Quantitative monitoring of WT1 expression in peripheral blood before and after allogeneic stem cell transplantation for acute myeloid leukemia - a useful tool for early detection of minimal residual disease.

Overexpressed Wilms tumor gene 1 (WT1) has been found in a majority of patients with acute myeloid leukemia (AML). The aim of this study was to confirm the applicability of WT1 expression measurement as a marker of minimal residual disease (MRD). The expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood (PB) according to European Leukemia Net (ELN) recommendations. The WT1 expression was related to the expression of a reference gene Abelson (ABL) and the results were calculated as a number of WT1 copies related to 104 copies of ABL gene. The upper normal limit of WT1 expression was set at 50 copies of WT1 to 104 copies of ABL. Morphological, flow cytometry and chimerism examinations were evaluated according to standard protocols.A total of 51 AML patients with overexpressed WT1 gene were analyzed. The median follow-up after transplantation was 14 (2-72) months. WT1 expression levels exceeding the upper normal limit were considered as a sign of impending hematological relapse, in accord with morphological, flow cytometry and chimerism data, as well as with the expression of the specific fusion genes. Moreover, in 7 patients the rise of WT1 expression preceded all other standard methods. Patients with high WT1 expression before allogeneic hematopoietic stem cell transplantation (allo-HSCT) had significantly worse outcome than patients with low WT1 level. Examination of WT1 expression in PB of patients with AML is a useful tool for MRD monitoring. Moreover, the WT1 gene expression before stem cell transplantation seems to be of prognostic significance.

WT1 expression in peripheral leukocytes of patients with chronic myeloid leukemia serves for the prediction of Imatinib resistance.

The determination of patient's resistance to a particular drug contributes to more efficient therapeutical approach. The aim of this study was to evaluate if the responsiveness of Chronic Myeloid Leukemia (CML) patients to Imatinib therapy was predictable from WT1 gene expression in peripheral blood leukocytes. To examine the resistance we implemented an in vitro cultivation of the primary cells of 48 CML patients with Imatinib. The effect of Imatinib was characterized not only by the expression of WT1 but also by BCR-ABL, and proliferative factor Ki-67.
Our results showed that leukocytes of CML patients, clinically responsive to Imatinib treatment, significantly decreased WT1 expression after in vitro incubation with Imatinib. It was accompanied by an inhibition of expression of Ki-67 but not BCR-ABL. In leukocytes of CML patients clinically resistant to Imatinib, the expression of WT1, Ki-67, and BCR-ABL remained unaffected. The presented results showed that in vitro testing using peripheral blood cells enabled clinicians to predict responsiveness of CML patients to Imatinib.

Immunological profiles of patients with chronic myeloid leukaemia. I. State before the start of treatment.

In view of the increasing interest in the immunotherapy of CML it seems highly desirable to broaden the present knowledge on the immune reactivity of CML patients. A group of 24 patients and 24 healthy controls were studied for the total of 15 immunological parameters, including the prevalence of antibodies against human herpesviruses and papillomaviruses. To clearly discriminate between changes associated with the disease and those induced by the therapy, all patients were enrolled prior to the start of any anti-leukaemic therapy. Statistically significant differences between patients and controls were found in the levels of IgA, C4 component of complement, CRP and IL-6, the production of Th1 cytokines in stimulated CD3 cells and the E. coli stimulatory index. The analysis of the interrelationship between the results obtained in the individual patients presented some unexpected findings, such as the lack of correlation between the CRP and IL-6 levels. It will be the purpose of a follow-up to determine whether and how the immune status of the patients prior to the treatment correlates with their response to therapy and how the individual immunological profiles change in the course of the disease. These observations will be utilized in the future immunotherapeutic studies to constitute the vaccine- and placebo-treated groups.

[The use of quantitative assessment of Wilms tumour gene 1 for monitoring of residual disease in acute myeloid leukemia patients]. / Uzití kvantitativního stanovení exprese genu wilmsova tumoru 1 pro monitorování reziduální nemoci pacientu s akutní myeloidní leukémií.

BACKGROUND: Despite a considerable effort, the majority of acute myeloid leukaemia (AML) patients do not have a suitable specific molecular marker for monitoring minimal residual disease (MRD). The results of some studies suggest the Wilms tumour gene (WT1) as a possible molecular marker of MRD. METHODS AND RESULTS: We measured the expression of WT1 at diagnosis and during treatment of the acute myeloid leukaemia (AML) patients. The expression of WT1 was measured by the quantitative real-time RT-PCR in peripheral leukocytes from 56 AML at diagnosis and 7 patients with AML transformed from myelodysplastic syndromes (MDS). The WT1 expression was significantly elevated (up to 3 orders of magnitude) in peripheral blood samples (PB) of AML patients at diagnosis compared to PB samples of healthy donors (P < 0.0001). The level of WT1 expression depends particularly on FAB AML subtype, with the highest being found in AML patients with subtypes M4, M1, M3 and AML transformed from MDS. Conversely, AML patients with M2 and with the presence of AML1/ET0 at presentation showed a significantly lower expression of the WT1 gene compared to the remaining AML patients at presentation (P = 0,005). Further, sequence samples of 12 AML patients under long-term surveillance were tested for the WT1 expression in parallel with the expression of specific MRD markers--fusion genes: AMLI/ETO, PML/RARalpha and CBFB/MYH11. The levels of WT1 gene expression and the above specific fusion genes significantly correlated. Moreover, 14 patients without the specific MRD marker were tested for the WT1 expression. The results show that haematological relapses were associated with the rise of expression of the specific fusion genes and with the WT1 gene expression. The rise of WT1 expression above the level seen in leucocytes from peripheral blood and/or bone marrow of healthy donors--in four patients under long-term surveillance the "molecular relapse" predicted ongoing haematological relapses as early as 2 months in advance. CONCLUSIONS: Our results, in accordance with some of the previously published ones, show that WT1 expression seems to be a suitable marker of minimal residual disease in AML patients.

[Frequency view on genome changes testing]. / Frekvencní pohled na vysetrení odchylek genomu.

Laboratories dealing with human genome, both inherited and acquired changes, dispose with similar methods and technology. The spectrum of genetic tests is relatively broad and the number of mutations or variants tested differs substantially. Also the number of examinations carried out in individual laboratories varies. Data presented in the tables come from the year 2004 and indicate the number of examinations requested and number of positive results. Many laboratories mentioned in the registry CZDDNAL (http://www.uhkt.cz/lab_a_vysetreni/nr lab_dna_diag/dna_lab_db) perform the same tests but there is also a great number of tests carried out by only one laboratory. Reasons of the request, cost-effectiveness and clinical utility of genetic testing is being discussed.

Studies on lectins. XXXVIII. Isolation and characterization of the lectin from black locust bark (Robinia pseudacacia L.).

The lectin of black locust (Robinia pseudacacia) bark was isolated by specific adsorption on formaldehyde-fixed human erythrocytes and elution with a borate solution. The lectin is homogeneous on disc electrophoresis and ultracentrifugation (s20,w = 5.8 S) but yields three bands on isoelectric focusing. It has a molecular weight of approximately 110,000 and consists of two types of subunit (mol. wt 29,000 and 31,500). Its pI is approximately 5.9; it contains high amounts of aspartic acid, threonine and serine, no cysteine and very little methionine. Also 7.2% of covalently bound neutral sugar and 0.47% of glucosamine are present. The lectin is nonspecific in agglutination of human erythrocytes, it is inhibited by high concentrations of N-acetyl-D-galactosamine and is mitogenic in rabbit lymph node lymphocytes.

Simple competitive two-step RT-PCR assay to monitor minimal residual disease in CML patients after bone marrow transplantation.

Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assessing the amount of transcripts of the BCR/ABL gene, the molecular marker of chronic myeloid leukemia (CML), is the only method sensitive enough for monitoring of minimal residual disease (MRD) in CML patients after bone marrow transplantation (BMT). In this study we present a simple modification of competitive Q-RT-PCR using natural competitors from cell lines K562 and BV173. The competitors were used in the form of unpurified RNA in cell lysates which ensured their high stability. Mixing competitors and samples before RNA extraction eliminated problems with quantification of cDNA or RNA entering the competitive reaction and with checking for the RNA quality and reverse transcription (RT) efficiency. The bulk of the malignant clone was expressed as the number of leukemic cells in 10(6) leukocytes when the overproduction of the BCR/ABL mRNA in the cell lines we used as competitors was taken into account. It was found to be 82-fold and 14-fold in K562 and BV173, respectively, in comparison with 100% Ph-positive CML standard. The assay reliability was verified by comparison of results with the mathematical model of competitive PCR. The assay is highly reproducible and sensitive (10(-5)). Its accuracy was proved to be excellent in a wide range of malignant cell concentrations. The method is demonstrated on three CML patients suffering from MRD after BMT. In conclusion, this method fulfills all criteria of competitive Q-RT-PCR. Because of its simplicity it is suitable for clinical laboratories and due to the high stability of the lysates used it may serve in the standardization of results between different laboratories.

Aleukemic granulocytic sarcoma with AML1/ETO fusion gene expression and clonal T cell populations.

We report a unique case of aleukemic granulocytic sarcoma of the neck, originally misdiagnosed as non-Hodgkin's lymphoma (NHL), though chloroma was also suspected due to a greenish macroscopic appearance and the presence of myeloid chloroacetate esterase (CAE)+ cells. The proof of clonal T cell receptor gamma chain (TcRgamma) gene rearrangements in the recurring tumor was deemed to confirm the initial diagnosis of T cell NHL. Altogether five distinct types of clonal TcRgamma gene rearrangements were found in the tumor, bone marrow and peripheral blood. Only retrospectively, using RT-PCR, did we detect the acute myeloid leukemia subset-specific fusion gene AML1/ETO in the frozen samples of the relapsed tumor, as well as in the otherwise unaffected bone marrow and peripheral blood (representing 'minimal initial disease' in the latter two samples). Simultaneous staining verified that the neoplastic CAE+ cells and CD45RO+ T cells were different populations.

Location of the BCR/ABL fusion genes on both chromosomes 9q34 in Ph negative chronic myeloid leukemia.

We present two patients with Ph-negative chronic myeloid leukemia (CML) and fusion signal BCR/ABL on both chromosomes 9, located in region 9q34. The first case was a 27 years old man with CML. Molecular studies (RT-PCR) revealed the rearrangement in the major-BCR region and expression of chimeric BCR/ABL mRNA of b3a2 configuration. By classical cytogenetic studies (G-banding) karyotype 46,XY was found in short-term cultivated bone marrow cells and phytohemagglutinin (PHA) stimulated peripheral lymphocytes. FISH studies revealed the BCR/ABL fusion signals on both chromosomes 9 and green BCR signals on both chromosomes 22 in all mitoses studied. Detection of the alleles of ABL1 intragenic STR locus by fluorescence PCR followed by fragmentation analysis in the patient and his parents provided no information about transmission of the ABL gene. Quantitative assessment of BCR/ABL transcript level by RT-PCR showed 60 and 70% BCR/ABL positivity in two peripheral blood samples at 6,5 and 10,5 months after diagnosis, respectively, which does not correspond to the expression from two identical BCR/ABL hybrid genes. Therefore, the possible mechanism of the origin of two BCR/ABL fusion signals present on both chromosomes 9 could not be resolved and remains speculative. The second case was a 53 years old male with diagnosis of chronic phase of CML, with first sign of acceleration one month after diagnosis and death because of sepsis in blastic phase within four months. The cytogenetic findings were identical to those in case No. 1., i.e. karyotype 46, XY by G-banding, two BCR/ABL fusion signals on both chromosomes 9 and RT-PCR molecular studies proved b3a2 breakpoints. It is generally accepted that prognosis of the patients with fused BCR/ABL gene located on chromosome 9 is poor. The presence of two fused genes could be anticipated as two Ph chromosomes in accelerated and blastic phases of the disease. However, in our study, quantitative findings of BCR/ABL transcripts did not corresponded to the expression of two BCR/ABL genes originating from duplication. If this assumption is correct then the expression of both fused genes BCR/ABL was in case No. 1 equally suppressed and total expression reached about the level of one BCR/ABL gene.

Acrylonitrile depletes glutathione without changing calcium sequestration in hepatic microsomes and mitochondria.

Acrylonitrile administered either in vivo or in vitro reduced the level of non-protein thiols, GSH and GSSG in rat liver (in vivo) and liver microsomes (in vitro). It neither influenced protein thiols nor calcium sequestration in the microsomes and mitochondria. The fact that the GSSG level was not increased indicates that a mere unoxidative depletion of GSH does not lead to impaired hepatocyte Ca homeostasis, which has been associated with decreased GSH:GSSG ratio. An opposite effect was caused by CCl4 which did not considerably change the protein and non-protein SH, but strongly decreased microsomal calcium sequestration.

Gene expression during camptothecin-induced apoptosis in human myeloid leukemia cell line ML-2.

Malignant cell proliferation and accumulation depends on the balance between the rates of cell production and cell death. Recent evidence indicates that apoptosis is important in the development of cancer. Apoptosis is strictly controlled by various regulators, which can take part in the apoptotic process, proliferation and differentiation alike. Apoptosis was induced in myeloid cell line ML-2 by camptothecin, an inhibitor of topoisomerase I. After 18 hours of induction by camptothecin 50% of cells were apoptotic. The apoptotic effect of CAM was reversible in the cells studied. The induction of apoptosis influenced the expression of apoptosis and cell cycle regulators as detected by cDNA arrays, RT-PCR or Western blotting. According to cDNA arrays e.g. bax, bfl1, bak, pRb2, c-jun, jun-B were upregulated, and cdk4, cyclin B1, wee1, CRAF1, DP1 were downregulated. A number of other regulators like p21 and cdc25A, as well as some other genes linked with apoptosis, as p53 and the bcl-2 family, were up- or down-regulated as determined by real-time PCR. Changes in gene expression were found not only in the group of regulators of apoptosis and the cell cycle, but also among regulators of differentiation.

Differentiation of human myeloid leukemia cell line ML-1 induced by retinoic acid and 1,25-dihydroxyvitamin D3.

Human myeloblastic leukemia cell line ML-1 was induced to differentiate by 1 mumol/l all-trans-retinoic acid (RA) or by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). After 4-6 days of the induction several differentiation-associated characteristics were observed: (1) Ability to stimulate respiration burst in the ML-1 induced cells as detected by nitroblue tetrazolium (NBT) test or by chemiluminescence (CL). (The percentage of the NBT-positive cells was up to 99% in the RA-induced cells and up to 85% in the 1,25(OH)2D3-induced cells.) (2) Substantially higher phagocytosis of colloid iron, latex or Staphylococcus particles was found in the induced cells. (3) The 1,25(OH)2D3-induced ML-1 cells expressed the monocytic enzyme NaF-inhibitable alpha-naphthyl butyrate esterase and the surface monocytic antigen CD-14. (4) A majority of the induced cells lost the morphological features of blast cells; while the 1,25(OH)2D3-treated cells acquired certain features of monocyte-macrophage differentiation, the RA-treated cells displayed several granulocytic characteristics. (5) Cytofluorometric DNA assay after treatment of the cells with colcemid showed that the decline observed in the growth rate of the induced cells was connected with their arrest in G1/G0 phase of the cell cycle. The obtained results indicate granulocytic differentiation of the RA-induced ML-1 cells and monocyto/macrophage differentiation of the 1,25-(OH)2-D3 induced cells.

Interaction of lectins with surface membrane receptors of animal cells. I. Factors responsible for agglutinability of human, rabbit, and sheep erythrocytes with concanavalin A.

A study of various factors influencing the Con A-mediated cell agglutinability failed to reveal any correlation between the agglutinability and the binding capacity of Con A-agglutinable and non-agglutinable human, rabbit, and sheep erythrocytes. Treatment with trypsin and neuraminidase on the one hand and chondroitin sulphate, polyvinylpyrrolidone and polylysine on the other hand made the agglutination of human erythrocytes possible, promoted the agglutination of rabbit erythrocytes, but had almost no effect on sheep erythrocytes, which agglutinated only after treatment with pronase or polylysine. The negligible effects of low temperature, NaN3, cytochalasine B and theophylline on cell agglutinability indicate that neither membrane fluidity, metabolic energy, cAMP, nor the microfilamentous apparatus are likely to play any important role in the Con A-mediated agglutination of erythrocytes. Differences in agglutinability between erythrocytes obtained from different animal species and subjected to different treatments with enzymes and polymers are explained as being due to alterations in the surface charge and the zeta potential.

The use of housekeeping genes (HKG) as an internal control for the detection of gene expression by quantitative real-time RT-PCR.

Quantitative real-time RT-PCR is a very useful technique for estimating gene expression at the mRNA level. The expression of a tested gene has to be compared with that of a control gene. Various housekeeping genes have been used as control genes in different systems. In our study we tested several housekeeping genes in the model of gene expression after induction of apoptosis and differentiation. The myeloid cell lines were incubated with phorbol esters, butyric acid and combination of TNFalpha and IFNgamma to induce differentiation. Camptothecin was used for induction of apoptosis. Tested control genes included beta2-microglobulin, GAPDH, 18S ribosomal RNA and abl. GAPDH was found to be the best control gene in the apoptotic system. Different control genes were suitable for different systems where differentiation or senescence was induced. Our results show that attention should be paid to the choice of an appropriate control gene of quantitative real-time RT-PCR for different experimental models and various experimental conditions.

In vitro responsiveness of lymphocytes of different mouse strains to phytohaemagglutinin.

Responsiveness of spleen and lymph node lymphocytes to in vitro stimulation by phytohaemagglutinin was investigated in several mouse strains. Considerable interstain differences in RNA and DNA synthesis were found both for PHA-stimulated and unstimulated cells; the latter component thus tends to contribute to the observed over-all differences in lymphocyte responsiveness to "specific" stimulation. Of the tested strains and F-1 HYBRIDS, THE HIGHEST RESPONDERS WERE A/Ph, its F-1 hybrids with a few other strains and BALB/C. The possibility that the interstrain differences in the rate of nucleic acid synthesis and hence in lymphocyte responsiveness could be due to different sensitivity of the lymphocytes to experimental treatment was not confirmed experimentally. Relatively high rate of synthesis of nucleic acids in PHA unstimulated cells and its great interstrain variation is interpreted in terms of variation in "spontaneous" transformability.

Changes in agglutination of cells with H-2 antibodies or lectins and in stimulation of lymphocytes as induced by certain polymers (chondroitin sulphate/PVP/PHA/Con A/capping of H-2 antigens).

The effect of certain polymers on the course of immune interaction has been well known although its mechanism at the cellular level remains obscure. We thus affected cells in vitro with two of such substances, CS and PVP, and followed their behaviour in various assays. It was shown that in this way the agglutinability of mouse erythrocytes with H-2 antibodies or of mouse lymphocytes with threshold doses of Con A could be enhanced, antibody-induced capping of H-2 antigens on lymphocytes accelerated and PHA-induced stimulation of mouse lymphocytes inhibited. The possible mechanisms of these changes putatively mediated through the effect on the cell membrane or cell coat are discussed.

Independent cyclic fluctuations of cell surface parameters: expression of H-2 antigens, rosetting capacity and thickness of the cell coat.

Cultures of a mouse X mouse cell hybrid (synchronized either by hydroxyurea or Colcemid) were tested for fluctuations during the cell cycle of several parameters characterizing the cell surface: expression of H-2 antigens, capacity of the cells to form rosettes with sheep red blood cells and thickness of the cell coat. H-2 antigenicity of cells collected at different phases of the mitotic cycle was assessed on the basis of their capacity to bind 125I-labelled antibodies as well as their sensitivity to complement dependent immune lysis. The latter parameters fluctuated more or less independently: around mitosis, their trends were even opposite and minimum antibody-binding capacity roughly coincided with maximum cytolytic sensitivity. The cyclic fluctuations of H-2 antigenicity were similar, but not identical for two antigens contributed to the cell hybrid by different parents and even the curves for two antigens of the same parental origin displayed a slight shift one from another. The frequency of cells forming large rosettes (i.e., of more than six SRBC) reached its peak roughly at the boundery of G1 and S phase. Mitotic (versus non-mitotic) cells had a significantly thicker cell coat (as visualized by ruthenium red staining and electron microscopy) and a high incidence of morphological abnormalities. The tested parameters thus seem to fluctuate independently of each other.

Membrane potential in human myeloid leukemia cell line ML-1: responsiveness of granulocytic and monocytic differentiated cells.

The membrane potential responsiveness of human myeloid leukemia cells (ML-1 line) was studied with the voltage sensitive fluorescent dye diS-C3-(5). The experimental procedure used in this study enabled us to assess the magnitude of the membrane potential change in cells treated with ouabain, 12-0-tetradecanoylphorbol-13-acetate (TPA) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), relative to the membrane potential in the untreated control. Inhibition of the Na, K-ATPase by ouabain was followed by a (20 +/- 4) mV depolarization. In undifferentiated homogeneous cell population TPA caused a (19.4 +/- 4.4) mV depolarization while FMLP had virtually no effect. Cells in which granulocytic or monocytic differentiation was induced by retinoic acid or 1,25-dihydroxyvitamin D3 exhibited under the effect of TPA a (57.8 +/- 7.1) mV and (34.8 +/- 10.9) mV depolarization, respectively. A very small transient depolarization was also observed up on treating of the cells with FMLP. The changes in the membrane potential responsiveness in the induced cells are obviously connected with the cell differentiation.

[Antibodies against granulocyte precursors in patients with neutropenia: use of the ML-1 myeloid line]. / Protilátky proti prekursorum granulocytu u nemocných s neutropenií: vyuzití myeloidní linie ML-1.

With the aid of indirect immunofluorescent test, the authors investigated antibodies in the serum of patients, which reacted against immature myeloid cells and mature granulocytes. Myeloid line ML-1, longitudinally maintained in vitro, was used as source of immature myeloid cells. 248 sera were examined and antibodies against the immature myeloid cells were detected in 8% of them. In 6 selected sera of the patients with neutropenia, the authors determined the Ig class of the identified antibody against the immature cells of the ML-1 myeloid line, against the myeloid cells of the ML-1 line differentiated with retinoic acid, respectively even the Ig class of the antibody against neutrophils. The IgM class antibody was found to have the highest frequency. Using absorption studies, the authors provided evidence that the antibody against the myeloid cells can be absorbed only by means of the cells of the myeloid line ML-1, which is demonstrated by the absence of the surface character in the mature neutrophils against which the antibody was directed. The authors discuss the outcome of their investigation aimed at making a more accurate diagnosis of immune neutropenia.

[Use of cytogenetic and molecular biology in the detection of chronic myeloid leukemia]. / Cytogenetický a molekulárne biologický prukaz chronické myeloidní leukémie.

The examination of the presence of Ph chromosome and of the fused gene BCR-ABL in patients with chronic myeloid leukemia (CML) is significant for the precise diagnosis and in some cases for the prognosis of the disease. We examined peripheral blood for the presence of BCR-ABL fused gene by polymerase chain reaction (PCR) in eight patients with CML consecutively cytogenetically studied before and after the bone marrow transplantation and in two patients treated with interferon. Southern blot analysis was performed before BMT in two patients and the molecular rearrangement of Ph chromosome was found. In all cases our results have proved that cytogenetic and recombinant DNA evaluations confirm each other. Due to the high sensitivity of PCR technique the minimal residual leukemia can be detected.