Leukotriene production by fresh human bone marrow cells: evidence of altered lipoxygenase activity in chronic myelocytic leukemia.

The metabolism of arachidonic acid through the lipoxygenase pathway was studied in suspensions of fresh human bone marrow cells from eight patients with chronic myelocytic leukemia (CML) and 10 normal controls. After the cells were incubated with the calcium ionophore A23187 and arachidonic acid, a technique including reverse- and straight-phase high-pressure liquid chromatography (HPLC) was employed to isolate and detect different lipoxygenase-mediated compounds. The detected compounds included leukotriene B4 (LTB4), with its two major nonenzymatic isomers 6-trans-LTB4 and 12-epi-6-trans-LTB4 5S,12S-DHETE, and the monohydroxy eicosatetraenoic acids 5-HETE, 12-HETE, and 15-HETE. The pattern of lipoxygenase-mediated products from the bone marrows was similar to that previously described from human peripheral blood. Of eight bone marrow samples from CML patients, five expressed values above 600 ng LTB4/10(8) nucleated cells, as compared to only one out of 10 controls. In contrast, the CML patients produced significantly lower amounts of both the double-dioxygenation product 5S,12S-DHETE (56.8 +/- 16.0 ng [mean +/- SE] versus 146.1 +/- 31.3 ng; p less than 0.05) and the monohydroxy acid 12-HETE (965 +/- 351 ng versus 4390 +/- 1801 ng; p less than 0.05), indicating a 12-lipoxygenase deficiency. The present results show that leukotrienes are formed by human bone marrow cells and further suggest the existence of altered lipoxygenase activity in CML.

Comparative absorption of calcium from carbonate tablets, lactogluconate/carbonate effervescent tablet, and chloride solution.

Intestinal absorption of calcium from three different labelled calcium preparations (all containing 500 mg elemental calcium) was determined using the whole body retention and urinary excretion of 47Ca in 14 normal subjects. Chewable calcium carbonate tablets showed a significantly (p less than 0.05) better mean minimum absorption of calcium (25.6% in exp. I, 22.8% in exp. II) than calcium given in the form of a lactogluconate/carbonate effervescent tablet, (17%), but similar to calcium in a chloride solution (24.7%). The minimum calcium absorption varied from 85 to 128 mg. All the preparations were taken with standardized low calcium test meals.

Phase I pharmacologic study of a new Vinca alkaloid: navelbine.

Navelbine (NVB) is a new semi-synthetic Vinca alkaloid selected on the basis of its affinity for tubulin. NVB inhibits the polymerisation of tubulin and it has significant antitumor activity on P388 and L1210 leukemias and some other experimental tumors. In the present study, 20 patients (9 carcinomas, 10 lymphomas and 1 blastic crisis of chronic myeloid leukemia) received a median of 4 weekly i.v. doses of NVB. Two patients at least received each dose level: 3.6 mg/m2 (1/10 of the LD10 dose/kg in BDF1 mice), 7.2, 12, 18, 32.4, 35 and 43 mg/m2 per week. A total of 89 doses were administered. All patients had been first heavily pretreated and 17 of them had received a Vinca alkaloid. Leukopenia (neutropenia) was the dose-limiting toxicity. There was no thrombocytopenia. Leukopenia was dose-related and first seen at 32.4 mg/m2 per week. The maximal tolerated dose appears to be about 43 mg/m2. At that dose, 2 out of 3 patients developed severe leukopenia and neutropenia. One localized allergic reaction, one case of transient hepatic dysfunction, and 2 reversible peripheral neuropathies were seen. Pharmacokinetics, studied with a radioimmunoassay (RIA) method, suggested an elimination half-life of 30 h and a plasma clearance of 75 l/h. Four patients with Hodgkin's disease and two patients with non-Hodgkin's lymphoma, all of them refractory to vincristine (VCR) and/or vinblastine (VBL), showed minor responses lasting 2-8 weeks. They had received between 4 and 12 doses of 30 and 43 mg/m2. We recommend for phase 11 trials the dose of 40 mg/m2 per week.

Growth of normal and malignant bone marrow cells.

Results of five different methods (heme breakdown, mitotic indices, total marrow cellularity and different methods of labelling indices) to determine bone marrow cell growth are reviewed. Normal immature erythroblasts and myeloblasts have generation times of about 5-10 h. Erythroblasts in neoplastic conditions (the myelodysplastic syndrome and polycythemia vera) have generation times which are significantly prolonged, by 12-75 per cent, but this may not be true for myeloblasts, since total labelling indices are increased (from normal 3.9 to 11.2 per cent) in the myelodysplastic syndrome. In contrast, generation times in aplastic anemia are significantly shortened, by about 70-80 per cent. Normal mature erythroblasts and promyelocytes have generation times of about 24 h, and the malignant prolongation may be even greater.

Autotransplants in leukemia: current state, future progress.

Autotransplants in leukemia are controversial; their rationale and results have been questioned. Here we consider several issues central to this debate: (1) Are there convincing data to suggest that more intensive therapy increases cures? (2) Are results post-autotransplant a consequence of the transplant, or do they reflect subject-selection and time-to-treatment (time censoring) biases? (3) Does leukemia relapse after an autotransplant develop from persisting leukemia cells in the subject or the graft? (4) Do autotransplants using hematopoietic stem cells from different sources have distinct outcomes? (5) Are immune-mediated anti-leukemia mechanisms likely to prevent relapse after autotransplants? and (6) Can comparably intensive therapy be given without an autotransplant?

Leukotrienes and lipoxins--new potential performers in the regulation of human myelopoiesis.

Leukotrienes and lipoxins are bioactive lipoxygenase products formed by leukocytes alone or in collaboration with other cells. While the physiological role of lipoxins remains to be clarified, accumulating evidence shows that leukotrienes are important mediators in asthma and inflammation. Consequently, recent clinical trials with leukotriene D4 receptor antagonists and 5-lipoxygenase inhibitors have demonstrated marked reduction of airway symptoms in asthmatic patients. In addition, both leukotrienes and lipoxins have been indicated as modulators of cell proliferation. This article reviews recent findings suggesting that these compounds may also participate in the regulation of human myelopoiesis. Such a role is conceivable since leukotrienes and lipoxins can be produced by bone marrow cells and potently modulate GM-CSF-induced myeloid stem cell proliferation.

Cytotoxic effects on viable human leukemic cells by combinations of lymphokine activated killer cells and monoclonal antibodies.

Earlier studies with individually phenotyped monoclonal antibody combinations and complement or lymphokine activated killer (LAK) cells showed that many acute myeloid leukemic cells were resistant to these cytotoxic agents when used singly. Therefore, a combination of both agents was studied. When the leukemic target cells were submitted to killer cells activated with 100 or 800 IU of recombinant interleukin-2 (rIL-2), only averages of 6.0 and 16.7% of the targets were killed respectively. When the remaining, refractory cells were confronted with a cocktail of individually phenotyped monoclonal antibodies and complement, an additional significant cell kill was obtained, but it amounted to only between 7.4 and 5.5% (for LAK-100 and LAK-800, respectively). In contrast, of the target cells initially refractory to the same cocktail of monoclonal antibodies, all were cross-resistant both to LAK-cells activated with 100 and to those activated with 800 IU of rIL-2. This cross-resistance was caused neither by sub-optimal LAK-cell activation, nor by antibody blocking of hypothetical LAK-cell receptors, since pre-incubation with monoclonal antibodies without complement did not inhibit LAK-cell cytotoxicity. Although only partial cross-resistance was found in the present study, it still remains that only a minority of the tumor cells could be killed. A higher in-vitro cell kill should be attempted prior to clinical trials in order to avoid clinical effects resembling those of a partial surgical tumor resection.

The quandary of cancer prevention.

The incidence and mortality due to the major cancers such as lung, breast, colon-rectum, prostate, and ovary have changed very little over the past 20-30 years, in spite of the introduction of important new treatments and apparent prolongation of survival of patients with these cancers. The new strategies focus on earlier detection and primary prevention of cancer. Three approaches for prevention are receiving increasing prominence as an approach to reducing the incidence of cancer: (1) control of common source environmental carcinogens, (2) modification of personal health behavior believed to increase or decrease the risk of cancer, and (3) identification of specific genotypes that increase the risk of cancer. All of these approaches offer some hope of reducing cancer incidence and morbidity. All will be costly and, therefore, require careful evaluation. It is likely that the changes in personal health behaviors will have the greatest overall impact on cancer incidence. Identification of specific genotypes will be of importance for high risk families. At present, it is unlikely that control of environmental common sources will substantially reduce cancer incidence without better measures of exposure and risk of disease.

Body composition and muscle constituents during weight loss: studies in obese patients following gastroplasty.

BACKGROUND: The influence of rapid and prolonged weight loss on body composition and muscle constituents in the obese patient is not well known. There are serious complications related to rapid and prolonged weight loss. It is of general interest to increase the understanding of the mechanisms and consequences of significant weight loss in man. METHODS: In 40 obese patients, the body composition and muscle constituents were studied before and during 1 year of weight loss following gastroplasty. The study was undertaken in two groups (A and B) of obese patients, comprising 32 women and eight men, body weight 82-175 kg and aged 24-49 years. Mean BMI in group A and B was 45 (W/H(2)) and 43 (W/H(2)) respectively. Body composition was assessed by total body potassium measurements and muscle constituents were determined by analyses of muscle specimens obtained percutaneously. RESULTS: The preoperative body composition was found to be equal parts of lean body mass and body fat. Preoperatively, muscle constituents revealed a higher protein content per cell and a lower potassium concentration related to fat-free solids. The loss of 18-28% body fat and lean body mass occurred in equal proportions during the first 3 postoperative months of rapid weight loss, followed by a continuous decrease of body fat but not of lean body mass. The concentrations of proteins and potassium per muscle cell revealed a reduction during the period of rapid weight loss. The RNA/DNA ratio 1 year after surgery was still reduced, indicating a low protein synthesis rate. CONCLUSIONS: Preoperatively mean body fat accounted for 50% of the body weight in obese patients. Following weight loss, body fat, lean body mass and concentrations of proteins were reduced compared to preoperative values. After the period of rapid weight loss, with reduction of lean body mass and body fat in parallel, a progressive reduction of body fat was observed whereas the lean body mass did not decrease further. Protein synthesis rate was still low 12 months after surgery.

Decreased retention of vinca alkaloids in chronic lymphatic leukemia cells from refractory patients.

Uptake and retention of vincristine (VCR), vinblastine (VB), and vindesine (VD) in isolated mononuclear cells from six healthy donors and in leukemic cells from 12 patients with chronic lymphatic leukemia (CLL) were studied: Three patients responded to VCR-containing regimens, whereas 4 patients were or became refractory and five patients did not receive VCR. Incubation of leukemic or normal cells with 1 microgram/ml vinca alkaloid for 1-24 h showed a steady state level after 1-2 h. Normal cells both took up and retained significantly more drug than those from patients, both responding and refractory. Cells from VCR-refractory patients had a significantly (P less than 0.01) lower drug retention than those from patients responding to or not receiving VCR. In contrast, the difference in uptake was not statistically significant.

Cardiomyopathy in leukemia, with reference to rubidomycin cardiotoxicity.

Sixteen patients who had died with leukemia were studied at autopsy between September 1975 and Februrary 1977. Special attention was given to degenerative changes in the heart. Five of the patients died of cardiac failure, all with no or only slight leukemic infiltration in various organs at autopsy. Five patients showed basophilic necroses in the myocardium, and two of these also showed necroses in the bone marrow. The myocardial lipofuscin was significantly (P less than 0.01) higher in the autopsies of leukemic patients (mean age 45 years) than in autopsies performed at the Department of Forensic Medicine in 18 cases of accidental death (mean age 36 years). No dose-response relationship could be found between the amount of myocardial lipofuscin and the total dose of rubidomycin. Eight of twelve patients with malignant lymphoma (mean age 45 years) also had increased amount of myocardial lipofuscin.

Treatment of acute nonlymphoblastic leukemia in adults with daunorubicin-DNA complex: a preliminary report.

Forty-four adult patients under 60 years of age with acute nonlymphoblastic leukemia were randomized for induction treatment with one of the following three regimens: R 1 = courses of daunorubicin on day 1 + ARA-C on days 1--5; R 2 = courses of daunorubicin on days 1 and 2 + ARA-C on days 4--8; R 3 = courses of daunorubicin-DNA complex on days 1--2 + ARA-C on days 4--8. Out of 14 patients, 9 went into remission on R 1, 6 out of 14 on R 2, and 8 out of 16 on R 3. The preliminary results suggest that daunorubicin-DNA complex has the same efficacy for inducing remission as daunorubicin alone, if the same time intervals and dosages are used.

Effect of Cytotoxic Cells on Minimal Residual Leukemia.

The presence of minimal residual disease is indicated by the high frequency of relapses after twin bone marrow transplants and after allogeneic bone marrow transplants without graft versus host disease (up to 75% and 45% of cases, respectively). The graft versus leukemia effect may be mediated by IL-2 activation of natural killer cells (CD16 +, CD56 +, CD3-, CD8+/-) or cytotoxic T cells (CD3 +, CD56+/-). These activated killer cells can bind to targets and cause their lysis, and then recirculate to kill other targets. Killing can be blocked by anti-perforin antibodies and enhanced by protein kinase C-activation of effectors There are several studies indicating that a high percentage of leukemic cells can be killed by LAK-cells. However even in the most sensitive cases, lysis of all the cells cannot be achieved. The finding that leukemic clonogenic cells are generally sensitive to both NK and LAK cytotoxicity provides a more hopeful possibility The lack of tumor specific antigents, leukemic cell-immunoheterogeneity and maturation asynchrony explains why antigen dependent T-cell mediated cytotoxicity is only partly effective in eradicating residual leukemic cells. Future work should therefore include more studies of the mechanism of resistance to LAK cells, possibilities of further enchancing cytotoxicity and the mechanism of graft-versus-leukemia effect.

Conjugate formation by leukemic blasts from acute myeloid leukemia with cytotoxic lymphocytes.

Conjugate formation by AML blasts with fresh peripheral blood lymphocytes (PBL) and lymphokine activated killer (LAK) effectors was studied by flow cytometry. Leukemic blasts formed very low numbers of conjugates with fresh PBL and were resistant to natural killer (NK) cytotoxicity. When LAK effectors were used a significant increase in conjugate formation was observed, which in the majority of cases was followed by an increased killing. There was a positive correlation between the percentages of conjugates formed by AML blasts with LAK effectors and the susceptibility to lysis. No significant difference in binding activity between the CD3+ and CD56+ LAK subpopulations was found. There was no correlation between the expression of ICAM-1, LFA-3 and Transferrin receptor (CD71) and the conjugate formation. The blocking of CD71 on the control K562 cell line reduced the conjugate formation with LAK effectors but no such effect could be observed with CD71+ AML blasts.

Blocking of MHC class I antigens on leukemic B-cells enhances their conjugate formation with cytotoxic lymphocytes and their susceptibility to lysis.

The role of major histocompatibility complex (MHC) class I antigens and adhesion molecules (AM) in the resistance of leukemic B-cells to cell-mediated cytotoxicity was investigated using cells from eight patients with B-chronic lymphocytic leukemia (B-CLL) and six patients with immunocytoma (IC). Both CLL and IC cells were completely resistant to natural killer (NK) and lymphokine activated killer (LAK) cytotoxicity and no binding to effector cells was observed, irrespectively of AM expression. Blocking of MHC class I antigens with monoclonal antibodies or their temporary elimination from leukemic B-cell surface by acid treatment resulted in a significant (p < 0.005) increase in both conjugate formation and susceptibility to lysis, thus suggesting the relevance of MHC class I expression on leukemic B-cells for the NK/LAK resistance phenomenon.

DNA image analysis in childhood acute lymphoblastic leukemia.

DNA index (DI) and percentages of cells in S and G2/M phase were determined in Feulgen stained nuclei of blasts from 31 cases of childhood ALL at diagnosis. In 6 cases the results of DNA analysis and cytogenetics were concordant showing hyperdiploidy. Two other cases with normal karyotype were revealed as DNA aneuploid with image analysis. Cases with cytogenetic abnormalities like translocation, deletion or presence of single or double supernumerary chromosomes had DI within normal ranges. Nine ALL cases (29%) were found to be DNA aneuploid--8 hyperdiploid and 1 hypodiploid. The percentages of cells in S and G2/M phase for blasts from bone marrow (mean 17.6%) were significantly higher than those estimated in the peripheral blood (mean 1.57%). We conclude that analysis by image cytometry can detect aneuploid DNA content even in cases, which showed a normal karyotype and provides new information concerning the biological aspects of leukemic blasts.

Staging of myeloma. A preliminary study of staging factors and treatment in different stages.

Attempts were made to find prognostic factors in myeloma. In 16 deceased patients, urinary light chains, skeletal lesions, and the quantity of the monoclonal protein fraction in the serum were correlated to prognosis, in contrast to the electrophoretic mobility of the monoclonal fraction, the hemoglobin, the serum creatinine value, the serum calcium, or the intestinal calcium absorption. Skeletal calcium uptake was only numerically higher in mild myeloma than in advanced myeloma. Since these findings partially agreed with the staging procedure previously proposed by Salmon, a modification of this procedure was used to stage 50 myeloma patients. Survival was statistically significantly shorter in stage III than in stage I. A differentiated treatement with melphalan-prednisone in stage I, cytoxan infusions in stage II, and vincristine-cytoxan-prednisone in stage III is proposed. A preliminary comparison of nine patients in stage II-III given intensive treatment with 23 given melphalan-prednisone suggests a numerically, but not as yet a statistically significant increase in survival in the intensively treated group, which seems to have an 80% 2-year survival.

Possible mechanisms of immunotherapy action in acute nonlymphocytic leukemia: macrophage production of colony-stimulating activity.

Earlier studies show that immunotherapy (IT) improves prognosis in acute nonlymphatic leukemia (ANLL), and that ANLL cells probably have tumor-associated antigens, for autologous lymphocytes can react to them. Also, IT seems to immunize against allogeneic ANLL cells, but there is no cross-immunity to autologous ones. Moreover, patients immunized against their ANLL cells have no better prognosis than patients not having lymphocytes reacting to their ANLL cells. It has also been suggested that IT causes nonspecific immunostimulation, but IT patient's lymphocytes actually react less than those of patients not given IT. The current hypothesis is macrophage activation: Lymphocyte suppression in IT could be explained by suppressor macrophages. Colony-stimulating activity, produced by bone marrow macrophages, decreases during remission in patients not given IT, but not in IT patients. Numerically, blood cells from patients given IT form more colonies than those from patients not given IT. Three of eight patients given IT had more colonies than the upper normal limit.

Immunotherapy versus chemotherapy of acute myeloid leukemia: response to PHA, allogeneic lymphocytes, and leukemic myeloblasts of remission lymphocytes from leukemia patients.

Lymphocytes were studied from 51 patients with acute myeloid leukemia (AML) in remission given chemotherapy (CT) maintenance alone or given chemoimmunotherapy (CIT) with BCG and viable allogeneic leukemic cells. CT lymphocytes reacted significantly more to PHA (P less than 0.05) if taken later than 100 days after remission than if taken earlier. CIT lymphocytes reacted less. Thus, the late CT lymphocytes reacted significantly more to nonspecific stimulators (PHA and allogeneic lymphocytes, P less than 0.01) than did late CIT lymphocytes or control lymphocytes. In consequence, the ratio of reactions to specific (leukemic myeloblasts) over nonspecific stimulators was significantly higher in CIT (P less than 0.01) than in CT lymphocytes. Results may indicate nonspecific immunostimulation during CT, and also some relative specific sensitization to allogeneic myeloblasts during CIT.

Can verapamil induce second response in patients refractory to vincristine?

Verapamil (240 mg daily orally) was tested in a phase II trial to restore vincristine sensitivity in 9 patients with myeloma, chronic lymphatic leukemia and immunocytoma. These tumors were selected because treatment response and tumor progression can easily be ascertained with the help of electrophoresis and marrow studies, blood counts and lymph node examination. All patients were clinically refractory to vincristine-cytoxan-prednisolone combinations, to which adriamycin had been added in 2 patients. One patient was refractory to adriamycin, VM 26, and prednisone. In 2/9 patients a side effect-free second response lasting 5-10 months was observed, with a doubtful response in two additional patients. It is suggested that occasional clinical responses can be seen, despite the fact that in vitro the mean verapamil concentration required to affect vincristine efflux from malignant lymphocytes in 5 mumols/1 and the mean in vivo serum concentration only 1 mumole. Hypothetically, the clinical response can be explained by an overlapping in some patients of an unusually high serum concentration with an unusually low verapamil requirement.