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Molecular Genetics of Myeloid Leukemia

Contact/Address

Prof. Dr. med. Konstanze Döhner

Department of Internal Medicine III

University Hospital of Ulm

Albert-Einstein-Allee 23

89081 Ulm

Germany
 

Phone:
Fax:
+49-(0)731-500-45543

+49-(0)731-500-45505


Principal Investigator

Prof. Dr. Konstanze Döhner

E-Mail: konstanze.doehner[at]uniklinik-ulm.de

Prof. Dr. Lars Bullinger

E-Mail: lars.bullinger[at]uniklinik-ulm.de


Scientific Lab Members

Dr. Andrea Corbacioglu

Dr. Verena Gaidzik

Dr. Frank Rücker

Dr. Frank Stegelmann

 

Technicians

Ursula Botzenhardt

Marianne Habdank

Patricia Scheible

Sabrina Skambraks

Olesia Sirik

Christa Wieland

 

Students

Andreas Anhalt

Montserrat Hoyos Colell

Raphael Kranz

Mia Kugler

Stephan Kurz

Martin Lochstampfer

Judith Mehner

Corinna Mendla

Christoph Stirner

Jana Vehma

Research Fields

(A)

Identification of novel gene mutations in AML and evaluation of their prognostic impact

Chromosome aberrations are one of the most important prognostic factors in acute myeloid leukaemia (AML) and are used for selecting postremission therapy. However, approximately 40 to 50% of all AML patients lack chromosomal aberrations and therefore risk-adapted therapy is difficult. Recently, mutations in a variety of genes have been identified and most of these gene rearrangements are of prognostic relevance.

The aim of our studies is to perform mutation analysis in genes that are known to be involved in hematopoietic differentiation and proliferation and to evaluate their prognostic impact. Therefore, AML samples (peripheral blood, bone marrow) from a large tumor bank will be analyzed for mutations in a large number of genes (e.g. MLL, NPM1, CEBPA, NRAS, WT1) using different molecular techniques.

 

(B)

Design and Evaluation of real-Time RT-PCR Assays for the Detection of Minimal Residual Disease in AML

One of the strategies being explored to predict clinical outcome in patients with acute myeloid leukemia (AML) exhibiting specific gene rearrangements is the detection of minimal residual disease (MRD) following achievement of hematologic remission. Real-time RT-PCR is a novel method that allows the identification of MRD by quantification of chimeric transcripts. Nevertheless, for most of the AML-specific gene fusions the prognostic value of MRD still has to be determined.

We are currently investigating the value of MRD Monitoring in AML exhibiting the AML-specific fusion types such as t(8;21), inv(16)/t(16;16), t(9;11), and t(15;17). In addition, based on more recent findings we are establishing MRD assays for the detection of novel molecular markers that have been detected in AML with normal karyotype.

The objective of this study is the design of real-Time RT-PCR assays for quantification of the common AML-associated fusions and to evaluate the predictive value of fusion transcript quantification for disease relapse.

 

(C)

Gene expression profiling based identification of biologically and prognostically relevant signatures in AML

Approximately 40 to 50% of adult patients with AML lack chromosomal abnormalities. For this subgroup of AML patients new molecular markers are warranted. Moreover, little is known about the pathogenic role of known AML-associated genes and their downstream targets. The objectives of this project are to identify new candidate genes and to elucidate the pathogenic role of known AML-associated genes and their downstream targets using DNA microarray technology.

Current projects encompass the detailed analysis of core binding factor leukemias, normal karyotype AML with FLT3 and/or NPM1 mutations, and the prospective analysis of gene expression profiles within our current multicenter treatment trials in order to further evaluate the prognostic impact of gene expression signatures in AML.

 

(D)

Molecular genetic and functional characterization of candidate genes located in the commonly deleted region at chromosomal bands 7q22-q31 in myeloid Leukemias

Loss of chromosome 7 (-7) or deletion of the long arm (7q-) are recurring chromosome abnormalities in myeloid leukemias. Recently, a commonly deleted region (CDR) of 2 to 3 Megabases at chromosomal bands 7q22-q31 was identified at the molecular level. Since that CDR provides a potential site harboring genes that might play a role in the pathogenesis of myeloid leukemias, construction of a detailed transcriptional map was initiated. Based on this map several ESTs and genes were identified. Genetic and functional analysis are being currently performed. In addition, FISH screening in a large number of AML samples exhibiting 7q aberrations as well as high resolution array-mapping (mCGH, SNP analysis) will be performed. This allows us to identify novel deletion and translocation breakpoints on the long arm of chromosome 7 that might harbour genes being involved in leukemogenesis.

 

(E)

Identification of genomic imbalances in AML with complex karyotype using matrix-based comparative genomic hybridization

Comparative genomic hybridization to microarrays (array CGH or matrix CGH) is a novel technique allowing genome-wide screening for genomic imbalances at high resolution and thus facilitating the identification of new regions harboring potential disease-related genes. Using an 8.0 k platform consisting of 8051 different bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) vectors with an average resolution of approximately 1 Mb, we are currently investigating in AML with complex karyotype and AML with normal karyotype. So far, several novel hot spot regions have been identified that serve as a starting point for further studies on leukaemia-associated genes.

In addition, we have established the use of SNP-chip technology to perform LOH analyses in AML with the intention to further explore the role of uniparental disomy in leukemogenesis.

 

(F)

Investigation of miRNA expression in AML – potential role in leukemogenesis?

Recently, the impact of miRNA expression on the regulation of genes not only during developmental processes, but also its significance in the development of cancer, has been impressively demonstrated. To further investigate the potential role of miRNAs in AML pathogenesis we are aiming to profile miRNA expression in a comprehensive set of AML cases in order to identify potential candidates for further functional exploration.

Techniques

  • Cell culture
  • Fluoresence in situ hybridization (FISH)
  • Fluorescence microscopy
  • DNA/RNA preparation
  • PCR techniques including quantitative real-Time RT-PCR (Taq Man, Light Cycler )
  • DNA cloning
  • Southern- ,Northern-, Western blotting
  • DNA sequencing
  • Sequence analysis
  • Genescan analysis
  • DHPLC-based mutation screening (WAVE System)
  • Matrix-CGH
  • LOH analysis (SNP microarrays)
  • Gene expression profiling using different platforms (cDNA, Affymetrix)
  • Profiling miRNA expression (oligo microarray, real-time PCR)

Grants/Funding

  • Medizinische Fakultät der Universität Ulm („Bausteinprogramm“)
  • Deutsche José Carreras Leukämie Stiftung
  • Else Kröner-Fresenius-Stiftung
  • Deutsche Forschungsgemeinschaft (DFG)
  • Wilhelm Sander Stiftung
  • Nationales Genomforschungsprojekt 2 (NGFN2)
  • Bundesministerium für Bildung und Forschung (BMBF)

Representative Publications

Fröhling S, Schlenk RF, Stolze I, Bihlmayr J, Benner A, Kreitmeier S, Tobis K, Döhner H, Döhner K:
CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: prognostic relevance and analysis of cooperating mutations.
J Clin Oncol. 2004;22:624-633.

 

Bullinger L, Döhner K, Bair E, Fröhling S, Schlenk RF, Tibshirani R, Döhner H, Pollack JR:
Use of gene-expression profiling to identify prognostic subclasses in adult acute myeloid leukemia.
N Engl J Med. 2004;350:1605-1616.

 

Döhner K, Schlenk RF, Habdank M, Scholl C, Rucker FG, Corbacioglu A, Bullinger L, Frohling S, Dohner H:
Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics - interaction with other gene mutations.
Blood. 2005;106:3740-3746.

 

Rücker FG, Bullinger L, Schwaenen C, Lipka DB, Wessendorf S, Fröhling S, Bentz M, Miller S, Scholl C, Schlenk RF, Radlwimmer B, Kestler HA, Pollack JR, Lichter P, Döhner K, Döhner H:
Disclosure of candidate genes in acute myeloid leukemia with complex karyotypes using microarray-based molecular characterization.
J Clin Oncol. 2006; 24:3887-3894.

 

Bullinger L, Rücker F, Kurz S, Du J, Scholl C, Sander S, Corbacioglu A, Lottaz C, Krauter J, Fröhling S, Ganser A, Schlenk RF, Döhner K, Pollack JR, Döhner H:
Gene expression profiling identifies distinct subclasses of core binding factor acute myeloid leukemia. Blood. 2007;110:1291-1300.

Collaborations

Clara D. Bloomfield, M.D.
Distinguished University Professor
William G. Pace III Professor of Cancer Research Cancer Scholar and Senior Advisor The Ohio State University Comprehensive Cancer Center and James Cancer Hospital and Solove Research Institute 320 West 10th Ave, A455 Starling-Loving Hall Columbus OH 43210

 

Prof. Dr. Peter Lichter
DKFZ, Department of Molecular Genetics, Heidelberg

 

Prof. Hans-Jörg Fehling
Department of Molecular Immunology, University of Ulm

 

Prof. Dr. Jon Pollack
Dept. of Pathology, Stanford University, California, USA

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