http://repub.eur.nl/res/aut/31421/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/34609/ 2011-11-24T00:00:00Z Clonality analysis is a critical tool for the diagnosis of suspect lymphoproliferative disorders. Amplification of the immunoglobulin and T cell receptor genes on genomic DNA from the suspect samples is followed by analysis of the PCR products to distinguish between polyclonal and clonal rearrangements. These analyses are based on electrophoresis in polyacrylamide gels after heteroduplex formation, or more frequently, GeneScan analysis is performed by capillary electrophoresis in automated DNA analysers, providing higher resolution and sensitivity. An alternative method for clonality analysis is the use of single-strand conformation analysis; however, this usually required labour intensive work with polyacrylamide gels and radioactive labelling. Within the EuroClonality group, we have developed a non-radioactive automated analysis based on capillary electrophoresis of single-strand PCR products that combines some of the benefits of heteroduplex and GeneScan analysis. This new method could be particularly suitable for challenging cases and could be implemented as an alternative to the more laborious heteroduplex analysis in standard gels in some instances. http://repub.eur.nl/res/pub/33391/ 2011-06-30T00:00:00Z We have recently reported inactivation of the tyrosine phosphatase PTPN2 (also known as TC-PTP) through deletion of the entire gene locus in ∼ 6% of T-cell acute lymphoblastic leukemia (T-ALL) cases. T-ALL is an aggressive disease of the thymocytes characterized by the stepwise accumulation of chromosomal abnormalities and gene mutations. In the present study, we confirmed the strong association of the PTPN2 deletion with TLX1 and NUP214-ABL1 expression. In addition, we found cooperation between PTPN2 deletion and activating JAK1 gene mutations. Activating mutations in JAK1 kinase occur in ∼ 10% of human T-ALL cases, and aberrant kinase activity has been shown to confer proliferation and survival advantages. Our results reveal that some JAK1 mutation - positive TALLs harbor deletions of the tyrosine phosphatase PTPN2, a known negative regulator of the JAK/STAT pathway. We provide evidence that down-regulation of Ptpn2 sensitizes lymphoid cells to JAK1-mediated transformation and reduces their sensitivity to JAK inhibition. http://repub.eur.nl/res/pub/33393/ 2011-06-16T00:00:00Z Cumulative evidence indicates that MYC, one of the major downstream effectors of NOTCH1, is a critical component of T-cell acute lymphoblastic leukemia (T-ALL) oncogenesis and a potential candidate for targeted therapy. However, MYC is a complex oncogene, involving both fine protein dosage and cell-context dependency, and detailed understanding of MYC-mediated oncogenesis in T-ALL is still lacking. To better understand how MYC is interspersed in the complex T-ALL oncogenic networks, we performed a thorough molecular and biochemical analysis of MYC activation in a comprehensive collection of primary adult and pediatric patient samples. We find that MYC expression is highly variable, and that high MYC expression levels can be generated in a large number of cases in absence of NOTCH1/FBXW7 mutations, suggesting the occurrence of multiple activation pathways in addition to NOTCH1. Furthermore, we show that posttranscriptional deregulation of MYC constitutes a major alternative pathway of MYC activation in T-ALL, operating partly via the PI3K/AKT axis through down-regulation of PTEN, and that NOTCH1mmight play a dual transcriptional and posttranscriptional role in this process. Altogether, our data lend further support to the significance of therapeutic targeting of MYC and/or the PTEN/AKT pathways, both in GSI-resistant and identified NOTCH1-independent/MYC-mediated T-ALL patients. http://repub.eur.nl/res/pub/24568/ 2009-04-24T00:00:00Z BCR-ABL fusion proteins show increased signaling through their ABL tyrosine kinase domain, which can be blocked by specific inhibitors, thereby providing effective treatment. This makes detection of BCR-ABL aberrations of utmost importance for diagnosis, classification and treatment of leukemia patients. BCR-ABL aberrations are currently detected by karyotyping, fluorescence in situ hybridization (FISH) or PCR techniques, which are time consuming and require specialized facilities. We developed a simple flow cytometric immunobead assay for detection of BCR-ABL fusion proteins in cell lysates, using a bead-bound anti-BCR catching antibody and a fluorochrome-conjugated anti-ABL detection antibody. We noticed protein stability problems in lysates caused by proteases from mature myeloid cells. This problem could largely be solved by adding protease inhibitors in several steps of the immunobead assay. Testing of 145 patient samples showed fully concordant results between the BCR-ABL immunobead assay and reverse transcriptase PCR of fusion gene transcripts. Dilution experiments with BCR-ABL positive cell lines revealed sensitivities of at least 1%. We conclude that the BCR-ABL immunobead assay detects all types of BCR-ABL proteins in leukemic cells with high specificity and sensitivity. The assay does not need specialized laboratory facilities other than a flow cytometer, provides results within ∼4h, and can be run in parallel to routine immunophenotyping.