaemia, acute and chronic myeloid leukaemia, non-Hodgkin lymphoma, carcinoma of the lung, stomach and liver, rhabdomyosarcoma and breast carcinoma. The results demonstrate outstanding chemosensitivity in the majority of childhood acute lymphoblastic leukaemias, medium chemosensitivity of adult haematopoietic malignant diseases and chemoresistance of solid tumour cells. Our preliminary data suggest a good correlation betweenin vitro MTT assay and clinical curability of individual malignant diseases.Abbreviations ALL acute lymphoid leukaemia - AML acute myeloid leukaemia - CML chronic myeloid leukaemia - LCS₅₀ 50% leukaemic cell survival     

系统性红斑狼疮患者血清MBL水平变化及其临床意义

目的：研究系统性红斑狼疮（SLE）患者外周血中甘露糖结合凝集素（MBL）水平与SLE的临床表现、经典的血清学指标及疾病活动性指数的相关性。方法：应用固相酶联免疫吸附（ELISA）法检测54例（其中活动期39例、缓解期15例）SLE患者和45名健康志愿者外周血MBL水平,同时检测SLE患者外周血抗dsDNA抗体、C3补体、白细胞计数及循环免疫复合物（CICs）水平,并记录SLE患者主要临床症状（肾脏系统受累、神经系统受累、严重感染等）,疾病活动度用SLEDAI记分。结果：系统性红斑狼疮患者外周血MBL水平显著低于健康志愿者（P〈0．01）;特别是活动期SLE患者血清MBL水平明显降低,与缓解组比较差别具有统计学意义（P〈0．01）;SLE患者血清MBL水平与SLEDAI、抗dsDNA抗体、CICs水平呈负相关,与白细胞计数及C3补体水平呈正相关;并发严重感染的SLE患者组血清MBL水平显著低于无受累组（P〈0．01）。结论：SLE患者血清MBL水平明显降低,血清MBL水平与SLEDAI、抗dsDNA抗体、C3水平、CIC水平、白细胞水平之间明显相关,血清MBL水平较低的患者更易发生比较严重的感染,血清MBL水平可能是SLE潜在生物学标记。     

Acute lymphoblastic leukemia: a study of 25 cases by scanning electron microscopy.

Cells from 25 cases of acute lymphoblastic leukemia (ALL) were studied under the scanning electron microscope (SEM). In 24 of the cases, the vast majority of circulating leukaemic cells had few microvilli. Villous cells were rarely encountered and prominent ridge-like profiles and ruffled membranes were not seen. Only six cases were studied by immunological techniques and four of the cases were of the null type while in two the cells bore detectable T-markers. It seems that ALL is almost always associated with the presence of cells with few microvilli in the peripheral circulation, differing in this respect from most cases of CLL. Although circulating leukaemic lymphocytes with few microvilli are sometimes seen in CLL, the most frequent cell type encountered is a more villous lymphocyte. Differences between leukaemic cells from patients with ALL, CLL and non-lymphoblastic leukaemias are discussed. It appears that SEM may help to distinguish lymphoblastic and nonlymphoblastic leukaemic cells in many instances and can be used as a useful adjunct to other modes of microscopy in the diagnosis of acute leukaemia.     

A novel CD44 antibody identifies an epitope that is aberrantly expressed on acute lymphoblastic leukaemia cells

Previous studies have shown that the antibody 7H9D6 identifies CD44, a glycoprotein receptor for hyaluronic acid. 7H9D6 recognizes an epitope of CD44 that is not always present on CD44 molecules. The 7H9D6 antibody bound to the hyaluronic acid binding domain of CD44 and inhibited cell adhesion to immobilized hyaluronic acid. However, the expression of the 7H9D6 epitope was not sufficient for hyaluronic acid binding. Immunofluorescent staining with 7H9D6 revealed a punctate surface staining pattern, suggesting that CD44 molecules recognized by 7H9D6 are located in clusters on the cell surface. In contrast, other CD44 antibodies produced a uniform staining pattern. Early bone marrow B cells were negative for 7H9D6 but reactive with other CD44 monoclonal antibodies. In contrast, leukaemic cells from 65% of patients (28 of 43) with B lineage acute lymphoblastic leukaemia bound 7H9D6. Patients expressing the 7H9D6 epitope on their leukaemic cells had an increased risk of death (HR 3.5 95% CI 1.1-10.9, P = 0.029) and of disease relapse (HR 3.2 95% CI 1.2-8.5, P = 0.017) when corrected for white cell count. This antibody may be useful for the detection of residual disease in B lineage acute lymphoblastic leukaemia and as a prognostic indicator and for the study of CD44 function.     

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.     

Granulocytopoiesis in children with acute lymphoblastic leukaemia.

Numbers, proliferative potential, and differentiative capacity of bone marrow granulocyte-macrophage precursor cells were studied in 130 children with acute lymphoblastic leukaemia (ALL), including 77 children in an acute phase of the disease and 53 in remission. Bone marrow samples from 65 children without haematopoietic abnormalities were used as controls. The numbers of clonogenic precursors were found to be below normal in all phases of ALL, particularly during the acute period when the bone marrow was heavily infiltrated with leukaemic cells. It is shown that the decreases in the numbers and proliferative potential of the precursor cells during the acute phases was associated with the effects of leukaemic blast cells, but that in remission the observed reduction in the precursor cell pool was due to the cytostatic effect of therapy. The differentiative capacity of clonogenic granulocyte and macrophage precursors was not altered in children with ALL.     

Mathematical model of acute myeloblastic leukaemia: an investigation of the relevant kinetic parameters

A simple model of acute myeloblastic leukaemia (AML) development is introduced, explicitly including cell growth, cell differentiation and cell-cell interaction. Each of these processes is described by a single model parameter. It is hypothesized that the leukaemic cell is characterized by an alteration of only one of these processes. The kinetic behaviour of the AML system is examined separately for possible alterations of each of the three parameters describing the three processes involved. It is shown that, on the basis of the existing data on AML kinetics, the alteration of the growth and cell-cell interaction parameters can be eliminated as a possible source of AML. Thus kinetic data support the modification of the differentiation process as the origin of the AML state. Further, the growth characteristics of normal and leukaemic cells in the presence of each other are analysed. It is shown how the initial growth of leukaemic cells depends on the difference in the differentiation of normal and leukaemic cells and how the same difference determines the decay of normal cells in the presence of the predominantly leukaemic population. Correlations between the kinetic parameters of the normal and leukaemic populations are suggested to characterize the leukaemic state.