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Mejstrikova E.,CLIP Childhood Leukemia Investigation Prague | Mejstrikova E.,University Hospital Motol | Fronkova E.,CLIP Childhood Leukemia Investigation Prague | Fronkova E.,University Hospital Motol | And 21 more authors.
Pediatric Blood and Cancer | Year: 2010

Background. Residual disease (RD) is an important prognostic factor in acute lymphoblastic leukemia (ALL). Flow cytometry (FC)-based RD detection is easy to perform, but interpretation requires expert analysis due to individual differences among patients. Procedure. We focused at the design of standardized and reproducible RD monitoring in ALL. RD was investigated by a uniform gating strategy, which was designed internationally and tested in one center by Ig/TCR rearrangements. Results. For each gate, positivity cutoff value was assigned using quantification of non-leukemic background. Comparing to Ig/TCR at 0.1% level, 80 of 103 specimens were correctly diagnosed by FC. The predictive value of FC RD at day 15 was then analyzed. In B lineage ALL, day 15 FC significantly correlated with Ig/TCR results at day 33 and/or week 12 (P<0.01). No significant correlation was found in T lineage ALL. Conclusions. Thus, FC with preset uniform gating at day 15 predicts PCR-detectable MRD in B precursor ALL. Presented data may be used to define new polychromatic cytometric diagnostics of MRD including semiautomatic assessment. © 2009 Wiley-Liss, Inc.

Hermanova I.,CLIP Childhood Leukemia Investigation Prague | Trka J.,CLIP Childhood Leukemia Investigation Prague | Starkova J.,CLIP Childhood Leukemia Investigation Prague
Transfuze a Hematologie Dnes | Year: 2010

Acute lymphoblastic leukaemia (ALL), the most common haematological malignancy in childhood, is treated by combined chemotherapy, which includes the enzyme L-Asparaginase (L-Asp). The cytotoxic effect of L-Asp consists in its ability to deplete extracellular asparagine and glutamine. The sensitivity of primary ALL cells to this depletion is traditionally explained by decreased activity of glutamine-dependent enzyme asparagine synthetase (ASNS). Despite the fact that increased ASNS level was indeed shown to be connected with L-Asp resistance, the exact relationship between ASNS expression and L-Asp sensitivity has not been elucidated so far. The gene expression and ASNS protein content was evaluated in 4 leukemic cell lines: Nalm6 (TEL/PDGFRB[+]); RS4;11 (MLL/AF4[+]); REH (TEL/AML1[+]) and UOCB6 (TEL/AML1[+]). ASNS protein levels reflected ASNS mRNA levels and these correlated negatively with L-Asp sensitivity. UOCB6 as the most resistant cell line had the highest expression of ASNS, followed by Nalm6, REH and RS4;11. Detection of protein content in primary ALL blasts was not possible due to significantly lower ASNS gene expression compared to cell lines. Gradient knock-down was performed in 2 ALL cell lines: REH with intermediate basal expression and RS4;11 with very low basal expression. A gradual silencing of ASNS gene in REH cell line led to gradual increase of L-Asp sensitivity. The reduction of ASNS did not potentiate L-Asp cytotoxicity in RS4;11 cell line. Our data demonstrate that in cells with very low ASNS expression, as shown in primary ALL blasts of various subtypes, the difference in ASNS levels is not relevant to the sensitivity to L-Asp. We suppose that glutamate dehydrogenase (GDH) is a new player in the response to cytotoxic effect of L-Asp. Silencing of GDH gene in TEL/AML1[+] REH cell line increased sensitivity to L-Asp. Furthermore, we suggest a relationship between ASNS and GDH based on our observations of increased GDH expression in cells with silenced ASNS gene.

Svojgr K.,CLIP Childhood Leukemia Investigation Prague | Svojgr K.,Charles University | Kalina T.,CLIP Childhood Leukemia Investigation Prague | Kalina T.,Charles University | And 6 more authors.
Experimental Hematology | Year: 2012

The biology of T-cell acute lymphoblastic leukemia (ALL) is characterized by functional pre-T-cell receptor (TCR) signaling. Non-T-cell activation linker (NTAL) is a nonenzymatic transmembrane adaptor molecule that is involved in the proximal signaling of lymphocytes. In our previous work, we found an association between high NTAL expression in T-cell ALL blasts and a favorable response to initial glucocorticoid treatment. In the present study, we confirm our previous observation in an experimental model. In addition, the molecular mechanism of the contribution of NTAL to malignant T-cell ALL blast signaling and to methylprednisolone-induced cell death is analyzed. In the in vitro experiments, we used the T-cell ALL Jurkat cell line (Jurkat/wt) and derived Jurkat cell line with stable NTAL expression (Jurkat/NTAL +). Cell signaling and cell death after methylprednisolone treatment and after TCR stimulation were analyzed using flow cytometry, Western blot, and quantitative polymerase chain reaction. Jurkat/NTAL + cells are significantly more sensitive to both methylprednisolone treatment and TCR-induced stimulation. In addition, after TCR stimulation, Jurkat/NTAL + cells show a higher level of intracellular extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and increased expression of the CD69 activation marker on the cell surface than the Jurkat/wt cells. The ERK inhibitor U0126 almost completely abrogates TCR-induced cell death and, importantly, reverses the sensitizing effect of the NTAL protein on methylprednisolone-induced cell death. In conclusion, NTAL acts as a tumor suppressor that enhances the proximal signaling of leukemic blasts. The key downstream molecule responsible for the biological effect of TCR signaling is ERK. Higher ERK phosphorylation leads to enhanced cell death after TCR stimulation and increases cell sensitivity to methylprednisolone-induced cell death. © 2012 ISEH - Society for Hematology and Stem Cells.

Kovac M.,CLIP Childhood Leukemia Investigation Prague | Petrackova D.,Mikrobiologicky ustav AV CR | Bezouskova S.,Mikrobiologicky ustav AV CR | Pelkova V.,CLIP Childhood Leukemia Investigation Prague | And 6 more authors.
Transfuze a Hematologie Dnes | Year: 2010

In acute leukemia (AL), several clinical symptoms may be caused by soluble factors secreted by AL cells into the bone marrow (BM) microenvironment. On the other hand, AL cells are often dependent on the microenvironment of the host, with most AL cells dying during first days after transferred to in vitro. This support of leukemic cells may be mediated by soluble factors as well. Attention is logically focused on malignant cells and the composition of soluble factors may be unjustly omitted. We aimed at identifying proteins in BM plasma of children with lymphoblastic AL (ALL), which may be responsible for ALL aggressiveness or for microenvironment-mediated survival of ALL cells. BM plasma and blood samples were analyzed by protein microarray and/or by two-dimensional electrophoresis (2D PAGE). We detected 23 proteins with a significantly different concentration in patients by Protein microarray. Before 2D PAGE, BM plasma was immuno-depleted from 12 abundant proteins by affinity chromatography. With this approach we succeeded to increase the number of spots that were analyzed by the PDQuest software.

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