Is the mdr 1 gene relevant in chronic lymphocytic leukemia?

Chronic lymphocytic leukemia (CLL) is a progressive disease in which chemotherapy may result in temporary suppression of the peripheral blood lymphocyte count, but cure is not usually possible. Drug resistance mechanisms and the multidrug resistant (MDR) phenotype may be relevant to the therapeutic response. We have studied 34 patients with CLL (seven untreated, 27 treated), screening both DNA and RNA with the mdr 1 gene probe. In pure lymphocyte populations from 10 normal subjects, low levels of mdr 1 RNA expression were found. Eighteen CLL patients (four untreated, 14 treated) had levels of mdr 1 RNA expression above the normal range. No evidence of mdr 1 gene amplification could be found in these patients. Sequential estimations of RNA levels in three patients suggest that malignant lymphocytes in CLL can increase mdr 1 expression in response to chemotherapy and return to basal levels on withdrawal of the treatment. Such data raise important questions about the type of timing of cytotoxic therapy in CLL.

Effective reversal of warfarin-induced excessive anticoagulation with low dose vitamin K1.

Reversal of the anticoagulant effect of warfarin in patients with no active haemorrhage can be achieved by administration of intravenous vitamin K1. Currently recommended doses of intravenous vitamin K1, for this purpose often result in subsequent difficulties in anticoagulation. We observed the response to low dose intravenous vitamin K1 in patients requiring reversal of anticoagulant therapy. Ten consecutive patients received 1 mg and 21 further patients received 0.5 mg of intravenous vitamin K1. In 50% of the patients who received 1 mg of vitamin K1 the INR (International Normalised Ratio) fell below 2 at 24 h whereas in patients who received 0.5 mg the INR fell below 5.5 in all subjects after 24 h and in none did it fall below 2.0. No patient had any thrombotic or haemorrhagic complications and no difficulty was encountered in re-establishing anticoagulant control after 24 h. We recommend 0.5 mg of vitamin K1 as an effective and convenient method of predictable and fine control of oral anticoagulant therapy.

In vitro chemosensitivity testing in chronic lymphocytic leukaemia patients.

Twenty-nine samples from eighteen patients with chronic lymphocytic leukaemia (CLL) were used in a direct comparison of in vitro response to chlorambucil measured in a metabolic (MTT) and dye exclusion (D.Ex) assay. Reduced ability to produce formazan corresponded to a reduced number of dye-excluding viable cells and a significant correlation was found between dose-response measured in the two assays. Initial low absorbance values obtained with untreated control cells in the MTT assay were effectively overcome by increasing both the cell seeding density and MTT exposure time with the consequent increase in assay sensitivity. The MTT assay provided qualitatively similar dose-response data to that obtained in the D.Ex assay. A wide range in in vitro response was seen for both pretreatment and treatment patient groups. In vitro dose-responses were seen to coincide with decreasing or stable white cell counts, taken around the time of sampling, in samples from 4 patients. Less marked dose-responses were observed for 2 patients considered clinically resistant to chlorambucil. The MTT assay would seem, therefore, to be applicable to in vitro assay of cell response in CLL.

Aplastic anaemia incidence in parts of the United Kingdom in 1985.

This paper describes the distribution of new cases of aplastic anaemia in parts of the UK in 1985. The age, sex and spatial pattern are described. Special attention is given to the investigation of five childhood cases which occurred close in space and time. The results are contrasted with similar data from elsewhere.

Serum ferritin concentration as an index of storage iron in rheumatoid arthritis.

Serum ferritin concentration has been compared with semi-quantitative histochemical estimates of bone marrow iron deposits in 60 anaemic patients with rheumatoid arthritis. There was considerable variation in the visual assessment of iron stores made by different observers. Serum ferritin appears to be a particularly sensitive index of iron status when stores are low. The best means of detecting iron deficiency in rheumatoid arthritis are discussed.

An improved method for the diagnosis of polycythaemia.

The red cell mass was measured in 44 normal subjects and showed a closer correlation with total body water or surface area than with body weight. The results obtained in a group of patients with polycythaemia, however, still overlap with the normal range. When the total number of circulating red cells is measured these patients form a group quite separate from the normals. The diagnostic value of this measurement is therefore considerably greater than results obtained with the red cell mass.

Granuloma formation in patients receiving BCG immunotherapy.

Eleven patients with acute myeloblastic leukaemia have received repeated intravenous injections of BCG containing 4-9 X 10(6) live organisms per millilitre. Non-caseating epithelioid granulomas, sometimes with giant-cell formation, have been demonstrated in eight bone marrow aspirates. Seven patients had granulomas in the liver, three in the lung, one in the spleen, one in lymph nodes, and one in a skin biopsy. One patient had a raised serum alkaline phosphatase, but none of the patients had any illness which could be related to the presence of granulomas. Granuloma formation appeared more extensive in four patients who were probably anergic before BCG treatment. Until the significance of this finding becomes clear great care should be taken when giving BCG by the intratumour of intravenous routes to potentially immunoincompetent patients.

Anticoagulation for three versus six months in patients with deep vein thrombosis or pulmonary embolism, or both: randomised trial.

OBJECTIVE: To determine the optimum duration of oral anticoagulant therapy after an episode of deep vein thrombosis or pulmonary embolism, or both. DESIGN: Multicentre, prospective, randomised study with follow-up for one year. SETTING: 46 hospitals in United Kingdom. PARTICIPANTS: Patients aged > or =18 with deep vein thrombosis or pulmonary embolism, or both. INTERVENTIONS: Three (n=369) or six months (n=380) of anticoagulation with heparin for five days accompanied and followed by warfarin, with a target international normalised ratio of 2.0-3.5. MAIN OUTCOME MEASURES: Death from deep vein thrombosis or pulmonary embolism; failure to resolve, extension, recurrence of during treatment; recurrence after treatment; and major haemorrhage during treatment. RESULTS: In the patients allocated to three months' treatment two died from deep vein thrombosis or pulmonary embolism during or after treatment, compared with three in the six month group. During treatment deep vein thrombosis or pulmonary embolism failed to resolve, extended, or recurred in six patients in the three month group without fatal consequences, compared with 10 in the six month group. After treatment there were 23 non-fatal recurrences in the three month group and 16 in the six month group. Fatal and non-fatal deep vein thrombosis or pulmonary embolism during treatment, and after treatment thus occurred in 31(8%) of those who had received three months' anticoagulation compared with 29 (8%) of those who had received six months' (P=0.80, 95% confidence interval for difference -3.1% to 4.7%). There were no fatal haemorrhages during treatment but there were eight major haemorrhages in those treated for six months and none in those treated for three months (P=0.008, -3.5% to -0.7%). Thus 31 (8%) of the patients receiving three months' anticoagulation experienced adverse outcomes as a result of deep vein thrombosis or pulmonary embolism or its treatment compared with 35 (9%) of those receiving six months' (P=0.79, -4.9% to 3.2%). CONCLUSION: For patients in the UK with deep vein thrombosis or pulmonary embolism and no known risk factors for recurrence, there seems to be little, if any, advantage in increasing the duration of anticoagulation from three to six months. Any possible advantage would be small and would need to be judged against the increased risk of haemorrhage associated with the longer duration of treatment with warfarin. TRIAL REGISTRATION: Clinical Trials NCT00365950 [ClinicalTrials.gov].

Iron metabolism and anaemia in pregnancy.

The mechanism by which anaemia develops in pregnancy is well understood: haemodilution causes a fall in the haemoglobin concentration during the first and second trimesters of normal pregnancies. Negative iron balance throughout pregnancy, particularly in the latter half, may lead to iron deficiency anaemia during the third trimester. The increase in iron demand is required to meet the expansion in maternal haemoglobin mass and to meet the needs of fetal growth. Fetal demand for iron results in a unidirectional flow of iron to the fetus against a concentration gradient regulated by fetal requirements for iron; this iron transfer occurs almost entirely irrespective of maternal iron status. The development of maternal iron deficiency during pregnancy may be detected by monitoring the haemoglobin concentration frequently; values falling to less than 11 g/dl should be regarded as abnormal, but specific red cell changes, such as microcytosis, may be lacking. A diagnosis of iron deficiency can be most conveniently confirmed by the serum ferritin concentration falling to less than 12 micrograms/l. Women at risk from iron deficiency anaemia can therefore be readily identified and corrective treatment instituted prior to the development of severe anaemia. A serum ferritin concentration of less than 50 micrograms/l in early pregnancy is an indication for iron supplements. Women in whom the serum ferritin concentration is greater than 80 micrograms/l at booking are unlikely to require iron supplements during pregnancy. This approach would eliminate the need for routine prophylactic iron therapy, which, in populations enjoying a good nutritional status, can no longer be justified in early pregnancy. Furthermore, any risk to the fetus from severe maternal anaemia would be avoided by prophylaxis and prompt treatment.

The inhibition of DNA synthesis in chronic lymphocytic leukaemia cells by chlorambucil in vitro.

The inhibition of 3H-thymidine incorporation into the DNA of mitogen-stimulated lymphocytes from patients with chronic lymphocytic leukaemia by chlorambucil was measured in vitro and the results related to clinical drug resistance. The assay proved to be both sensitive and specific showing a clear separation of those patients with responsive disease from those with disease resistant to treatment. There was evidence of primary drug resistance in untreated patients. In almost all patients who received treatment this led to increasing resistance to chlorambucil in vitro. The assay is predictive of clinical responsiveness and provides a potential means whereby new therapeutic agents and treatment modifiers may be investigated.

Erythropoiesis in the anaemia of rheumatoid arthritis.

Erythropoietic activity and mean red-cell lifespan were measured using 59Fe-transferrin in 32 anaemic patients with active rheumatoid arthritis (RA) and in 20 haematologically normal subjects. Marrow iron turnover (MIT) in the patients was normal and in only 11 was red-cell lifespan less than 70 d. Ineffective iron turnover (IIT) was significantly increased in those patients in whom there was evidence of iron deficiency (serum ferritin less than 12 microgram/l) but in the remaining patients IIT was significantly less than in the normal subjects. In a further 19 patients with simple iron deficiency anaemia IIT was significantly increased. The marrow response to chronic inflammatory disease is clearly distinct from that seen in simple iron deficiency and this suggests different pathogenic mechanisms for these two anaemias.

Parenteral iron therapy in the anaemia of rheumatoid arthritis.

Thirty anaemic patients with active rheumatoid arthritis were each given 800 mg of iron , as iron dextran, intramuscularly over an interval of four weeks. The haemoglobin concentration rose significantly within two months in 26 of the patients but this was followed by a significant fall to the pre-treatment level nine months after treatment. The response to iron therapy was not related to the initial haemoglobin concentration, serum iron concentration, transferrin saturation nor to the amount of storage iron, whether assessed by bone marrow stainable iron or the serum ferritin concentration. There was an unexpected fall in the serum ferritin concentration within the first two months after treatment in half of the patients and this was followed by a rise towards the pre-treatment level during the following seven months, such that there was no apparent addition to the amount of storage iron over the period of the study. The possible mechanisms for these findings are discussed. A response to parenteral iron therapy in patients with active rheumatoid arthritis should not be regarded as evidence of iron deficiency and only by correction of the underlying inflammatory process will lasting improvement in the anaemia be obtained.

The apoptotic pathway: a target for therapy in chronic lymphocytic leukemia.

Cell division and apoptosis (programmed cell death) are the two major physiological processes which control the size of cell populations. Chronic lymphocytic leukaemia arises as a result of the clonal expansion of, usually B-, lymphocytes in which a dysregulation of apoptosis leads to prolonged cell survival. The same process becomes exaggerated with increasing drug resistance, the usual cause of treatment failure in this condition. The identification of points in the apoptotic pathway at which dysregulation occurs is beginning to open up new therapeutic opportunities where the conventional cytotoxic chemotherapy approach is found to fail. Although these strategies are still in their infancy they may increase understanding of the pathogenesis of the disorder and overcome the problem of drug resistance.

Accumulation of storage iron in patients treated for iron-deficiency anaemia.

The repletion of iron stores after treatment was studied in 38 patients with uncomplicated iron-deficiency anaemia. The serum ferritin concentration rose significantly when oral treatment was continued for two months after the attainment of a normal haemoglobin concentration. Patients treated with a total-dose infusion of iron dextran had thehighest final serum levels, which were significantly greater than in patients given Ferro-Gradumet. Oral ferrous sulphate was almost as effective as parenteral iron in producing iron stores.

High-dose etoposide with autologous bone marrow transplantation as initial treatment of small cell lung cancer--a negative report.

Seven patients with small cell lung cancer were treated with high-dose etoposide (1400-2400 mg/m2), given as a single course over 3 days, in conjunction with autologous bone marrow transplantation. Five patients achieved a partial response and two had no response. All patients required further treatment. The lack of any complete responder or good partial responder dissuaded us from entering further patients into the study, and high-dose, single-agent etoposide cannot be recommended in the initial treatment of small cell lung cancer.

When is autologous bone marrow transplantation safe after high-dose treatment with etoposide?

Etoposide (VP16-213) is widely used in the treatment of malignant disease and increasingly high doses are now used in conjunction with autologous bone marrow transplantation. After treatment with etoposide, bone marrow should be reinfused as soon as the plasma etoposide concentration has fallen to a level which will not prove toxic to the small number of pluripotential stem cells present in the reinfused marrow, but this level has not been previously defined. Nine patients were studied of whom five received 1400 mg etoposide/m2 and four received 2400 mg etoposide/m2 intravenously over 3 days. Bone marrow was reinfused 64-136 h after finishing chemotherapy. Haemopoietic recovery occurred in all patients within 16 days of autologous bone marrow reinfusion performed at a time when plasma etoposide concentrations ranged from 0-2.42 micrograms/ml. However the clinical and in vitro data presented suggest that bone marrow reinfusion after treatment with high-dose etoposide should be delayed until the plasma etoposide concentrations have fallen to less than 0.4 microgram/ml although haemopoietic recovery may occur after bone marrow reinfusion at higher concentrations.

Bcl-2/Bax ratios in chronic lymphocytic leukaemia and their correlation with in vitro apoptosis and clinical resistance.

The bcl-2 gene is overexpressed in the absence of gene rearrangements in most cases of B-cell chronic lymphocytic leukaemia (B-CLL) and the proto-oncogene product Bcl-2 has been shown to be a regulator of apoptosis. The activity of this protein is opposed by Bax, a homologous protein that accelerates the rate of cell death. B-lymphocyte Bcl-2 and Bax protein levels were found to be significantly altered in B-CLL and increased Bcl-2/Bax ratios were observed in both the treated and untreated patients compared with those of normal controls. These alterations were particularly pronounced in those treated patients found to be clinically unresponsive to chemotherapy. In order to determine whether Bcl-2/Bax ratios affected cell survival via an anti-apoptotic mechanism, cell death was induced in B-CLL cells in vitro using chlorambucil, and apoptosis was monitored by Annexin V and propidium iodide staining. Confirmation that the labelled cells were apoptotic was achieved by morphological assessment of cytospin preparations of cell-sorted populations. Drug-induced apoptosis in B-CLL cells was inversely related to Bcl-2/Bax ratios.

A method for the investigation of reticuloendothelial iron kinetics in man.

A colloidal suspension of hydrolysed radio-iron of high specific activity has been developed for the investigation of reticuloendothelial (RE) iron kinetics in man. Following intravenous injection this material is cleared rapidly by the RE system and the iron released intn to the endogenous RE iron load, and it has proved possible to measure RE iron release without disturbing the normal iron flow.