Entwicklung einer computerlesbaren zytogenetischen Notation zur Detektion minimal rekurrenter Aberrationen bei Patienten mit hämatologischen Neoplasien

Ergebnisse tumorzytogenetischer Analysen werden nach gültiger ISCN dokumentiert. Diese Nomenklatur ist nur sehr eingeschränkt computerlesbar. Die Metaanalyse großer Datensammlungen mit den oft komplexen Karyotypen von Tumorpatienten wird dadurch erschwert. Es wurde eine vereinfachte computerlesbare zytogenetische Notation (SCCN) und entsprechende Analyse-Programme entwickelt. Die im Karyotyp enthaltene qualitative und quantitative Metainformation wird in zwei Datenfeldern gespeichert. Die Metaanalyse der Bruchpunkte und chromosomalen Imbalancen erfolgt getrennt. Ergebnisse werden grafisch und tabellarisch dargestellt. Die SCCN und Programme wurden an einer Datensammlung von Karyotypen von 94 Patienten mit akuter lymphatischer Leukämie, t(9;22)(q34;q11) und Zusatzaberrationen getestet. Die Metaanalyse des Patientenkollektivs bestätigte die bekannten Literaturwerte

PAX5 fusion genes in t(7;9)(q11.2;p13) leukemia: a case report and review of the literature

BACKGROUND: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by recurrent genetic alterations including chromosomal translocations. The transcription factor PAX5, which is pivotal for B-cell commitment and maintenance, is affected by rearrangements, which lead to the expression of in-frame fusion genes in about 2.5% of the cases. RESULTS: Using conventional cytogenetics, fluorescence in situ hybridization (FISH), and molecular methods, an additional case with a der(9)t(7;9)(q11.23;p13) resulting in the expression of a PAX5-ELN fusion gene was identified. Furthermore, a general review of leukemia harboring a t(7;9)(q11.2;p13) or der(9)t(7;9)(q11.2;p13), which occurs more often in children than in adults and shows a remarkably high male preponderance, is given. These cytogenetically highly similar translocations lead to the expression of one of three different in frame PAX5-fusions, namely with AUTS2 (7q11.22), ELN (7q11.23), or POM121 (7q11.23), which constitute the only currently known cluster of PAX5 partner genes. CONCLUSION: Our report underlines the recurrent involvement of PAX5 in different fusion genes resulting either from t(7;9)(q11.2;p13) or der(9)t(7;9)(q11.2;p13), which cannot be distinguished cytogenetically and whose discrimination requires molecular analysis

Computer aided analysis of additional chromosome aberrations in Philadelphia chromosome positive acute lymphoblastic leukaemia using a simplified computer readable cytogenetic notation

BACKGROUND: The analysis of complex cytogenetic databases of distinct leukaemia entities may help to detect rare recurring chromosome aberrations, minimal common regions of gains and losses, and also hot spots of genomic rearrangements. The patterns of the karyotype alterations may provide insights into the genetic pathways of disease progression. RESULTS: We developed a simplified computer readable cytogenetic notation (SCCN) by which chromosome findings are normalised at a resolution of 400 bands. Lost or gained chromosomes or chromosome segments are specified in detail, and ranges of chromosome breakpoint assignments are recorded. Software modules were written to summarise the recorded chromosome changes with regard to the respective chromosome involvement. To assess the degree of karyotype alterations the ploidy levels and numbers of numerical and structural changes were recorded separately, and summarised in a complex karyotype aberration score (CKAS). The SCCN and CKAS were used to analyse the extend and the spectrum of additional chromosome aberrations in 94 patients with Philadelphia chromosome positive (Ph-positive) acute lymphoblastic leukemia (ALL) and secondary chromosome anomalies. Dosage changes of chromosomal material represented 92.1% of all additional events. Recurring regions of chromosome losses were identified. Structural rearrangements affecting (peri)centromeric chromosome regions were recorded in 24.6% of the cases. CONCLUSIONS: SCCN and CKAS provide unifying elements between karyotypes and computer processable data formats. They proved to be useful in the investigation of additional chromosome aberrations in Ph-positive ALL, and may represent a step towards full automation of the analysis of large and complex karyotype databases

Entwicklung einer computerlesbaren zytogenetischen Notation zur Detektion minimal rekurrenter Aberrationen bei Patienten mit hämatologischen Neoplasien

Ergebnisse tumorzytogenetischer Analysen werden nach gültiger ISCN dokumentiert. Diese Nomenklatur ist nur sehr eingeschränkt computerlesbar. Die Metaanalyse großer Datensammlungen mit den oft komplexen Karyotypen von Tumorpatienten wird dadurch erschwert. Es wurde eine vereinfachte computerlesbare zytogenetische Notation (SCCN) und entsprechende Analyse-Programme entwickelt. Die im Karyotyp enthaltene qualitative und quantitative Metainformation wird in zwei Datenfeldern gespeichert. Die Metaanalyse der Bruchpunkte und chromosomalen Imbalancen erfolgt getrennt. Ergebnisse werden grafisch und tabellarisch dargestellt. Die SCCN und Programme wurden an einer Datensammlung von Karyotypen von 94 Patienten mit akuter lymphatischer Leukämie, t(9;22)(q34;q11) und Zusatzaberrationen getestet. Die Metaanalyse des Patientenkollektivs bestätigte die bekannten Literaturwerte

Quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) using amplicon-fusion-site polymerase chain reaction (AFS-PCR)

Abstract The amplification of putative oncogenes is a common finding within the genome of various cancer types. Identification and further targeting of specific junction sites within the sequence of genomic amplicons (amplicon fusion sites, AFS) by PCR (AFS-PCR) is suitable for quantification of minimal residual disease (MRD). This approach has recently been developed and described for MYCN amplified neuroblastomas. To compare AFS-PCR directly to routinely used MRD diagnostic strategies, we mapped the amplified genomic regions (ampGR) of an iAMP21-amplicon in high resolution of a patient with acute lymphoblastic leukemia (ALL). Successfully, we established AFS-PCR covering junction sites between ampGR within the iAMP21-amplicon. Quantification of MRD by AFS-PCR was directly comparable to IgH/TCR based real time quantitative PCR and fluorescence activated cell sorting (FACS) analysis in consecutive bone marrow (BM) specimens. Our data give an additional proof of concept of AFS-PCR for quantification of MRD. The method could be taken into account for ALL patients with genomic amplifications as alternative MRD diagnostic, if no or qualitatively poor Ig/TCR-PCRs are available.</p

MicroRNAs Distinguish Cytogenetic Subgroups in Pediatric AML and Contribute to Complex Regulatory Networks in AML-Relevant Pathways

<div><h3>Background</h3><p>The role of microRNAs (miRNAs), important post-transcriptional regulators, in the pathogenesis of acute myeloid leukemia (AML) is just emerging and has been mainly studied in adults. First studies in children investigate single selected miRNAs, however, a comprehensive overview of miRNA expression and function in children and young adults is missing so far.</p> <h3>Methodology/Principal Findings</h3><p>We here globally identified differentially expressed miRNAs between AML subtypes in a survey of 102 children and adolescent. Pediatric samples with core-binding factor AML and promyelocytic leukemia could be distinguished from each other and from MLL-rearranged AML subtypes by differentially expressed miRNAs including miR-126, -146a, -181a/b, -100, and miR-125b. Subsequently, we established a newly devised immunoprecipitation assay followed by rapid microarray detection for the isolation of Argonaute proteins, the hallmark of miRNA targeting complexes, from cell line models resembling core-binding factor and promyelocytic leukemia. Applying this method, we were able to identify Ago-associated miRNAs and their targeted mRNAs.</p> <h3>Conclusions/Significance</h3><p>miRNAs as well as their mRNA-targets showed binding preferences for the different Argonaute proteins in a cell context-dependent manner. Bioinformatically-derived pathway analysis suggested a concerted action of all four Argonaute complexes in the regulation of AML-relevant pathways. For the first time, to our knowledge, a complete AML data set resulting from carefully devised biochemical isolation experiments and analysis of Ago-associated miRNAs and their target-mRNAs is now available.</p> </div

Ago-associated miRNAs and - mRNAs using the PAR-CLIP-Array method.

<p>(A) Western Blot analysis of immunoprecipitates of human Ago1-4 from AML cell lines, KASUMI-1 with t(8;21) and NB4 carrying t(15;17). The immunoprecipitates show a specific band of the Argonaute protein (∼97 kDa; ←) in contrast to the isotype control antibody (rat IgG2a) and empty-bead control. A representative sample of the biological triplicate is shown. Please note that more material was loaded for Ago3 and Ago4 since these two Ago proteins are much lower expressed as was also validated by qRT-PCR (not shown). Antibodies were tested for specificity for detection of native and denatured protein prior to this experiment with cell lines overexpressing tagged Ago protein (not shown) (B) Validation of miRNA- and mRNA-enrichment in immunoprecipitation experiments. Argonaute proteins (black bar) are compared to the isotype (white bar) and empty bead controls (grey bar) using TaqMan qRT-PCR assays for microRNA- (upper panel) and SYBR Green qRT-PCR assays for mRNA-quantification (lower panel). Shown are the measured levels (2^−CT) of six and five miRNAs of KASUMI-1 cells (upper left panel) and NB4 cells (upper right panel), respectively. Immunoprecipitation experiments as well as cDNA synthesis were each done in triplicates and the mean value of the nine values as well as one standard deviation is depicted. miRNAs differentially expressed in patient samples between the t(8;21) and t(15;17) were selected together with ubiquitously expressed miR-16. Please note that calculation of ΔC_T-values is not possible due to the lack of a housekeeping gene bound to Argonaute proteins. Six Ago-associated mRNAs in the KASUMI-1 cells (lower left panel) and NB4 cells (lower right panel), covering the whole range from low to high enrichment over isotype control according to microarray data, were selected for qRT-PCR validation. Graphs are centered around a C_T-value of 29.9 cycles (2^−CT = 0⁻⁹).</p