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Molecular Genetics of Myeloproliferative Neoplasms

Contact/Address

Prof. Dr. med. Konstanze Döhner
Dr. med. Frank Stegelmann

 

Department of Internal Medicine III

University Hospital of Ulm

Albert-Einstein-Allee 23

89081 Ulm

Germany

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

E-Mail: frank.stegelmann[at]uniklinik-ulm.de
 

Phone:
Fax: 

+49-(0)731-500-45521
+49-(0)731-500-45525 

 
Project Leader

Dr. Frank Stegelmann

 

Technicians

Susanne Kuhn
Rebecca Matyl
Anita Szmaragowska

Research Fields

(A)
Molecular diagnosis of Myeloproliferative Neoplasms (MPN)

Myleoproliferative Neoplasms (MPN) comprise Various chronic leukemias with myeloid origin. Among them, the molecular cause of chronic myeloid leukemia (CML) is defined most precisely. Typical CML is represented by the activating tyrosine kinase fusion protein BCR-ABL that results from the recurrent chromosome translocation t(9;22). At diagnosis, the most frequent BCR/ABL fusion transcripts that occur in CML (e1a2, b2a2 and b3a2) are detectable by Reverse transcriptase- (RT-) Multiplex-PCR. In the course of disease, Minimal residual disease- (MRD-) monitoring is predictive for the clinical outcome. Therefore, internationally standardized PCR assays are used to monitor response in BCR-ABL positive CML patients under tyrosine kinase inhibitor treatment.

More recently, genetic alterations were also identified in BCR/ABL negative MPN. In particular, the activating JAK2 V617F point mutation has improved the understanding of BCR-ABL negative MPN. In 2005, JAK2 V617F has been discovered as a single-site, clonal, gain-of-function mutation in myeloid cells in the majority of patients with Polycythemia vera (PV, >95%), Essential thrombocythemia (ET, 50-60%) and primary myelofibrosis (PMF, 60%). Though less frequent, JAK2 Exon 12 mutations (e.g JAK2 K539L) occur in V617F negative PV patients, and the MPL W515L/K point mutation is present in about 5%-10% of PMF and ET patients, respectively. In 2013, frameshift mutations in the CALR gene have been described in 20-30% of ET-and PMF patients, now allowing to prove clonality in the vast majority MPN cases since CALR mutations occur mutually exclusive from JAK2 and MPL. Furthermore, the FIP1L1-PDGFRA fusion gene and the KIT D816V mutation are detectable in a subset of patients suffering from Hypereosinophilic syndrome (HES) and Systemic mastocytosis (SM), respectively. For molecular based detection of the above mentioned genetic alterations, PCR-based analyses are applied.

 

(B)
Clinical impact of the JAK2 V617F mutation

In the mouse model, JAK2 V617F leads to a myeloproliferative phenotype resembling PV with marked elevation of erythrocytes and granulocytes. To date, little is known on the pathogenic value of JAK2 V617F in ET and PMF. In addition, it is controversially discussed whether the mutations are relevant for the clinical course by increasing the risk of vascular events, secondary myelofibrosis or transormation into acute myeloid leukemia. The aim of our study is to analyze a large cohort of clinically well-defined MPN patients for the presence of JAK2 V617F. Moreover, the proportion of the mutant allele (homozygous vs. heterozygous) is determined in JAK2 V617F mutated patients by quantitative Real-time PCR. Samples from primary diagnosis and during follow-up (3-6 months intervals) are analyzed prospectively to explore the evolution and prognostic impact of JAK2 V617F in ET, PV and PMF by correlating clinical and molecular data. In addition, JAK2 V617F is used as a marker for sensitive minimal residual disease (MRD) monitoring in patients showing a decline of the allele burden under treatment (e.g. under Interferon-α or after allogeneic stem cell transplantation).

 

(C)
Detailed genomic characterization of MPN

Recent findings show that a subset of MPN patients harbor various other mutations in genes that have been implicated in leukemogenesis (e.g. TET2, DNMT3A or ASXL1). In contrast to JAK2, MPL or CALR, these mutations are less specific for MPN as they also occur in MDS and AML patients. However, these findings reflect the genomic complexity of MPN and are likely relevant for the heterogeneous clinical course as well as for disease evolution. We therefore screen clinically well-defined MPN patient cohorts using modern molecular techniques such as high-resolution ´single nucleotide polymorphism´ (SNP) arrays and ´Next-Generation-Sequencing´ for detailed genomic characterization beyond JAK2, MPL or CALR. These data allow links between the molecular basis and the clinical phenotype by correlating genetic and patient data. In addition, novel hot spot regions harboring candidate genes can be identified serving as a starting point for further functional studies using cell culture and siRNA techniques.

Techniques

  • Cell isolation techniques (Ficoll Paque® centrifugation, MACS® Cell Separation)
  • DNA/RNA preparation
  • PCR techniques including quantitative Real-time PCR (QuantStudio™, LightCycler®)
  • Sanger sequencing and software-supported analyses
  • GeneScan™-based mutation screening (Applied Biosystems®)
  • High-resolution SNP array analysis (Affymetrix®)
  • Next-generation sequencing' (NGS) on HiSeq™ and MiSeq™ (Illumina)

Grants/Funding

  • Else Kröner-Fresenius Stiftung

Selected Publications

 

Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis.
Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, Boggon TJ, Wlodarska I, Clark JJ, Moore S, Adelsperger J, Koo S, Lee JC, Gabriel S, Mercher T, D'Andrea A, Fröhling S, Döhner K, Marynen P, Vandenberghe P, Mesa RA, Tefferi A, Griffin JD, Eck MJ, Sellers WR, Meyerson M, Golub TR, Lee SJ, Gilliland DG.
Cancer Cell. 2005 Apr;7(4):387-97.


High-resolution single-nucleotide polymorphism array-profiling in myeloproliferative neoplasms identifies novel genomic aberrations.
Stegelmann F
, Bullinger L, Griesshammer M, Holzmann K, Habdank M, Kuhn S, Maile C, Schauer S, Döhner H, Döhner K.
Haematologica. 2010 Apr;95(4):666-9


DNMT3A mutations in myeloproliferative neoplasms.
Stegelmann F
, Bullinger L, Schlenk RF, Paschka P, Griesshammer M, Blersch C, Kuhn S, Schauer S, Döhner H, Döhner K.
Leukemia. 2011 Jul;25(7):1217-9.


Myeloproliferative neoplasm (MPN) symptom assessment form total symptom score: prospective international assessment of an abbreviated symptom burden scoring system among patients with MPNs.
Emanuel RM, Dueck AC, Geyer HL, Kiladjian JJ, Slot S, Zweegman S, te Boekhorst PA, Commandeur S, Schouten HC, Sackmann F, Kerguelen Fuentes A, Hernández-Maraver D, Pahl HL, Griesshammer M, Stegelmann F, Döhner K, Lehmann T, Bonatz K, Reiter A, Boyer F, Etienne G, Ianotto JC, Ranta D, Roy L, Cahn JY, Harrison CN, Radia D, Muxi P, Maldonado N, Besses C, Cervantes F, Johansson PL, Barbui T, Barosi G, Vannucchi AM, Passamonti F, Andreasson B, Ferrari ML, Rambaldi A, Samuelsson J, Birgegard G, Tefferi A, Mesa RA. J
Clin Oncol. 2012 Nov 20;30(33):4098-103.


MPN patients harbor recurrent truncating mutations in transcription factor NF-E2.
Jutzi JS, Bogeska R, Nikoloski G, Schmid CA, Seeger TS, Stegelmann F, Schwemmers S, Gründer A, Peeken JC, Gothwal M, Wehrle J, Aumann K, Hamdi K, Dierks C, Kamar Wang W, Döhner K, Jansen JH, Pahl HL.
J Exp Med. 2013 May 6;210(5):1003-19.


Effect of mutation order on myeloproliferative neoplasms.
Ortmann CA, Kent DG, Nangalia J, Silber Y, Wedge DC, Grinfeld J, Baxter EJ, Massie CE, Papaemmanuil E, Menon S, Godfrey AL, Dimitropoulou D, Guglielmelli P, Bellosillo B, Besses C, Döhner K, Harrison CN, Vassiliou GS, Vannucchi A, Campbell PJ, Green AR.
N Engl J Med. 2015 Feb 12;372(7):601-12.


Genetic variation at MECOM, TERT, JAK2 and MYB predispose to myeloproliferative neoplasms.
Tapper W, Jones A, Kralovics R, Harutyunyan A, Zoi K, Leung W, Godfrey A, Guglielmelli P, Callaway A, Ward D, Aranaz P, White H, Waghorn K, Lin F, Chase A,  Baxter J, Maclean C, Nangalia J, Chen E, Evans P, Short M, Jack A, Wallis L, Oscier D, Duncombe A, Schuh A, Mead A, Griffiths M, Ewing J, Gale R, Schnittger S, Haferlach T, Stegelmann F, Döhner K, Grallert H, Strauch K, Tanaka T, Bandinelli S, Giannopoulos A, Pieri L, Mannarelli C, Gisslinger H, Barosi G, Cazzola M, Reiter A, Harrison C, Campbell P, Green A, Vannucchi A, Cross NCP.
Nat Commun. 2015 (in press).

Collaboration Partners

  • Prof. Christine Chomienne, Hopital Saint Louis, Paris, France
  • Prof. Nicholas Cross, Wessex Regional Genetics Laboratory, Salisbury, UK
  • Prof. Anthony Green, University of Cambridge, Cambridge, UK
  • Prof. Ruben Mesa, Mayo Clinic, Scottsdale/Arizona, USA
  • Prof. Heike Pahl, University Hospital Freiburg, Center for Clinical Research, Freiburg, Germany

Forschungsbereiche

Molecular Genetics of Myeloid Leukemia
Prof. Dr. Konstanze Döhner / Prof. Dr. Lars Bullinger

Molecular Genetics of Myeloproliferative Disorders
Dr. Frank Stegelmann / Prof. Dr. Konstanze Döhner

Molecular Pathogenesis and Progression of Lymphoproliferative Disorders
Prof. Dr. Stephan Stilgenbauer

Mechanisms of Leukemogenesis
PD Dr. Daniel Mertens

Molecular Pathogenesis and Prognostic Markers in Monoclonal Gammopathies
PD Dr. Christian Langer

Tumor Immunology Group (TIG)
Prof. Dr. Jochen Greiner

Haemostaseology
PD Dr. Christian Langer
 

Molecular Hematopoiesis
PD Dr. Dr. Florian Kuchenbauer

 

Emmy Noether Research Group

Dr. Jan Krönke

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