Symptomatic epilepsy associated with intracranial calcifications in children with acute lymphoblastic leukemia (ALL)

Acute and long-term sequels of central nervous system (CNS) prophylaxis with irradiation and intrathecal chemotherapy in children suffering from acute lymphoblastic leukemia (ALL) include vasculopathies, leucoencephalopathies, intracranial calcifications, intellectual and neurological impairment. We report two children at the age 5 and 8 years who manifested partial motor or complex seizures and intracranial calcifications 2-4 years after the diagnosis of ALL had been established. The occurrence of these disorders was much earlier than reported in the literature. Both children received prophylactic CNS treatment with irradiation and intrathecal methotrexate (MTX). Their brain CT scans and EEG had been normal before the first epileptic seizure was registered. Children are now seizure free on carbamazepine, and a boy with complex partial and myoclonic seizures is also on valproate and vigabatrine. Symptomatic epilepsy associated with intracranial calcifications and persisting EEG changes might occur as side effects of ALL treatment.     

Comparison of enzyme-cytochemical findings and immunological marker investigations in acute lymphatic leukemia (ALL).

APh-activity and PAS-positive deposits were studied in 50 cases of ALL, classified as T-ALL and O-ALL according to immunological marker investigations. Correlation between morphological features of the cells and APh and PAS reactions, as well as between morphology and immunological markers was not detected. APh-activity in general was stronger in T-ALL (R+ and R-), while PAS-content was more pronounced in O-ALL. The results suggest that cytochemical methods, especially APh and PAS reaction, are valuable to distinguish T-ALL from O-ALL but not reliable enough to replace immunological marker investigations.     

A new chromosome anomaly in acute lymphoblastic leukemia (ALL)

Summary A new chromosome anomaly in acute lymphoblastic leukemia (ALL) is reported. Three, possibly four, patients showed an identical karyotype anomaly, characterized by a (4;11)(q13;q22) reciprocal translocation. This anomaly has not so far been found in lymphoproliferative disorders other than ALL. Two of the patients had congenital leukemia, but the anomaly described appears to be more characteristic of ALL than of congenital leukemia, and may help the clinician in establishing the diagnosis of ALL.     

Prefinal clinical findings and results of autopsy in 82 children with acute lymphatic leuckemia (ALL) under various courses of therapy]

At present 80...90 % of the patients with acute lymphatic leukaemia die in the blastic crisis. About 10% will come ad finem during full remission caused by side effects of the treatment and their complication. The leukaemic terminal crisis may be accompanied or overlapped by a number of complications, the most frequent among own patients being acute bleeding in the terminal phase. First of all the source of bleeding is to be found in the gastro-intestinal tract (80%). Other authors found infections to be the most frequent final cause of death. It is only under autopsy that leukaemic infiltrates, infections and bleeding are completely recognized to their full extent. After polychemo-therapy the patients showed a significant increase of complications including pulmonary oedema and a marked insufficiency of the bone-marrow with leukocytopenia and granulocytopenia in the peripheral blood. Among the biochemical parameters only a generally significant increase of alpha2 and gamma globulins could be found in the serum. A correlation towards a form of therapy could not be ensured.     

Assessment of circulating biochemical markers and antioxidative status in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients

Various circulating biochemical markers are indicators of pathological state in leukemia and its subtypes. Increased oxidative stress and decreased antioxidant factors portray clear image associated with malignancies during subtypes of leukemia. In this research work we investigated the inter-relationship among the subtypes of leukemia with circulating biochemical markers and oxidative stress in the Pakistani population. This research work was conducted on a total number of 70 subjects in which 20 were control participants and 50 were suffering from leukemia and divided into two subtypes (ALL and AML). Various circulating biomarkers were investigated including hematological, hepatic and renal profiles as well as oxidative stress markers, electrolytes and vitamins C and E. Results show that vitamin E was found to be decreased in diseased sub-types (P < 0.05). Malondialdehyde (MDA) levels were very high in disease sub-types (ALL-B = 8.69 ± 1.59; ALL-T = 8.78 ± 0.97; AML = 8.50 ± 1.29) compared to controls (1.22 ± 0.10; P < 0.05) while the levels of antioxidants [superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GSH), catalase (CAT)], platelets, as well as electrolytes (Ca and Mg) were reduced in patients suffering from leukemia (sub-types). Enhanced levels of oxidative stress (MDA) and decreased levels of enzymatic and non-enzymatic antioxidants reflect the pathological state and impaired cell control in patients suffering from leukemia (subtypes) and show a strong correlation with oxidative stress, indicating that patients’ biological systems are under oxidative stress.     

DNA aneuploidia in cells of acute leukemia non B non T type in children

In 31 children with acute lymphoblastic leukemia of non B and non T type the cellular DNA amount using cytophotometric method was estimated. Parallely the computer microscopic image analysis was performed. These investigations were made using Morphoquant of C. Zeiss, Jena, GDR. The cells were studied before the introduction of therapy, after obtaining the first phase remission, during remission and at the relapse. DNA-diploidal type in 13 cases, aneuploidio-poliploidal in 8 cases and hipoploidal type in 13 cases. The survival time of investigated children was the largest in the case with diploidal and hyperploidal types of leukemic cells. The children with DNA-aneuploidopoliploidal type cells had the shortest survival time. Besides the estimation of the cellular DNA amount the defining of the percentage of the cells being in S phase seems to be very important. In differentiating between the normal and leukemic lymphocytes, containing the same amount of DNA as non neoplastic lymphocytes, it may be advisable to analyse the morphological picture, particularly of such parameters as chromatin compactness degree and the presence of nucleoli.     

C-type Natriuretic Peptide: a novel biomarker of steroid induced bone toxicity in children with acute lymphoblastic leukemia (ALL)

Impaired bone growth and mineralization, and osteonecrosis are significant and common long-term sequelae of chemotherapy for childhood acute lymphoblastic leukemia (ALL). Here we have evaluated the relationship between linear bone growth during chemotherapy for ALL and bone derived C-type Natriuretic Peptide (CNP). CNP is known to be critical to normal endochondral bone growth in both rodents and humans, and plasma concentration of the amino terminal pro CNP (NTproCNP) is strongly correlated with concurrent height velocity in children. Plasma NTproCNP and CNP were measured by radio-immunoassay in 12 children aged 2-9 years during induction and maintenance chemotherapy for children with ALL. Height velocity was calculated from stadiometer readings at intervals of 3-12 months and related to plasma NTproCNP during each growth interval. Plasma NTproCNP was markedly suppressed in all subjects during induction chemotherapy. Brief periods of NTproCNP decline and rapid rebound during maintenance treatment coincided with the use of dexamethasone but not with other chemotherapeutics. Height velocity was markedly reduced during ALL induction but unaffected in maintenance phase, and these changes in growth were strongly correlated with plasma NTproCNP concentration. Plasma NTproCNP has potential use as a biomarker of glucocorticoid-induced bone toxicity.     

Identification of prognostic protein biomarkers in childhood acute lymphoblastic leukemia (ALL)

Early response to 7 days of prednisolone (PRED) treatment is one of the important prognostic factors in predicting eventual outcome in childhood acute lymphoblastic leukemia (ALL). Using proteomic tools and clinically important leukemia cell lines (REH, 697, Sup-B15, RS4; 11), we have identified potential prognostic protein biomarkers as well as discovered promising regulators of PRED-induced apoptosis. After treatment with PRED, the four cell lines can be separated into resistant (REH) and sensitive (697, Sup-B15, RS4;11). Two dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS identified 77 and 17 significantly differentially expressed protein spots (p<0.05) in PRED-sensitive and PRED-resistant cell lines respectively. Several of these were validated by Western blot including proliferating cell nuclear antigen (PCNA), cofilin 1, voltage-dependent anion-channel protein 1 (VDAC1) and proteasome activator subunit 2 (PA28β). PCNA is a promising protein because of its important roles both in cell cycle regulation and survival control. We subsequently validated PCNA in 43 paired bone marrow samples from children with newly diagnosed ALL (Day 0) and 7 days after PRED treatment (Day 8). ROC curve analysis confirmed that PCNA was highly predictive of PRED response in patients (AUC=0.81, p=0.007) and most interestingly, independent of the molecular subtype, providing a promising universal prognostic marker.     

Decreased hormone content of immune cells in children during acute lymphocytic leukemia (ALL) - effect of treatment

Histamine, serotonin and triiodothyronine (T3) content of different circulating lymphocyte subsets of leukemic (acute lymphocytic leukemia, ALL) and non-leukemic (control) children were investigated by multicolor flow cytometry. The hormone contents of the cells were followed from the time of diagnosis till the end of treatment. Each hormone could be detected in every time in the investigated cell types, although the amounts of them changed during the treatment.T lymphocytes: Significantly lower amount of serotonin was found in each T cell subsets (Th, Tc and activated T lymphocytes) of leukemic children compared to the healthy control group at the time of diagnosis and it was permanently low during the maintenance therapy. The decreased amount of serotonin could be demonstrated in Tc and Th cells even at one year after the end of treatment. However, there was no alteration in the histamine and T3 content of T cell subsets in the time of diagnosis, but significant decrease was detected during the maintenance therapy and after treatment.NK cells: The serotonin and T3 contents of NK cells (both NK and NKT subsets) were significantly lower at the time of diagnosis and during the maintenance therapy. Similar decrease was detected in the case of serotonin in B cells. Although there was no difference in the T3 content of B cells at the time of diagnosis, significantly lower amounts could be detected during the therapy compared to the healthy control group. The serotonin concentration remained low for years after the end of treatment, both in B and NK cells. These observations might have diagnostic and prognostic importance.     

CXCL12 and TP53 genetic polymorphisms as markers of susceptibility in a Brazilian children population with acute lymphoblastic leukemia (ALL)

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Genetic polymorphisms in the 3′UTR region of the CXCL12 (rs1801157) and TP53 codon 72 (rs1042522) genes may contribute to susceptibility to childhood ALL because they affect some important processes, such as metastasis regulation and tumor suppression. Thus the objective of the present study was to detect the frequency of two genetic polymorphisms in ALL patients and controls and to add information their impact on genetic susceptibility and prognosis. The CXCL12 and TP53 polymorphisms were tested in 54 ALL child patients and in 58 controls by restriction fragment length polymerase chain reaction and allelic specific chain reaction techniques, respectively. The frequencies of both allelic variants were higher in ALL patients than in the controls and indicated a positive association: OR = 2.44; 95 % CI 1.05–5.64 for CXCL12 and OR = 2.20; 95 % CI 1.03–4.70 for TP53. Furthermore, when the two genetic variants were analyzed together, they increased significantly more than fivefold the risk of this neoplasia development (OR = 5.24; 95 % CI 1.39–19.75), indicating their potential as susceptibility markers for ALL disease and the relevance of the allelic variant combination to increased risk of developing malignant tumors. Future studies may indicate a larger panel of genes involved in susceptibility of childhood ALL and other hematological neoplasias.     

Proteomic profile of pre - B2 lymphoblasts from children with acute lymphoblastic leukemia (ALL) in relation with the translocation (12; 21)

Background

Until now, the major prognostic factors for pediatric acute lymphoblastic leukemia (ALL), age, white blood cell count and chromosomal alterations are initially taken into account for the risk stratification of patients. In the light of protein marker studies to classify subtypes of Acute Myeloblastic Leukemia efficiently, we have compared the lymphoblastes proteome in Childhood ALL in accordance with the presence of t(12;21), indicator of good prognosis, usually.

Methods

Protein expression in pre-B2 lymphoblastic cells, collected from residual bone marrow cells after diagnostic procedures, was analyzed using two dimensional gel electrophoresis protocol. Protein spots whose average normalized volumes were statistically different in the two patients groups (n = 13; student t test p < 0.01), were excised. Tryptic peptides were then analyzed using a nano-LC1200 system coupled to a 6340 Ion Trap mass spectrometer equipped with a HPLC-chip cube interface. The tandem mass spectrometry peak lists extracted using the DataAnalysis program, were compared with the protein database Mascot Daemon.

Results

We focused on twelve spots corresponding to sixteen identified candidate proteins among the 26 found differentially expressed (p ≤ 0.05) regarding the presence of t(12;21). Among over expressed proteins, two proteins were implicated in cellular growth arrest (i.e. calponine 2, p ≤ 0.001 and phosphatidylinositol transfer protein beta, p ≤ 0.001) in accordance with good prognosis, while two other proteins favored cell cycle proliferation (i.e. methionine adenosyl transferase 2β, p ≤ 0.005 and heterogeneous nuclear ribonucleo-proteins A2 p ≤ 0.01) and could therefore be good marker candidates of aggressiveness. Level of expression of proteasome subunit beta type-2 (p ≤ 0.01) and protein casein kinase 2α (p ≤ 0.01) which both favored apoptosis, deubiquitinating enzyme OTUB1 (p ≤ 0.05) and MLL septin-like fusion protein MSF-B, septin 9 i4 (p ≤ 0.01) were in accord with a good prognosis related to t(12;21) lymphoblasts.

Conclusion

By drawing up the protein map of leukemic cells, these new data identified marker candidates of leukemic aggressiveness and new t(12;21) patients subgroups. These preliminary results will be in the near future confirmed by using a larger sample of pre-B2 childhood ALLs from national lymphoblastic cell collections.     

Mitogen activation of human lymphocytes from normal and acute lymphocytic leukemia (ALL) subjects

Lymphocytes from acute lymphocytic leukemia (ALL) subjects were converted by mitogens to blast-like cells whose microscopic appearance and rate of formation was indistinguishable from those in mitogen incubated control lymphocytes. In ALL lymphocytes, however, pokeweed mitogen (PWM) failed to stimulate GGT expression; the mean increase it caused in thymidine kinase (TK) activity and thymidine incorporation was normal, though there were appreciable individual variations. These variations were also apparent with concanavalin A (Con A) but, in most ALL cases, TK and thymidine incorporation rose to much higher levels than in Con-A-treated control lymphocytes. The results indicate that evaluation of the response to mitogens by quantitative biochemical criteria provides a sensitive method for revealing functional impairments in microscopically normal ALL lymphocytes.     

Thrombocytopenia after autologous bone marrow transplantation in in acute lymphatic leukemia]

There is reported about the treatment of refractory thrombocytopenia in a 9 years old boy following the autologous bone marrow transplantation for acute lymphoblastic leukaemia. The megakaryocytes were found diminished in the bone marrow smears. Controls of the thrombocyte count and the kinetics with radioactively labeled platelets of a donor spoke in favour of immunothrombocytopenia. Threatening bleeding complications challenged the use of all treatment possibilities. The irradiation of the spleen was without any success. After the splenectomy the thrombocyte count increased slowly, but after a remarkable lag phase, however. A diminished reproduction capacity of the bone marrow graft for special cell sorts has to be taken into account in such cases. The usual cytodynamics after splenectomy cannot be expected at all.     

Comparison of the cytomorphological classification with the cytochemical differentiation of acute lymphatic leukemia in children]

In 100 children with acute lymphatic leukaemia the cytomorphological subclassification of the pathological cell type was made according to Mathé and the French-American-British Co-operative group (FAB). In addition, all cases of leukaemia were differentiated according to their cytochemical type. Lymphoblasts from 10 cases of leukaemia could be subclassified immunologically. From 71 children will ALL the survival rates of those cases of leukaemia subclassified cytomorphologically and the cytochemical reactions were compiled and partially compared. Microlymphoblastic leukaemia could be found to be the most frequent type of ALL at children's age. Prolymphocytic leukaemias were characterized by a favourable survival rate and the highest percentage of ALL with the PAS type. Macrolymphoblastic and microlymphoblastic cases of leukaemia revealed no essential differences of survival rate, but significant differences of cytochemical reactions.     

Mitotic index in the bone marrow of children during the clinical course of acute lymphoblastic leukemia (ALL)]

From 31 children with acute lymphoblastic leukaemia the mitosis index in the bone-marrow was determined before the onset of therapy and during the clinical progress. Initially, the mean white mitosis index lay with 3.4% below that of the normal test persons, it rose significantly in the hematologic full remission and showed a decreasing tendency with a great range of dispersion in the recidive. The most lowered mitosis index was found in the final stage. Strong shifts in the kariologic distributions make a remaining in the prophase of the mitosis evident. The influence of polychemotherapy on the mitosis index and the phases of mitosis is discussed. Correlations between the mitosis index and clinical as well as paraclinical parameters were only to be found with respect to granulocytes and lymphoblasts. The considerable ranges of fluctuations of the mitosis index and the lack of congruity with the clinical progress of ALL allow no ensured assertions to be made for the single patient. It seems to be important in eosinophilia and in leukaemoid reactions.     

Impulse cytophotometric studies of bone marrow and blood cells in children with acute lymphoblastic leukemia (ALL). 1. Bone marrow cells

1. Compared with the peripheral blood lymphocytes of healthy children the cell fractions in the S- and G2 + M-phase are significantly higher in the bone-marrow of those children affected with ALL. This increase was proved in the SR- and MR-group irrespective of the cytomorphological subtype and cytochemical reaction. In patients with relapses the percentage of S-phase cells is below 6%. 2. In about 30% of our patients (mainly in the SR-group with L1-morphology) an initial DNA-aneuploidy was identified. As the risk of relapse is higher in children without DNA-aneuploidy, this symptom has a pretherapeutical-prognostic significance. 3. In the phase of hematological full remission, DNA-frequency distribution correlates with the proliferative activity of normal hematopoiesis. It provides no additional information about the pretherapeutical risk.     

Impulse cytophotometry studies of bone marrow and blood cells in children with acute lymphoblastic leukemia (ALL). 2. Lymphatic cells in blood

The DNA-content of mononuclear cells of the peripheral blood of infantile and juvenile ALL patients was investigated using Pulse Cytophotometry. The fraction of cells in S- and G2 + M-phase is significantly increased in comparison with samples of healthy probands. The fraction of DNA-synthesising cells (S-phase) of both peripheral blood (mononuclear cells) and bone marrow of leukemia patients cannot be significantly distinguished by mathematical methods. On the other hand, the fraction of cells in later phases of cell cycle (G2 + M-phase) is significant enhanced in the bone marrow in comparison with the peripheral blood. A high correlation was found between the number of leukocytes and fraction of G2 + M-phase cells in the peripheral blood of SR- and MR-patients. No correlation was found between the number of leukocytes and S-phase-fraction. The occurrence of aneuploid cell populations in the mononuclear fraction of peripheral blood in the acute state of ALL could be of importance for prognosis and regime of therapy.     

Acute lymphoblastic leukemia (ALL) antigens detected with antisera to E rosette-froming and non-E rosette-forming ALL blasts.

Based on the presence or absence of erythrocyte receptors(E) a T cell marker, acute lymphocytic leukemia (ALL), can be divided into E+ALL and E-ALL. We studied cell surface antigens on blasts from 12 children with untreated ALL: eight with E-ALL and four with E+ALL. Heterologous antisera were raised against thymus cells, E+ and E-ALL blasts, appropriately absorbed and tested by immunofluorescence and a radiolabeled antibody assay with normal and leukemic lymphoid cells. By both methods, anti-thymus and anti-E+ALL sera reacted with human thymocytes. Specific binding of anti-E+ALL serum to T antigens was indicated by the fact that a single absorption with thymocytes abolished its binding to allogenic thymocytes, and the reactivity of anti-E+ALL serum with thymus, blood and bone marrow lymphocytes was similar to that of anti-thymus serum. After exhaustive absorption with blood leukocytes, anti-E+ALL and E-ALL sera were negative against normal lymphocytes and bone marrow cells from children with ALL in remission. Anti-thymus and anti-E+ALL sera reacted with blasts from patients with E+ALL, but not with E-ALL. In contrast, anti-E+ALL serum reacted with 40 to 96% of blasts from all children with E-ALL, whereas of the four patients with E+ALL, two were negative and two had the lowest percentage of immunofluorescent cells (10 to 22%). These results were confirmed with the radiolabeled antibody assay. Patients with active E-ALL had cells bearing E-ALL antigen(s) in the peripheral blood and bone marrow, but the number of immunofluorescent cells was lower in blood. Cells reactive with anti-E-ALL serum did not react with thymus cells, blood lymphocytes, remission bone marrow cells, Raji cells, PWM and PHA-induced blasts and CLL cells bearing mIg (uk). These data suggest that the antigen detected on E-ALL blasts by anti-E-ALL serum is neither a HLA-related nor a cell differentiation antigen. Thus, by using antiserum to E+ALL blasts, we have confirmed the presence of a T cell-specific antigen(s) on E+ALL cells. This antiserum did not recognize other leukemia-associated antigens common to E+ and E-ALL. We have also demonstrated an antigen(s) which is regularly expressed on E-ALL blasts and is either not detectable or is present in a lower proportion of E+ALL blasts.