Correlation of clinical and anatomical findings in childhood meningitis]

Clinical and anatomical findings of 76 prophylactically not irradiated children with acute leukaemia were compared. In the group of 17 children (22%) with clinically manifest symptoms of CNS involvement in the course of the illness leukaemic infiltrates were identified in the CNS at the autopsy and during histopathological examinations even in 70% in spite of clinical healing and normalization of the cerebrospinal fluid finding. In children who had either no clinical symptoms of CNS leukaemia (42 children) or only terminal symptoms in the last 24 hours of their life (17 children) leukaemic infiltrations were found in 25.4% of the cases. Non-myeloid types showed 44.5% of histopathological changes, myeloid types 22.7%. Clinical main symptoms and anatomical findings were correlated to the corresponding groups of patients and typical illustrations were added.     

Remission-induction of acute lymphoblastic transformation of chronic myeloid leukaemia, followed by long-term maintenance therapy

Acute lymphoblastic transformation of chronic myeloid leukaemia (LT-CML) of 7 consecutive patients was treated according to a remission-induction and maintenance therapy used for de novo acute lymphoblastic leukaemia (ALL). All 7 patients achieved a second chronic phase with a median remission duration of 15 months. Two patients showed an isolated central nervous system (CNS) relapse. This did not occur in the 4 patients, who received prophylactic therapy with intrathecal methotrexate. The second transformation was nonlymphoblastic in nature in 4 out of the 5 patients. A treatment protocol similar to that of a bad risk ALL, including long-term maintenance therapy and CNS-prophylaxis is indicated in lymphoblastic transformation of CML.     

Haematological complications in polycythaemia vera and thrombocythaemia patients treated with radiophosphorus (32P).

We have evaluated 230 patients with myeloproliferative disorders treated in the last 15 years with 32P. None of the patients affected by essential thrombocythaemia developed haematological complications. In the larger group of polycythaemia patients (214 subjects) only 38 patients (17 males and 21 females) developed complications. 60.5% of these subjects had a minor complications: 1.8% showed a thrombocytopenia lower than 100.10e9/lt, 2.3% anaemia with Hb lower than 10 g%, 2.6% leukopenia lower than 40.10e9/lt and 2.3% a pancytopenia. All these complications were transient and eventually treated with limited blood transfusions. We could not identify a correlation between the dose used and the development of such complications. We noted only that the occurrence of anaemia, given a similar dose, was more frequent in females. Only 7% of all patients presented a major complication after 32P administration. In this case too, there was no correlation with the dose administered. Myelofibrosis and chronic myeloid leukaemia resulted to be the more frequent complication (9 out of 15) but we could not clarify if they represented a natural evolution of polycythaemia vera or were due to the treatment with 32P. Acute leukaemia developed only in 5 patients and again we could not recognized a correlation with the dose administered. Moreover, the time from the diagnosis of polycythaemia vera the onset of acute leukaemia ranged widely. 32P has a definite effect on the prevention of thrombotic and haemorrhagic complications in polycythaemia patients since it prolongs their life but it also increases the incidence of acute leukaemia.     

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.     

Immunoreactive calcitonin in leukaemia.

A radioimmunoassay was used to measure concentrations of immunoreactive human calcitonin (HCT) in plasma and leucocytes from patients with various leukaemic and myeloproliferative disorders. Plasma immunoreactive HCT concentrations were increased in 32 out of 33 patients with chronic granulocytic leukaemia (CGL) and in all eight patients with acute myeloid leukamia (AML) at presentation or in relapse. Out of 11 patients with other myeloproliferative disorders, eight had increased plasma immunoreactive HCT concentrations. Buffy-coat-cell extracts and culture media from peripheral leucocytes of patients with CGL also contained increased immunoreactive HCT concentrations. In contrast, plasma from patients with chronic lymphocytic leukaemia, acute lymphoblastic leukaemia, and AML in remission had low or undetectable immunoreactive HCT concentrations. Increased plasma and cellular concentrations of immunoreactive HCT may be a consequence of abnormal proliferation of myeloid cells and might prove to be valuable in predicting relapse in patients with myeloid leukaemias.     

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.     

Stem cell defects in Philadelphia chromosome negative chronic myeloproliferative disorders: a phenotypic and molecular puzzle?

Philadelphia chromosome-negative chronic myeloproliferative disorders (Ph(-) CMPD) comprise a group of heterogenous haematological stem cell disorders. These diseases harbour a pathological bone marrow stem cell which overwhelms normal stem cells due to sustained and uncontrolled proliferation. By clonal evolution, acute leukaemia or bone marrow fibrosis evolve in a proportion of cases with as yet unknown underlying mechanisms. Previously, groundbreaking investigations in Ph(-) CMPD detected an acquired mutation in the Janus kinase 2 (JAK2) in the majority of patients with polycythaemia vera (PV) and in up to 50% of patients with essential thrombocythaemia (ET) and chronic idiopathic myelofibrosis (CIMF). Unlike the stem cell defect in Philadelphia chromosome-positive chronic myeloid leukaemia only a subfraction of clonally proliferating haematopoiesis may be affected by the JAK2 mutation. More recently, another mutation in the juxtamembrane domain of the thrombopoietin receptor Mpl was discovered in about 5% of patients with CIMF and ET. In accordance with the uncontrolled Abl kinase activity in Ph(+) chronic myloid leukaemia these mutations in Ph(-) CMPD apparently represent a key to unlock some of the as yet unknown basic molecular defects and this raises hope for an upcoming efficient targeted therapy. However, neither the JAK2(V617F) nor the Mpl(W515L/K) provide the initiating molecular events. Moreover, apart from distinction between reactive and neoplastic lesions, detection of these mutations does not allow a clear-cut discrimination between the particular subtypes. This review will focus on previous and recent findings in the field of molecular defects in Ph(-) CMPD.     

Decreasedin vitro chemosensitivity of tumour cells in patients suffering from malignant diseases with a poor prognosis

The aim of the study was to assess the predictive value of MTTin vitro assay for evaluation of tumour cell resistance/sensitivity to cytotoxic drugs. We analyzed 105 samples of malignant cells of different origin. The study included patients with a diagnosis of acute and chronic lymphatic leukaemia, acute and chronic myeloid leukaemia, non-Hodgkin lymphoma, carcinoma of the lung, stomach and liver, rhabdomyosarcoma and breast carcinoma. The results demonstrate outstanding chemosensitivity in the majority of childhood acute lymphoblastic leukaemias, medium chemosensitivity of adult haematopoietic malignant diseases and chemoresistance of solid tumour cells. Our preliminary data suggest a good correlation betweenin vitro MTT assay and clinical curability of individual malignant diseases.Abbreviations ALL acute lymphoid leukaemia - AML acute myeloid leukaemia - CML chronic myeloid leukaemia - LCS₅₀ 50% leukaemic cell survival     

Bacterial glutaminase in treatment of acute leukaemia.

A glutaminase-asparaginase enzyme from Achromobacter sp has antitumour activity in vitro and in animals. Glutaminase was administered in doses of 3500-20 000 IU/m2 body surface area/day to six patients with acute lymphoblastic leukaemia (ALL) and three patients with acute myeloid leukaemia (AML). The enzyme had a blood half life of 80 minutes but depletion of blood glutamine persisted for 12 hours after single doses. Seven patients, including four (two with AML and two with ALL) resistant to asparaginase, received repeated doses of glutaminase. Antileukaemic effects were observed in all seven; one elderly patient developed metabolic acidosis. Study of this new antileukaemic agent in patients with acute leukaemia at an earlier stage of their disease is now justified.     

Invasive Geotrichum clavatum Fungal Infection in an Acute Myeloid Leukaemia Patient: A Case Report and Review

Invasive Geotrichum clavatum fungal infections are extremely rare and unusual, occurring nearly exclusively in patients experiencing prolonged neutropenia during the treatment for acute myeloid leukaemia. Several groups of cases of fatal G. clavatum infection were reported in France between 2011 and 2012, but the ecological niche has not yet been identified. We report a case of a 32-year-old patient with acute myeloid leukaemia who developed G. clavatum sepsis with primary peritonitis, hepatic nodular lesions, and multivisceral failure during aplasia after induction followed by salvage chemotherapy. He was treated with voriconazole and is still alive 1 year after with controlled disease. We then discuss the epidemiological, clinical, and therapeutic features of these serious fungal infections compared to the published data.     

Disturbances in iron utilization in acute leukemia and preleukemia]

With Prussian blue reaction nonhaemoglobin iron in the erythroblasts is demonstrable. Three pathological sideroblast types are recorded separately: abnormal intermediate type I and II sideroblasts and ring sideroblasts, representing increasing levels of sideroachrestic disturbance. This permits the classification of sideroachrestic disturbances into four degrees of seriousness. The frequency of a sideroachrestic disturbance in 47 untreated patients with acute myeloid leukaemia was 87%. Among 11 patients with preleukaemic condition, 8 had a disturbance of iron utilisation. In both preleukaemia and leukaemia mainly intermediate sideroblasts were present. All patients with preleukaemia developed leukaemia within 1-20 months. In the course of preleukaemic condition a slight increase of iron misutilisation was obvious when terminating in overt leukaemia. This could be of prognostic importance. After treatment, pathological sideroblasts disappeared only in 2 out of 15 patients with complete remission. There was no correlation between effect of therapy and course of iron misutilisation.     

Angiogenesis and the role of bone marrow endothelial cells in haematological malignancies

Increased microvessel density (MVD) has been observed in the bone marrow (BM) of patients with multiple myeloma (MM), acute lymphoblastic leukaemia, acute myeloid leukaemia, and myelodysplastic and myeloproliferative syndrome. The MVD is the net result of cumulative phases of angiogenesis and angio-regression and is as such not an indicator of the ongoing angiogenesis at the time of biopsy. There is, therefore, a need for additional methods that allow the estimation of ongoing angiogenesis. Double immunostainings for CD34 and Ki-67 can be used on paraffin-embedded tissue to determine the endothelial proliferation fraction. The BM endothelial cells, as a component of the BM stroma, have a close interaction with the malignant cells. In MM, for example, they are involved in the specific homing and are a source of paracrine growth factors. Targeting the BM microvessels will not only influence the nutrient and oxygen supply, but will in addition reduce the growth stimuli provided by the EC.     

The mll-AF9 gene fusion in mice controls myeloproliferation and specifies acute myeloid leukaemogenesis.

The MLL gene from human chromosome 11q23 is involved in >30 different chromosomal translocations resulting in a plethora of different MLL fusion proteins. Each of these tends to associate with a specific leukaemia type, for example, MLL-AF9 is found mainly in acute myeloid leukaemia. We have studied the role of the Mll-AF9 gene fusion made in mouse embryonic stem cells by an homologous recombination knock-in. Acute leukaemias developed in heterozygous mice carrying this fusion as well as in chimeric mice. As with human chromosomal translocation t(9;11), the majority of cases were acute myeloid leukaemias (AMLs) involving immature myeloblasts, but a minority were acute lymphoblastic leukaemia. The AMLs were preceded by effects on haematopoietic differentiation involving a myeloproliferation resulting in accumulation of Mac-1/Gr-1 double-positive mature myeloid cells in bone marrow as early as 6 days after birth. Therefore, non-malignant expansion of myeloid precursors is the first stage of Mll-AF9-mediated leukaemia followed by accumulation of malignant cells in bone marrow and other tissues. Thus, the late onset of overt tumours suggests that secondary tumorigenic mutations are necessary for malignancy associated with MLL-AF9 gene fusion and that myeloproliferation provides the pool of cells in which such events can occur.     

Gonadal function in men treated for acute leukaemia.

Gonadal function was assessed in eight men in remission of leukaemia who had completed treatment eight months to eight years previously. All four men treated for acute myeloid leukaemia had normal sperm counts and motility, compared with only one of the four with acute lymphatic or undifferentiated leukaemia. Hormonal studies indicated that the sterility resulted from gonadal failure rather than pituitary dysfunction after cranial irradiation. These findings are important in the counselling of patients with leukaemia.     

The immune receptor Tim-3 mediates activation of PI3 kinase/mTOR and HIF-1 pathways in human myeloid leukaemia cells

The T-cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated protein that is highly expressed in human acute myeloid leukaemia cells. As an acute myeloid leukaemia antigen, it could therefore be considered as a potential target for immune therapy and highly-specific drug delivery. However, a conceptual understanding of its biological role is required before consideration of this protein for therapeutic settings. Here, we reveal the detailed mechanism of action underlying the biological responses mediated by the Tim-3 receptor in myeloid cells. Our studies demonstrate that Tim-3 triggers growth factor type responses in acute myeloid leukaemia cells by activating a phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway. In addition, the receptor activates hypoxic signalling pathways upregulating glycolysis and pro-angiogenic responses. These findings suggest that Tim-3 could be used as a potential therapeutic target for immune therapy and drug delivery in human acute myeloid leukaemia cells.     

Prolonged remission maintenance in acute myeloid leukaemia.

Twenty-five patients with acute myeloid leukaemia were treated with three quadruple drug combinations in predetermined rotation: TRAP (thioguanine, daunorubicin, cytarabine, prednisolone); COAP (cyclophosphamide, vincristine, cytarabine, prednisolone); and POMP (prednisolone, vincristine, methotrexate, mercaptopurine). Fifteen patients (60%) achieved complete remission and five (20%) partial remission. For maintenance, five-day courses of drugs were administered every 14 to 21 days and doses were increased to tolerance. The median length of complete remission was 66 weeks. In eight patients remission maintenance treatment was discontinued and some remained in complete remission for over two years. In this series the remission induction rate was comparable with that reported for other regimens and complete remission lasted longer with this intensive maintenance regimen than with others. Nevertheless, the TRAP programme must still be regarded as only palliative treatment for acute myeloid leukaemia.     

Fibronectin and factor VIII-related antigen in acute leukaemia

The glycoprotein fibronectin is, as well as by various other cells, also produced in leucocytes and is said to play an important role in malignant transformation of cells. Therefore, the behaviour of plasma fibronectin and of factor VIII R:AG was investigated in acute leukaemia in order to prove their significance as prognostic and therapeutic markers (method: electroimmunoassay). In patients with acute myeloid leukaemia (n = 29) and acute lymphoblastic leukaemia (n = 11) no significant changes in fibronectin concentration could be evaluated. Fibronectin levels declined significantly only during therapy with asparaginase in patients with acute lymphoblastic leukaemia, probably as a result of disturbed synthesis in the liver. Using crossed immunoelectrophoresis against fibronectin antiserum, one normal and one slower migrating antigen (FN:C) could be observed in nearly all plasma samples in patients with acute leukaemia. By means of in vitro tests with highly purified substances and intermediate gel electrophoresis it could be shown that FN:C represents fibronectin which has bound fibrinogen, probably crosslinked by activated factor XIII. Factor VIII R:AG was found to be greatly raised in patients with acute leukaemia--up to 1400% of the normal level. Increased levels correlated well with a worsening of the disease. The protein seems to be suitable for estimating the activity and prognosis of acute leukaemia.     

Epigenetic inactivation of the hsa-miR-203 in haematological malignancies

miR-203 is a tumour suppressor microRNA (miRNA). We studied the methylation of hsa-miR-203 in 150 samples including acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL) and non-Hodgkin's lymphoma (NHL) by methylation-specific PCR, and miRNA expression by stem-loop RT-qPCR. hsa-miR-203 promoter was unmethylated in normal controls but homozygously methylated in two AML and four lymphoma cell lines, in which 5-Aza-2'-deoxycytidine treatment led to promoter demethylation and miR-203 re-expression. Restoration of miR-203 expression in lymphoma cells inhibited cellular proliferation and increased cell death, suggesting an inherent tumour suppressor activity. In primary samples, hsa-miR-203 methylation was absent in CML but detected in 5.0% ALL, 10.0% AML, 42.0% CLL and 38.8% of NHL (including six [60.0%] natural killer-cell, nine [40.9%] B-cell and four [23.5%] T cell NHL). Moreover, hsa-miR-203 methylation was associated with hypermethylation of hsa-miR-34a, -124a and -196b in NHL but not CLL. In CLL, hsa-miR-203 methylation was associated with a higher presenting Hb level (P = 0.033). The projected 10 year overall survival of the CLL patients was 58.2%, which was impacted by Rai stage and high-risk karyotypes but not hsa-miR-203 methylation. hsa-miR-203 was more frequently methylated in lymphoid than myeloid malignancies (P = 0.002). In conclusion, miR-203, a tumour suppressor gene, was hypermethylated in a tumour-specific manner with gene silencing. hsa-miR-203 was more frequently hypermethylated in lymphoid than myeloid malignancies. In NHL, hsa-miR-203 methylation was associated with concomitant methylation of other tumour suppressor miRNAs. The frequent hsa-miR-203 methylation in lymphoid malignancies suggested a pathogenetic role of hsa-miR-203 methylation.     

Scanning electron microscope cytochemistry of normal and leukaemic leukocytes

Backscattered Electron Imaging (BEI) is a particular technique which permits to study cytochemical reactions with the Scanning Electron Microscope (SEM). The BEI data pertaining to specific enzymatic activities can be directly correlated to the surface morphology of each individual cell. Leukocytes from 5 normal individuals, 14 patients with acute nonlymphoblastic leukaemia (ANLL), 7 patients with chronic myeloid leukaemia (CML) and 3 patients with acute lymphoblastic leukaemia (ALL) were studied for myeloperoxidase activity, acid phosphatase localization, silver staining of the nuclei and phagocytosis of iron carbonyl in the BEI mode of SEM. Some normal peripheral blood leukocytes which cannot be distinguished by their surface morphology alone were satisfactorily identified with the BEI technique. Leukaemic myeloid cells can be recognized in many cases because of their positive myeloperoxidase reaction, while monocytic elements can be characterized by the presence of surface ruffles, acid phosphatase activity and active phagocytosis. The usefulness of the BEI technique in identifying different blood cell types with the SEM and its possible application to the diagnosis of certain cases of leukaemia are discussed.