The pathomorphological diagnosis of acute leukaemias: cytology, cytochemistry and immunocytology

In 74 cases of acute leukaemia and of the blastic phase of chronic granulocytic leukaemia (CML), monoclonal antibodies of the "VI" series and a few commercially available antibodies proved to be of valuable help in establishing a definite diagnosis. In 4 out of 35 cases of AML (FAB-M 1-5), and in 2 out of 16 of the blastic phase of CML only the use of monoclonal antibodies secured the diagnosis. In the group of acute lymphoid leukaemias subtypes corresponding to various levels of differentiation, were defined. The blasts of 3 patients out of the 74 did not express any of the markers studied. Two additional cases were investigated for platelet peroxidase and studied with the antiplatelet antibodies VIPL 1, 2 and 3 by the electron microscope. These cases proved to be acute megakaryoblastic leukaemias (acute myelofibrosis).     

Prognostic significance of some clinical, morphological and cytogenetic findings in refractory anaemia (RA) and RA with sideroblasts

In 27 patients initially diagnosed as refractory anaemia (RA) or RA with sideroblasts (RA-S) according to the FAB-classification a number of clinical, morphological and cytogenetic parameters were correlated for prognostic significance. From these correlations it emerged that severe cytopenia is centrally positioned with regard to clinical course in RA and RA-S. Positive correlations were found to initial diagnosis, clonal cytogenetic abnormalities, progression to RA with an excess of blasts (RAEB) or acute myeloid leukaemia (AML), the percentage of bone marrow blast cells and prolonged half life for radioactively labeled iron. The degree of peripheral blood granulocytopenia, alone, was correlated to bone marrow hypoplasia. Moreover, the frequency of abnormal karyotypes was inversely correlated to bone marrow cellularity and proportional to the frequency of bone marrow blast cells. From these relationships it may be proposed that chromosome abnormalities are associated with prolonged blast cell generation times and inhibition of blast cell maturation resulting in reduced marrow cellularity and blast cell accumulation, and, in the peripheral blood, falling percentages of neutrophil granulocytes. With the blast cell accumulation the bone marrow cellularity again becomes hyperplastic and the preleukaemic condition is transformed into RAEB or AML.     

Liberation of anti-heparin activity during platelet aggregation in patients with blastic leukosis and blastic crisis of chronic myelosis]

The activity of platelet factor 4 (FP4) was examined in 37 patients affected with blastic leukaemia, in 16 patients with blastic crisis in chronic myeloid leukaemia and in 2 patients with Willebrand's syndrome. The real activity of FP4 determined by modifying the method according to NIEWIAROWSKI after a complete lysis of granular membrances by means of triton X-100 was found to be lowered in 9 patients affected with blastic leukaemia, in 5 patients with blastic crisis and in two patients with Willebrand's syndrome. Presuming that a maximal FP4 release of the irreversible platelet aggregation must be obtained, which corresponds to the real activity, the author has examined the apparent activity of FP4 released from the aggregated platelets with the help of her own method. In this way the quality of the release reaction from the platelets can indirectly be characterized with their extremely important role for haemostasis. Whereas in exacerbated myeloid leukaemia and Willebrand's syndrome the apparent activity will correspond to the real one, there is a severe specific disturbance of the platelet release response in blastic leukaemia. The platelet aggregation is incomplete caused by the derailment of the energy metabolism and the disturbance of the adenine nucleotides, thus causing an apparent as well as a real FP4 deficiency which can be brought into the same line with pathogenesis of thrombopathy in blastic leukaemia.     

Cell markers in the recognition of acute myeloblastic leukaemia subtypes

The diagnosis of acute myeloblastic leukaemia (AML) is based on cell morphology, cytogenetic and molecular changes, cell markers and clinical data. Our aim was to establish whether morphology and cell markers are comparable in the evaluation of AML. Bone marrow smears were analysed, and flow cytometry and monoclonal antibodies were used to determine cell type and maturity. Morphology and cell markers correlated differently in different AML subtypes.     

Immunoreactive calcitonin: a tumor marker for myelogenous leukemias

In a series of 59 patients with chronic or acute myelogenous leukemia (CML, AML) we investigated whether circulating immunoreactive human calcitonin (i-hCT) levels correlate with diagnosis, response to therapy and clinical course. I-hCT was detectable in plasma samples of 88% of patients with CML in the chronic phase and in 100% of patients with CML in blastic transformation. In the AML patients, a significant relation was observed between the cytological subtype and i-hCT levels at diagnosis. In sequentially studied patients the i-hCT plasma concentration was related to the overall mass of leukemic cells, being lower when complete remission was achieved than at diagnosis and increasing at time of recurrence. These data suggest that circulating i-hCT levels can serve as a "tumor marker" in human myelogenous leukemias.     

Cytogenetic analysis and clinical significance of chromosome 7 aberrations in acute leukaemia

The analysis was performed on bone marrow cells derived from 96 patients with acute leukaemia (AL): 76 with acute myelogenous leukaemia (AML) and 20 with acute lymphoblastic leukaemia (ALL). Aberrations of chromosome 7 were revealed in 20 (21%) of 96 analysed cases: in 14 (18%) with AML and in six (30%) with ALL. Structural aberrations, present in 13 patients (eight with AML and five with ALL), were unbalanced and led to partial monosomy (12 cases) or trisomy (four cases) of chromosome 7. Twelve (86%) out of 14 AML and all the ALL patients with chromosome 7 aberrations had complex karyotypes in their bone marrow cells. Monosomy 7 and 7q losses were frequently observed in the AML group, whereas, in the ALL group, gains in 7q and losses in the short arms constituted most chromosome 7 aberrations. The occurrence of monosomy, or of losses in 7q, results in a worse response to induction therapy in AML patients. The complete remission (CR) rate was significantly lower in this group in comparison to the group of AML patients with a normal karyotype (p = 0.01) in bone marrow cells.     

Acute myeloid leukaemia: a paradigm for the clonal evolution of cancer?

Acute myeloid leukaemia (AML) is an uncontrolled clonal proliferation of abnormal myeloid progenitor cells in the bone marrow and blood. Advances in cancer genomics have revealed the spectrum of somatic mutations that give rise to human AML and drawn our attention to its molecular evolution and clonal architecture. It is now evident that most AML genomes harbour small numbers of mutations, which are acquired in a stepwise manner. This characteristic, combined with our ability to identify mutations in individual leukaemic cells and our detailed understanding of normal human and murine haematopoiesis, makes AML an excellent model for understanding the principles of cancer evolution. Furthermore, a better understanding of how AML evolves can help us devise strategies to improve the therapy and prognosis of AML patients. Here, we draw from recent advances in genomics, clinical studies and experimental models to describe the current knowledge of the clonal evolution of AML and its implications for the biology and treatment of leukaemias and other cancers.KEY WORDS: Acute myeloid leukaemia, Cancer, Clonal evolution, In vivo models of leukaemia, Mutation     

Plasma transcobalamins in haematological disorders

Plasma UBBC-B12 and transcobalamins were measured in 112 patients suffering from different haematological disorders. The data showed different patterns of changes in plasma transcobalamin profile in different haematological disorders. Plasma UBBC-B12 and transcobalamins were significantly higher than normal in untreated chronic myeloid leukaemia, acute promyelocytic leukaemia, nutritional megaloblastic anaemia and in refractory anaemias with hypercellular marrow. Normal levels of these proteins were noted in chronic lymphatic leukaemias, in primary and secondary hypereosinophilic states and in multiple myeloma. Subnormal levels of these proteins were observed in hypoplastic anaemia and acute lymphoblastic leukaemia. Chronic myeloid leukaemia patients during blast crisis and acute myeloid leukaemia patients except those suffering from acute promyelocytic leukaemia showed varying pattern of plasma transcobalamins depending on type of blast crisis or FAB subtype of AML. The significance of these changes in plasma transcobalamins have been discussed along with the experience of other workers in this field.     

Studies of the geographic patterns of c-myc expression in bone marrow

C-myc expression was studied semi-quantitatively in bone marrow biopsies obtained from normal individuals, patients with non-malignant haematological disorders and patients with various haematological malignancies. In normal bone marrow and in the bone marrow of patients with non-malignant haematological disorders, cells containing c-myc protein are present in small clones (average 7 +/- 2.5 cells/clone) located in the centre of the histotopographic region of the biopsy. In contrast, c-myc-containing cells are diffusely distributed in the bone marrow of patients with acute myelogenous leukaemia (AML). In the marrow of patients with myelodysplastic syndromes evolving to AML and in patients with AML in early relapse, the clones of cells containing c-myc are larger than those present in normal marrows (average clone size = 17.5 +/- 3.5 cells). Additionally, the proportion of the cells in normal bone marrow which express c-myc protein is less than that present in AML marrows (23.3 +/- 10.17 v. 60.2 +/- 6.17) and the intensity of staining is also less. Non-Hodgkin's lymphoma patients with bone marrow involvement had distribution of c-myc positive cells similar to those with leukaemic infiltration.     

Day-6 bone marrow aspirate for the prediction of response to remission induction therapy for acute myelogenous leukaemia

Seventy-two adults were treated for acute myelogenous leukaemia (AML). Forty-two had previously untreated AML and 30 had AML after a preleukaemic phase, refractory AML or relapsed AML. The previously untreated patients received a 7-day course of cytosine arabinoside (100 or 200 mg/m2 daily), daunorubicin and vincristine while the remaining patients received a 7-day course of cytosine-arabinoside (1 g/m2 q 12h for 6 days) and amsacrine (on day 7). The percentage of malignant cells and the reduction in the percentage of malignant cells were determined by means of bone marrow aspirates taken on day 6 of the chemotherapy course and at the time of diagnosis. Both variables correlated significantly with the ultimate treatment outcome; the reduction in the percentage of malignant cells correlated even more significantly than the absolute percentage malignant cells in the day-6 bone marrow. By means of multiple regression analysis it became possible to calculate the probability of achieving complete remission for the individual patient; this is given by the equation: probability = 1.9-0.009X (% malignant cell reduction). In addition, the mean percentage of malignant cells in the day-6 bone marrow was significantly higher for patients who failed to achieve than those who entered complete remission. Eighty-six per cent of the patients with less than 20% malignant cells on day 6 entered remission, while 75% of the patients with more than 21% malignant cells failed to achieve complete remission (p less than 0.001). Although all of these calculations support the predictive value of the day-6 bone marrow aspirate, the 95% confidence intervals are too large to allow reliable and safe predictions; therefore more patients must be studied to demonstrate the reliability of this test.     

Similarities in long-term cultures of blood and bone marrow from patients with acute myelogenous leukemia

Dexter-type long-term cultures (LTC) were initiated with peripheral blood (PB) and/or bone marrow cells from 11 patients with acute myelogenous leukemia (AML), and 2 with myelodysplastic syndrome in blastic transformation. Marrow and PB cells from normal subjects served as controls. Assessment of nucleated cells and clonogenic progenitors in the adherent and nonadherent fractions of LTC revealed active hemopoiesis for greater than 5 wks in 4 of 8 cultures of AML blood, and 4 of 7 of AML marrow. The morphology and kinetics of nucleated cells and progenitors with putative normal (granulocyte-macrophage colony-forming units or CFU-gm), and abnormal (blast) phenotype in LTC from AML blood were similar to those from AML marrow, and adherent cells positive for collagen I and III and vimentin were found in both types of LTC. Growth of CFU-gm colonies ceased by wk 5-8 in AML cultures, significantly earlier than in LTC of normal marrow cells (survival of greater than 10 wks), which may indicate derivation of the CFU-gm from a transformed clone or deficiency of stromal function in the leukemic state. In most AML blood and AML marrow LTCs, growth of abnormal (blast) colonies continued until wk 4-6. This study demonstrates certain similarities of morphology and function between LTC of AML blood and AML marrow cells. LTC may provide a useful model for further analysis of circulating primitive hemopoietic progenitor cells in leukemic states.     

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.     

Osteolytic lesion in chronic myelogenous leukaemia

Destructive bone lesion of the femur was observed in one patient from a series of 103 patients with CML. Numerous myeloblasts were seen on touch preparations of the fresh surgical specimen obtained by open biopsy of the affected area. Bone marrow and peripheral blood differential count were compatible with chronic phase of CML at this time. 5 months later blastic crisis developed. Local radiotherapy produced effective palliation of pain but did not prevent blastic transformation of CML. The treatment of blastic crisis was unsuccessful and patient died 3 years after diagnosis of CML, 6 months after the first clinical evidence of bone infiltration.     

Cell kinetics after high dose cytosine arabinoside in patients with acute myelocytic leukemia

In this study 10 patients with acute myelocytic leukemia (AML) each received a rapid intravenous injection of high dose cytosine arabinoside (HD Ara-C; 1 g/m2). Bone marrow aspirates were obtained before and after Ara-C administration to determine the percentage of cells in S-phase measured by flow cytometry. In 5 out of 10 cases synchronization of the leukemic cells in S-phase of the cell cycle was observed. However, the time of maximum synchronization turned out to be difficult to predict. Therefore, the strong correlation between percentage of cells in S-phase at diagnosis and the time of maximal accumulation of S-phase cells after Ara-C administration, as observed by others in childhood AML, could not be confirmed for adult AML patients. Although synchronization of AML cells after in vivo Ara-C administration could be demonstrated in at least half of the patients, the practical consequences are such that clinical application was hampered.     

Cell-surface MMP-9 regulates the invasive capacity of leukemia blast cells with monocytic features

The metalloprotease 9 (MMP-9), a known mediator of tumour invasion, is secreted as a 92kDa pro-form but a non-secreted variant of 85 Kda has been described. The importance of this variant pro-form in tumour progression remains poorly defined. We previously showed that the DNA repair protein Ku interacts at the cell surface of leukaemia cell lines with the 85 Kda pro-form of MMP-9 and these Ku/MMP-9 complexes regulates cell invasion, highlighting their importance in haematological malignancies. We demonstrate here that all samples of acute myeloid leukaemia (AML) blasts purified from bone marrow of 16 affected patients express the 85 Kda form of MMP-9. However, only AML that display monocytic lineage markers (AML4/5) express this form at the cell surface with co-expression of the membrane associated form of Ku. Blocking antibodies directed against Ku or MMP-9 specifically inhibited cell invasion of those expressing Ku/MMP-9 on the cell surface. The membrane form of Ku might represent an important factor in the exposition to the cell surface of this specific MMP-9 pro-form in AML with monocytic features. These results might have important functional significance in the occurrence of extra-medullar infiltrates of leukaemia cells that occurs frequently during the onset of monocyte-related AML sub-types.     

Test for cryopreservation efficiency of human acute myelogenous leukaemia cells relevant to clinical requirements

Cryopreserved AML cells from eight patients when thawed retained radioactive chromium better when they were labelled with the chromium after they had first been grown in short-term in vitro culture to establish proliferative cell populations. Such labelled cells are required for immunological studies of patients with acute leukaemia, so the ability to proliferate is a new test, more relevant to immunology than other available methods, for designing optimum cryopreservation conditions for these AML cells.Fifty-six populations of cryopreserved AML cells (from 53 patients) were cultured in vitro, and the growth pattern of the cell populations fell into three classes: (i) rapid death of some of the cells followed by proliferative growth and an increase in cell number (14 populations); (ii) cultures in which the cell number remained constant after an initial fall (6 populations), and (iii) rapid death of all the cells (36 populations). Although morphological studies showed maturation of some of the leukaemia blast cells into polymorphs during proliferative culture, leukaemia cells persisted in all cultures. In addition, in two cases it was shown that cells from the cultures had the same karyo-type abnormalities as those found in the donor's bone marrow and so it was concluded that the proliferating cells were of leukaemic origin rather than from normal cells.Using the proliferative capacity of AML cells from nine patients as a test of factors influencing cryopreservation efficiency, it was shown that the longer AML cells were kept in DMSO (5, 7.5, and 10%) for periods of up to 120 min prior to controlled slow freezing (+4 to −30 °C at l °C/min), the poorer was their capacity to grow.     

Blast crisis of Philadelphia chromosome-positive chronic myelocytic leukemia (CML). Treatment results of 69 patients

We have studied the clinical courses of 69 patients with blastic crises of Philadelphia chromosome positive CML to identify parameters that were associated with an increased response rate or survival. Cytogenetic analysis at the time of blastic transformation revealed additional chromosome changes in 70% of the patients tested. Bone marrow fibrosis was detected in 58% of evaluable patients. Lymphoblastic transformation was seen in 28% of the patients tested with cell surface marker analysis. The value of 5'-nucleotidase as a marker for distinguishing lymphoid from non-lymphoid blast crisis was confirmed. Of 57 evaluable patients, 23 (40%) responded to therapy (CR/PR longer than 14 days). Median survival was 75 days. Longer survival was related to the following factors: Ph1-chromosome as the only detectable cytogenetic abnormality; lymphoblastic transformation; no bone marrow fibrosis; high percentage of blasts and promyelocytes in the bone marrow, and response to therapy. No prognostic significance was associated with age, sex, Tdt, LDH, spleen size, duration of the chronic phase of the disease, white blood cell count, Hb, platelet count and percentages of basophils, eosinophils, erythroblasts and blasts and promyelocytes in the peripheral blood. These data confirm the poor prognosis of patients with blastic crisis of CML treated by conventional chemotherapy.     

Lytic activity against primary AML cells is stimulated in vitro by an autologous whole cell vaccine expressing IL-2 and CD80

Despite being of the myeloid lineage, acute myeloid leukaemia (AML) blasts are of low immunogenicity, probably because they lack the costimulatory molecule CD80 and secrete immunosuppressive factors. We have previously shown that in vitro stimulation of autologous peripheral blood mononuclear cells (PBMCs) with primary AML cells modified to express CD80 and IL-2 promotes proliferation, secretion of Th1 cytokines and expansion of activated CD8⁺ T cells. In this study, we show that allogeneic effector cells (from a healthy donor or AML patients) when stimulated with IL-2/CD80 modified AML blasts were able to induce the lysis of unmodified AML blasts. Effector cells stimulated with IL-2/CD80AML blasts had higher lytic activity than cells stimulated with AML cells expressing CD80 or IL-2 alone. Similarly, AML patient PBMCs primed with autologous IL-2/CD80 AML cells had a higher frequency of IFN-γ secreting cells and show cytotoxicity against autologous, unmodified blasts. Crucially, the response appears to be leukaemia specific, since stimulated patient PBMCs show higher frequencies of IFN-γ secreting effector cells in response to AML blasts than to remission bone marrow cells from the same patients. Although studied in a small number of heterogeneous patient samples, the data are encouraging and support the continuing development of vaccination for poor prognosis AML patients with autologous cells genetically modified to express IL-2/CD80.     

MUC1‐C drives myeloid leukaemogenesis and resistance to treatment by a survivin‐mediated mechanism

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with an unmet need for improved therapies. Responses to standard cytotoxic therapy in AML are often transient because of the emergence of chemotherapy‐resistant disease. The MUC1‐C oncoprotein governs critical pathways of tumorigenesis, including self‐renewal and survival, and is aberrantly expressed in AML blasts and leukaemia stem cells (LSCs). However, a role for MUC1‐C in linking leukaemogenesis and resistance to treatment has not been described. In this study, we demonstrate that MUC1‐C overexpression is associated with increased leukaemia initiating capacity in an NSG mouse model. In concert with those results, MUC1‐C silencing in multiple AML cell lines significantly reduced the establishment of AML in vivo. In addition, targeting MUC1‐C with silencing or pharmacologic inhibition with GO‐203 led to a decrease in active β‐catenin levels and, in‐turn, down‐regulation of survivin, a critical mediator of leukaemia cell survival. Targeting MUC1‐C was also associated with increased sensitivity of AML cells to Cytarabine (Ara‐C) treatment by a survivin‐dependent mechanism. Notably, low MUC1 and survivin gene expression were associated with better clinical outcomes in patients with AML. These findings emphasize the importance of MUC1‐C to myeloid leukaemogenesis and resistance to treatment by driving survivin expression. Our findings also highlight the potential translational relevance of combining GO‐203 with Ara‐C for the treatment of patients with AML.     

Effect of AML serum on normal human myeloid differentiation and hexamethylene bisacetamide-induced granulocytic differentiation of the human HL-60 promyelocytic leukaemic cell line.

The effect of serum from 32 AML patients on the normal human myeloid differentiation and the hexamethylene-bisacetamide induced granulocytic differentiation of HL-60 promyelocytic leukaemic cell line was studied. Nonadherent normal mononuclear marrow cells were cultured in vitro at a concentration of 5 x 10(5) cells/ml for 6 days with each of the 32 AML sera. Ten normal human AB sera were used as control. The results showed an inhibitory activity on both morphological and functional differentiation of normal human myeloid immature marrow cells by 29 out of the 32 AML sera tested. These 29 AML sera were added to cultures of HL-60 (2.5 x 10(5)/ml) leukaemia cell line which incorporated 2 mM hexamethylene-bisacetamide for 6 days. The results showed no significant inhibition of hexamethylene-bisacetamide induced granulocytic differentiation by any of the 29 AML sera. The efficacy of hexamethylene-bisacetamide in inducing differentiation in the presence of inhibitory factors suggests a possible role in the treatment of AML patients.