Factors in Pathogenesis of Central-nervous-system Leukaemia

Eighty-three (50%) of 165 children with acute lymphoblastic or acute stem-cell leukaemia presenting during 1958-70 developed leukaemia of the central nervous system (C.N.S.). The rate of incidence of this complication is fairly constant throughout the first two-and-a-half years of the disease, but falls thereafter. The incidence of C.N.S. leukaemia is inversely correlated with the platelet count at the time of initial diagnosis of leukaemia, and directly correlated with the total leucocyte count and the presence of lymph-node enlargement. The major effect of initial leucocyte count is on the time of onset of clinical symptoms. It is suggested that leukaemic cells usually enter the C.N.S. from the blood as a result of intracranial petechial haemorrhage occurring around the time of initial diagnosis of leukaemia, and that the time for subsequent development of symptoms of C.N.S. disease is largely determined by the number and replication rate of leukaemic cells which gain access to the C.N.S. at that time. The increasing frequency of diagnosis of C.N.S. leukaemia in recent years is not wholly explained by increasing survival, and may in part be related to changes in the pattern of antileukaemic therapy.Prophylaxis for C.N.S. leukaemia should be instituted as early as practicable after diagnosis; the identification of a high-risk group may permit this to be done selectively.     

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.     

Unfavourable clinical implications for HLA-G expression in acute myeloid leukaemia

Human leukocyte antigen-G (HLA-G) molecule exerts multiple immunoregulatory functions that have been suggested to contribute to the immune evasion of tumour cells. Studies on HLA-G expression in malignant haematopoietic diseases are controversial, and the functions of HLA-G on this context are limited. In the current study, HLA-G expression was analysed in different types of patients: de novo acute myeloid leukaemia (AML, n = 54), B cell acute lymphoblastic leukaemia (B-ALL, n= 13), chronic myeloid leukaemia (CML, n= 9) and myelodysplastic syndrome (MDS, n= 11). HLA-G expression was observed in 18.5% cases of AML, 22.2% in CML and 18.2% in MDS, but not in B-ALL patients. In AML, HLA-G-positive patients had a significant higher bone marrow leukaemic blast cell percentage when compared with that of HLA-G-negative patients (P < 0.01). Total T-cell percentage was dramatically decreased in HLA-G-positive patients (P < 0.05). Cytogenetic karyotyping results showed that all HLA-G-positive AML patients (n= 5) were cytogenetically abnormal, which was markedly different from that of HLA-G-negative patients (P < 0.01). Ex vivo cytotoxicity analysis revealed that HLA-G expression in AML leukaemic cells could directly inhibit NK cell cytolysis (P < 0.01). These findings indicated that HLA-G expression in AML is of unfavourable clinical implications, and that HLA-G could be a potential target for therapy.     

Ultrastructural cytochemistry of chronic T-cell leukaemias. A study with four acid hydrolases

The ultrastructural localization of four acid hydrolases (acid phosphatase, beta-glucuronidase, beta-glucosaminidase and alpha-naphthylacetate esterase) has been studied in lymphocytes from 16 patients with three types of chronic T-cell leukaemia, namely, T-prolymphocytic leukaemia (T-PLL), T-chronic lymphocytic leukaemia (T-CLL) and adult T-cell lymphoma leukaemia (ATLL). Different patterns of enzyme distribution were observed in the leukaemic T-cells from these disorders. In T-PLL, reactivity for the four acid hydrolases was confined to single or a few large granules. Gall bodies were reactive for beta-glucuronidase, b-glucosaminidase and alpha-naphthylacetate esterase but apparently unreactive for acid phosphatase. In T-CLL, scattered small- to medium-size cytoplasmic granules and many parallel tubular arrays were strongly reactive for acid phosphatase, beta-glucuronidase and beta-glucosidase but showed no reactivity for alpha-naphthylacetate esterase. Intermediate features were observed in ATLL. The observed differences in enzyme reactivity reflect a different content of lysosomal granules in the various types of leukaemic T-cells. They also suggest that similar differences may be found in normal T-lymphocyte subsets.     

Non-random chromosomal aberrations in a complex leukaemic clone of a Bloom's syndrome patient

Summary Bloom's syndrome is one of the congenital disorders known to have increased frequency of acute leukaemia. The complex cytogenetic findings in the leukaemic cells of a 39-year-old male with Bloom's syndrome are described. These included a translocation t(7;17), missing 7q and 17p, a reciprocal translocation t(4;22); del 3q, del 8q22, del 20q, missing 12 and missing Y. In the same patient a missing Y had been noted 10 years previously in 15% of his peripheral blood lymphocytes.     

The cerebral sensitivity to "meningosis-prophylaxis" with 198Au radiogold according to EEG findings]

35 children (16 girls and 19 boys) at the age of 1 11/12 to 16 11/12 with acute leukaemia were injected intrathecally with 198Au-radiogold colloids (HOECHST-BEHRING) for "prophylaxis of meningosis". The colloid size of the isotope amounted to 5 or 30 nm, the applied activity lay between 1.4 and 3.12 mCi. According to a dosage estimation made with the help of LOEWINGERS formula 1 mCi of radiogold corresponds to approximately 1200 rad. Clinical observations, such as headaches, vomiting or fever up to 39 degrees C, could only be found in 6 children (17.1%) during the first 24 hours. All symptoms subsided quickly and without any sequels. Even retarded complications could not be detected. An electroencephalogram was made from all children before and after applying radiogold (1-8 d afterwards). After the injection of radiogold the majority of children had no change of findings in the electroencephalogram, 11 children even showed a tendency towards an improvement up to normalisation. Only 4 children had a deterioration of findings with unspecific disorders or appearances suspected of peak potential discharges. Simultaneously an accumulation of clinical complaints could be found. Judging from the clinical and electroencephalographic behaviour of our patients no absolute neurotoxity of radiogold could be ensured.     

Combination chemotherapy for acute lymphoblastic leukaemia in adults.

Fifty-one adults with acute lymphoblastic leukaemia were entered into a trial of intense initial chemotherapy and early "prophylaxis" of the central nervous system (CNS). Initial treatment with OPAL (Oncovin (vincristine), prednisolone, adriamycin (doxorubicin), and L-asparaginase (colaspase)) followed by craniospinal or cranial irradiation and intrathecal methotrexate produced remission in 36 patients (71%). Seventeen of these patients relapsed three to 18 months after the start of remission; the remainder had been in remission for 12 to 52 months by the end of the study. The predicted median duration of complete remission was 18.5 months. None of the four patients who initially had clinical evidence of CNS disease, three of whom also had leukaemic cells identical to those found in Burkitt''s lymphoma, achieved remission. Those patients who initially had hepatomegaly or splenomegaly had a shorter remission than those without. The predicted median survival was 27 months in those who achieved complete remission, one month in those who did not, and 21 months overall. The addition of colaspase and doxorubicin to vincristine and prednisolone and the use of early CNS treatment clearly improved the remission rate among adults with acute lymphoblastic leukaemia, though the presence and length of remission was affected by the extent of disease at presentation. Burkitt-like leukaemia, which had a poor prognosis, is probably a separate disease and may benefit from a different therapeutic approach.     

Binding of lectins to human leukaemic cells

The interactions of five different lectins: peanut (PNA), lentil (LEN), wheat germ (WGA), soybean (SBA), Asparagus pea (FBP) with leukaemic cells obtained from 31 children: 25 with acute lymphoblastic leukaemia (ALL) and 6 with acute myeloblastic leukaemia (AML) were examined in this study. The relationship of lectin-binding ability to cells cytomorphological, cytochemical and immunological features and its potential clinical application were investigated. It has been shown that PNA and LEN receptors were found in the majority of blast cells. The SBA reacting cells were found only in few patients and FBP binding was not found in studied ALL and AML cells. There was a clear difference in the WGA binding capacity in ALL cells with L1 and L2 characteristics respectively. No differences were found in PNA. WGA and LEN reactivity between PAS negative and PAS positive leukaemic cells. Only PNA of all studied lectins seemed to differentiate T- from B-ALL blast cells. Only WGA binding of ALL cells showed the positive correlation to the risk index value.     

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.     

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.     

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.     

Auer bodies in acute lymphocytic leukaemia and B-cell lymphoma: evidence for a common progenitor of myeloid and lymphoid cells?

In a patient with acute lymphocytic leukaemia (pre-T ALL) and another patient with leukaemic generalization of B-cell lymphoma Auer bodies were found in a few immature cells. The diagnosis in both cases was based on clinical grounds, morphology, cytochemistry, and immunological marker analysis of the blasts. Auer bodies are known to be a marker of high significance for acute non-lymphocytic leukaemias. Therefore the findings described suggest mixed leukaemias with either T-cell or B-cell predominance. It provides further evidence for the existence of a common progenitor of myeloid and lymphoid cells.     

Clinical, morphological, biochemical, and neuroradiological features of mitochondrial encephalomyopathies. Presentation of 19 patients

Nineteen patients (9 females, 10 males) with mitochondrial encephalomyopathies (ME) were studied. The diagnosis was established according to clinical and histopathological criteria. Leading clinical features were chronic progressive external ophthalmoplegia (CPEO) and muscle weakness in 95% of the patients. Pigmentary retinopathy was seen in 63%, and was always associated with CPEO. Hypacusis was present in 47% and cerebellar ataxia in 63% of patients. Clinical or electrophysiological signs of involvement of the central nervous system (CNS) were found in 21% of the patients. In muscle biopsy ragged red fibers were the predominant histopathological findings (100% of the patients), while COX-negative fibers were seen in 74%, deletions of the mitochondrial DNA in 42%, and defects of the respiratory chain in 32% of the patients. Increased blood lactate levels were found in 79% of the patients. Needle electromyography revealed myopathic features in 74%, features of denervation in 16%, and w as normal in the remainder. Imaging studies showed cerebral atrophy in 58%, cerebellar atrophy in 16%, and hyperintense lesions of the white matter, pyramidal tract or extrapyramidal system in 16% of the cases. It is concluded that the clinical manifestations of ME can be very variable. Diagnosis of ME should be always considered in young patients presenting with CPEO and muscle weakness. In most cases, diagnosis can be made by a few selected investigations, while detection of genetic abnormalities may lead to the diagnosis in the remaining cases. (Mol Cell Biochem 174: 297–303, 1997)     

Clinical results of the treatment of bacterial infections in children with ceftazidime

Ceftazidime (Fortum-Glaxo) was administrated to 19 children with urinary tract infections and to 21 children with pneumonia. Clinical symptoms of the infections resolved in 17 children (89%) with the urinary tract infections treated with ceftazidime within 4 days. The remaining children (11%) recovered after 7 days of therapy. Clinical symptoms of pneumonia resolved in 19 children (90%) within 7 days of the treatment. A significant improvement was achieved in the remaining 2 children after 10 days of the treatment.     

Clonal Growth of Leukaemic Cells In Vitro

Human leukaemic cell specimens were obtained from patients and directly plated into soft agar (t= 0) or cultured for 1 week in liquid phase and then plated in soft agar. Growth for 1 week in liquid phase allowed the clonal growth in agar of leukaemic specimens which were unable to clone at t= 0. Clonal growth after liquid culture consisted of the usual leukaemic type of cluster-colonies, growth of a new type of ‘syncytial’ cell colony or a mixture of colony types. In addition, marrow from a patient with acute lymphocytic leukaemia produced normal-appearing colonies after 1 week of growth in liquid phase. These studies suggest a similarity in the growth requirements of some leukaemic cells and normal CFUd cells.     

T-cell-antigen positive, E-rosette negative acute lymphoblastic leukaemia (author's transl)]

The lymphoblasts from 100 patients with acute lymphocytic leukaemia were investigated for the expression of receptors for sheep erythrocytes (E) and of a specific heterologous T cell antigen (T). In 17 cases, both T cell markers were expressed simultaneously on the leukaemic cells. In 13 cases only T antigens could be demonstrated on the lymphoblasts. A quantitative analysis of T antigens by immunoautoradiography revealed that the T expression of E-T+ -lymphoblasts was in general like that of E+T+-lymphocytes in the blood of normal persons, in several cases even higher. Therefore, the failure of E-rosette formation cannot be correlated to a decrease of the other T cell differentiation marker. In 7 out of 9 tested cases, a strong acid phosphatase reaction product located paranuclearly could be demonstrated. Complement-receptors were expressed in 3 of 5 cases which were also demonstrated in some cases of the E+T+-ALL group. The latter group was characterized by a T antigen expression like that of thymocytes. 4 cases of the E-T+ALL group were adults. Since the leukaemia cells of 2 cases were negative for acid phosphatase, PAS and all surface markers including cALL antigen, the T antigen can classify undifferentiated and otherwise unclassificable leukaemias. The clinical signigicance of the E-T+-ALL seems to be important since 5 out of 9 children with this type of ALL died soon after diagnosis.     

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.     

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.     

Increased cellular hypoxia and reduced proliferation of both normal and leukaemic cells during progression of acute myeloid leukaemia in rats

The microenvironmental changes in the bone marrow, spleen and liver during progression of the transplantable promyelocytic leukaemia in the Brown Norwegian rat (BNML) have been studied. We used flow cytometry to estimate cellular hypoxia and proliferation based on in vivo pulse-labelling with a mixture of 2-nitroimidazole linked to theophylline (NITP) and bromodeoxyuridine (BrdUrd). The leukaemic cells were identified with the RM124 antibody. In rats inoculated with leukaemic cells the fraction of RM124+ cells was significantly increased from day 20 onwards in the spleen and from day 27 in the bone marrow and liver, reaching a level of 65-87% in these organs at day 32. At day 32, the NITP+ fraction of RM124+ cells had increased significantly in the bone marrow and spleen to 88% and 90%, respectively. The corresponding fractions of NITP+ normal cells reached 63% and 65%, respectively. From day 13 to day 32, the DNA-synthesizing (BrdUrd+) fraction of RM124+ cells in the bone marrow decreased significantly from 52% to 25%, and of normal cells from about 20% to 6%. In the bone marrow and spleen at day 27 and 32, the S-phase and G2/M-phase fractions according to DNA content were higher for the NITP+ than for the NITP- cells. This could partly be explained by an impaired cell cycle progression due to hypoxia. Nevertheless, we found indications of leukaemic cells that were simultaneously labelled with NITP and BrdUrd, in the bone marrow and spleen. These latter findings suggest that in contrast to normal cells some of the leukaemic cells can proliferate even during hypoxia, and this subpopulation may consequently renew and expand the leukaemic cell load.     

The effect of exogenous alkalization on cytokinetics and on the survival rate of mice with lymphoblastic leukemia]

In mice vaccinated with two forms of lymphoblastic leukaemia and alkalized with intravenous administration of sodium bicarbonate, the survival rate, the extent of leukaemic infiltration and the proliferative capacity of cells in the bone-marrow, thymus, spleen, lymphnodes, liver and lungs were investigated. The survival rate in the TAL leukaemia of the AKR stem producing an endogenous acidosis could be significantly prolonged in a statistical way by alkalization. Yet an accelerated expiring rate could be observed after exogenous alkalization in L-1210 leukaemia of the DBA/2J stem producing an endogenous alkalosis. By means of cytological and impulse-cytophotometrical investigations the exogenous alkalization of both forms of leukaemia could be proved to have a direct bearing on the proliferative kinetics. In TAL leukaemia the leukaemic proliferation was inhibited by the exogenously involved correction of the acid-base balance; in the L-1210 leukaemia, however, the pH disturbances were enhanced, thus accelerating the leukaemic proliferation. Consequently, the disturbances of the acid base balance seem to be an essential cofactor in the leukaemia genesis. The exogenous direction of the acid-base balance may be important as a means of treating leukaemia.