Myeloid leukaemic cells can lyse fibrin directly

Purified preparations of circulating leukaemic blast cells from patients with acute myeloid (M1-7) or acute lymphoblastic leukaemia, and the myeloid or lymphoid cells from patients with chronic myeloid or lymphocytic forms of leukaemia, were incorporated into clots prepared from fibrinogen and plasminogen. Patterns of lysis were followed and measured by light transmission in a microtitre plate reader. Mature polymorphonuclear and mononuclear cell fractions from normal individuals were studied concurrently for comparison. Blast cells from the myeloid forms of acute leukaemia (M2-4) and 'myeloid' cell fractions from patients with chronic myeloid leukaemia were capable of lysing plasminogen-containing clots; this activity was neutralized by addition of immunoglobulin against urokinase plasminogen activator (u-PA), but not by anti-tissue plasmogen activator (t-PA). Mature polymorphonuclear and mononuclear cells from normal individuals lacked lytic activity, as did the leukaemic cells from patients with acute lymphoblastic or chronic lymphocytic leukaemia. Lysed blast cells showed the presence of free plasminogen activator on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with overlay zymography, also neutralized by anti-u-PA, whereas normal polymorphonuclear and mononuclear cells did not. These observations suggest that mechanisms underlying some forms of severe bleeding in acute myeloid leukaemias have a critical fibrinolytic component generated by the blast cells themselves.     

Molecular evidence for a common leukaemic progenitor in acute mixed lymphoid and myeloid leukaemia

De novo acute leukaemia presenting with a mixed lymphoid and myeloid leukaemic population has rarely been described. We have used the consensus Ig heavy chain primers and DNA isolated from these two distinct populations of cells in polymerase chain reactions. We demonstrated that both populations of cells exhibited Ig heavy chain gene rearrangements. Cloning and subsequent DNA sequencing of the amplified products showed a common V-D-J junctional nucleotide sequence. This work therefore provides the first evidence that the leukaemic cells in de novo acute leukaemia with a mixed lymphoid and myeloid population are derived from a common progenitor clone and may offer an explanation for the poor prognosis in these patients.     

Expression and functional role of urokinase-type plasminogen activator receptor in normal and acute leukaemic cells

Urokinase-type plasminogen activator receptor (UPA-R-CD87) is a GPI-anchored membrane protein which promotes the generation of plasmin on the surface of many cell types, probably facilitating cellular extravasation and tissue invasion. A flow cytometric quantitative analysis of expression levels for UPA-R was performed on fresh blast cells from patients with acute myeloid leukaemia (AML, n = 74), acute lymphoblastic leukaemia (ALL, n = 24), and biphenotypic leukaemia (BAL, n = 3) using two CD87 monoclonal antibodies (McAbs) (3B10 and VIM5). Peripheral blood and bone marrow (BM) cells from 15 healthy adults served as controls. Using 3B10 McAb, UPA-R was expressed (>99%) by blood monocytes, neutrophils, and BM myelomonocytic precursors in controls, whereas resting T and B lymphocytes, and CD34⁺ cells were UPA-R negative. We also attempted to clarify whether UPA-R has a role in mediating neutrophil functions. Oriented locomotion induced by different chemotaxins and lysozyme release by granules stimulated with fMLP or PMA were significantly decreased when UPA-R was neutralized by CD87 McAb. In contrast, the anti-UPA-R McAb had no effect on superoxide anion generation of normal neutrophils. Blasts from AML showed a heterogenous pattern of expression for the UPA-R McAbs, with reactivity strictly dependent on FAB subtype. The highest UPA-R expression was seen in the M5 group: all patients tested (n = 20) showed strong positivity for the UPA-R McAb whereas only 12% (3/24) of ALL patients were CD87 positive, and 2/3 of BAL patients showed a dim expression for CD87. The number of receptors expressed by blast cells in 6/74 (8.1%) AML patients was higher than those of normal samples; in addition, since co-expression of UPA-R and CD34 was not found in normal haemopoietic cells, it may be postulated that CD87 can be used alone (when overexpressed) or in combination with CD34 for the detection of minimal residual disease. Results also indicated that patients with UPA-receptors >12 × 10³ ABC/cell, irrespective of FAB subtype, had a greater tendency for cutaneous and tissue infiltration and a higher frequency of chromosome abnormalities, thus suggesting the concept that cellular UPA-R content positively correlates with the invasive potential of AML cells. The combination of higher UPA-R positivity, abnormalities of chromosome 11, and M5 FAB morphology may identify a peculiar subset of AML, characterized by a more aggressive clinical course.     

Clearance of leukaemic blasts from peripheral blood during standard induction treatment predicts the bone marrow response in acute myeloid leukaemia: a pilot study

Although several parameters are useful for risk stratification of patients with acute myeloid leukaemia (AML), there are no firm criteria for predicting response to induction treatment of individual patients. Daily flow cytometry (FC) analysis, carried out during induction treatment in 30 AML patients, showed that the clearance of blasts from peripheral blood (PBC) correlated closely with response, as assessed by bone marrow FC on day 14, and by morphologic analysis at haematopoietic recovery. Therefore, a major treatment outcome can be predicted very early in AML patients, thus providing an opportunity for tailoring treatment modalities from the outset.     

Changing bone marrow micro-environment during development of acute myeloid leukaemia in rats

The Brown Norwegian rat transplanted with promyelocytic leukaemic cells (BNML) has been used as a model for human acute myeloid leukaemia. We have previously shown that both the blood supply to the bone marrow and the metabolic rate decrease in relation to the leukaemic development in these rats. Here we have investigated how the development and progression of this leukaemia affect oxygenation, pH and proliferation of normal and leukaemic cells in vivo . Bone marrow pH was measured by a needle electrode. Nitroimidazol-theophylline (NITP) was used to identify hypoxic cells, and we applied bromodeoxyuridine (BrdUrd) to identify DNA replicating cells.   The leukaemia progressed slowly until day 27 after which a rapid deterioration could be observed leading to severe changes over the following 5 d. In whole blood there was evidence of progressing metabolic acidosis. In bone marrow the fraction of leukaemic cells increased to > 90% and the pH dropped to about 6.5. The fraction of NITP⁺ cells increased to > 80% in bone marrow and to about 40% in blood. The fraction of BrdUrd⁺ cells was unchanged in blood, but decreased in bone marrow both for normal cells (from about 20% to 5%), and for leukaemic cells (from about 45% to 25%), evidently as a result of the severely changed micro- environment. In this study we have demonstrated in vivo the development of an acidic and hypoxic bone marrow hampering normal haemopoiesis during leukaemic growth. Our data support the notion of BNML as a valuable tool for studying leukaemogenesis.     

Childhood acute myeloid leukaemia

Although acute myeloid leukaemia (AML) has long been recognized for its morphological and cytogenetic heterogeneity, recent high-resolution genomic profiling has demonstrated a complexity even greater than previously imagined. This complexity can be seen in the number and diversity of genetic alterations, epigenetic modifications, and characteristics of the leukaemic stem cells. The broad range of abnormalities across different AML subtypes suggests that improvements in clinical outcome will require the development of targeted therapies for each subtype of disease and the design of novel clinical trials to test these strategies. It is highly unlikely that further gains in long-term survival rates will be possible by mere intensification of conventional chemotherapy. In this review, we summarize recent studies that provide new insight into the genetics and biology of AML, discuss risk stratification and therapy for this disease, and profile some of the therapeutic agents currently under investigation.     

Bone marrow trephine findings in acute myeloid leukaemia with multilineage dysplasia

Acute myeloid leukaemia (AML) with multilineage dysplasia (MD) is one of the four main categories of AML in the World Health Organization (WHO) classification. The role of bone marrow trephine biopsy (BMTB) histology and immunohistochemistry in the diagnosis of AML-MD is currently unclear. BMTBs were studied in 11 cases of AML-MD and two cases of myelodysplasia that subsequently transformed to AML. Among them, six cases showed trilineage dysplasia and seven showed bilineage dysplasia. With respect to conforming to the WHO definition of AML, documentation of an increased proportion of immature myeloid cells was possible on morphology and counting of immature cells following immunostaining with CD34, CD117 or HLA-DR antibodies. Recognition and quantification of dysplastic features in the haemopoietic lineages was made easier by immunohistochemistry with antibodies to ret40f (glycophorin C), myeloperoxidase, CD61 and/or CD42b, CD34, CD117 and HLA-DR. Based on this relatively small series of cases we show the utility of BMTB and immunohistochemistry as an aid to the diagnosis of AML-MD. This has to be seen not just in light of its utility at diagnosis, but also the role the diagnostic BMTB would play for purposes of comparison when follow-up BMTBs are submitted in this group of patients.     

Spontaneous remission in acute myeloid leukaemia

A summary of the literature of adult patients with acute myeloid leukaemia (AML) who have undergone spontaneous remission (SR) is presented together with a report of a patient whose SR was accompanied by cytogenetic remission. There are less than 20 reports of SR since the 1980s. SR is by no means synonymous with cure, since the average duration of the remission is only 7·1±9·2 months. Neither infections nor transfusions are absolute requirements of SR.     

Ultrastructural features in 100 cases of acute myeloid leukaemia

Ultrastructural features thought to be significant in acute myeloid leukaemia (AML) have been examined in 100 cases entered into the Medical Research Council 9th AML Trial. It is concluded that, of the features examined, only rarely is there a striking correlation with diagnosis which could be usefully employed with confidence. This is a consequence of there being both a large overlap of similar ultrastructural features between the different sub-types of the disease and also wide variations in ultrastructure within the conventionally diagnosed sub-types of AML. Thus the similarities between cells from different cases which allow them to be classified by light microscopy do not necessarily extend to the ultrastructural level. However, it is suggested that a classification on ultrastructural grounds might have a prognostic significance which would only be revealed by a long-term study.     

Immunocytochemical and flow cytometric detection of proteinase 3 (myeloblastin) in normal and leukaemic myeloid cells

Summary. Proteinase 3 (P3) is a serine proteinase present in the primary granules of neutrophils. We have investigated the expression of this protein in samples of bone marrow from healthy individuals and patients with different types of leukaemias by using immunocytochemical staining and flow cytometric quantitation. In normal bone marrow the enzyme was found in promyelocytes, myelocytes, metamye-locytes, band forms and polymorphonuclear neutrophils, correlating with the synthesis of neutrophil serine proteinases during myeloid maturation. No staining was found within the lymphoid, erythroid and megakaryocytic lineage. In the leukaemic samples, only those of acute myeloid and chronic myeloid leukaemia patients were labelled with the antiproteinase 3 antibody. Cases of acute lymphoblastic and chronic lymphocytic leukaemia, as well as other malignant lymphomas, were consistently negative, indicating that P3 may be used as a specific marker for the discrimination between myeloid and lymphoid leukaemias. In addition. immunoreactivity of myeloperoxidase (MPO) was investigated and the expression of P3 and MPO correlated with the French-American-British (FAB) classification. P3 was not detected in minimally differentiated MO and Ml cases but was in predominantly labelled cells of M2 and M3 subtypes plus half of the M4 and one out of six M5 cases but not those of M6. These findings correspond to the differentiation stage in which P3 is expressed and stored in the primary granules. Therefore the enzyme may also be used as an adjunct to the classic morphological and cytochemical methods to elucidate further the stage at which the differentiation arrest of the leukaemic clone has occurred.     

Single agent thalidomide in patients with relapsed or refractory acute myeloid leukaemia

Thalidomide is a putative anti-angiogenesis agent that has significant anti-tumour activity in haematological malignancies with increased bone marrow angiogenesis, including multiple myeloma (MM) and myelodysplastic syndromes (MDS). Increased levels of the mitogen for angiogenesis, vascular endothelial growth factor (VEGF), correlate with worse survival in acute myeloid leukaemia (AML). A phase II trial of thalidomide was conducted in patients with relapsed- or refractory-AML previously treated with cytarabine-containing regimens. A total of 16 patients with refractory- or relapsed-AML were treated with thalidomide 200-800 mg orally daily (median dose 400 mg daily) for a median of 27 d (range, 3-94 d). Overall, one patient (6%) achieved complete remission (CR) lasting for 36 months, and two patients had a transient reduction in marrow blasts from 8% and 7% to less than 5% in both cases. There was no correlation between reduction in levels of angiogenesis markers and response. Toxicities related to thalidomide were significant, and precluded dose escalation beyond 400 mg orally daily in most patients. Although there appears to be some evidence of biological activity, single agent thalidomide is not an optimal choice of therapy for salvaging patients with relapsed- or refractory-AML. Thalidomide analogues with more potent immunomodulatory activities and more favourable toxicity profiles may offer more promise as anti-AML therapy.     

P-glycoprotein and multidrug resistance-associated protein, but not lung resistance protein, lower the intracellular daunorubicin accumulation in acute myeloid leukaemic cells

The in vitro intracellular daunorubicin accumulation (IDA) of blast cells from 69 patients with newly diagnosed acute myeloid leukaemia (AML) was correlated with the expression and functional activity of the multidrug resistance (MDR) proteins, P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP) and lung-resistance protein (LRP). An inverse and significant association was found between IDA and Pgp-related efflux activity (r = -0.31, P = 0.01) and also MRP (r = -0.25, P = 0.04) but not with LRP (r = -0.13, P = 0.28). Coexpression of the MDR proteins had an additive effect in further lowering of IDA levels, suggesting that the clinical MDR phenotype is dependent on the sum of multiple MDR factors available to the leukaemic cell. Thus, the median IDA of leukaemic cells without any MDR proteins was significantly higher than that of blasts carrying two MDR proteins (0.466 vs. 0.296, P = 0.046). Seven patients with no expression of Pgp, MRP and LRP still had low IDA levels, suggesting the presence of efflux MDR mechanisms other than those studied. The relation of IDA to clinical parameters known to be associated with poor prognosis, such as age, secondary AML, karyotype, peripheral blood blast and CD34 counts, was also studied, but no significance was found on multifactorial analysis. There was a non-significant trend for earlier relapse in patients with low IDA levels (leukaemia-free survival of 16.3 months compared with 21.1 months in patients with high IDA levels). Our data suggest that, while the IDA assay is a quick and relatively easy test for the combined efflux MDR phenotype, it is unable to detect other MDR mechanisms, such as LRP, which may be important to the clinical outcome of patients with AML.     

The concept of leukaemic stem cells in acute myeloid leukaemia 25 years on: hitting a moving target

The concept of leukaemic stem cells (LSCs) was experimentally suggested 25 years ago through seminal data from John Dick's group, who showed that a small fraction of cells from acute myeloid leukaemia (AML) patients were able to be adoptively transferred into immunodeficient mice. The initial estimation of the frequency was 1:250 000 leukaemic cells, clearly indicating the difficulties ahead in translating knowledge on LSCs to the clinical setting. However, the field has steadily grown in interest, expanse and importance, concomitantly with the realisation of the molecular background for AML culminating in the sequencing of hundreds of AML genomes. The literature is now ripe with contributions describing how different molecular aberrations are more or less specific for LSCs, as well as reports showing selectivity in targeting LSCs in comparison to normal haematopoietic stem and progenitor cells. However, we argue here that these important data have not yet been fully realised within the clinical setting. In this clinically focused review, we outline the difficulties in identifying and defining LSCs at the individual patient level, with special emphasis on intraclonal heterogeneity. In addition, we suggest areas of future focus in order to realise the concept as real-time benefit for AML patients.     

Isolated gingival relapse in acute myeloid leukaemia

A 47-yr-old man with acute myeloid leukaemia had been in complete remission for 18 months when he developed a gingival tumour. Histological examination revealed leukaemic infiltrate and, at this stage, no other signs of relapse could be demonstrated. Chemotherapy and local radiotherapy resulted in the disappearance of the mass; however, histological abnormalities persisted. First relapse of acute myeloid leukaemia presenting as a gingival tumour has not been previously reported.     

Absence of leukaemic CD34+ cells in acute myeloid leukaemia is of high prognostic value: a longstanding controversy deciphered

Primary resistance and relapses after initial successful treatment are common in acute myeloid leukaemia and therefore outcome remains poor. More accurate risk group stratification and effective personalized risk adapted treatment are necessary to improve outcome. In the last two decades, controversial results have been published concerning the prognostic relevance of CD34 expression. In this study of 706 acute myeloid leukaemia patients, we established a new flow cytometric‐based CD34‐definition, without use of cut‐off values. We discriminated CD34‐positive (n = 548) and CD34‐negative (n = 158) patients by the presence or absence of neoplastic CD34+ cells, respectively. CD34‐status was defined using aberrant immunophenotypes and validated using molecular phenotypes. This new definition of CD34 enables strong prediction of treatment outcome in the entire patient group and in several risk subgroups. Previously observed discrepancies in prognostic impact of CD34 protein expression using cut‐offs (5–20%) can now entirely be explained by considering the number of CD34‐negative cases. In the total patient group, the absence of neoplastic CD34‐positive cells is paralleled by low levels of minimal residual disease, suggesting relative therapy sensitivity and explaining longer survival. Overall, we present CD34 surface expression as a relatively simple, powerful and independent predictor of clinical outcome, now warranting incorporation in acute myeloid leukaemia risk stratification.