Mole­cu­lar Gene­tics of Mye­lo­pro­li­fe­ra­tive Neo­plasms

Pro­ject Lea­der

Con­tact/Address

Depart­ment of Inter­nal Medi­cine III
Uni­ver­sity Hos­pi­tal of Ulm
Albert-​Einstein-Allee 23
89081 Ulm
Ger­many
Phone: +49-(0)731-500-45521
Fax: +49-(0)731-500-45525

Tech­ni­ci­ans

Susanne Kuhn

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

Repre­sen­ta­tive Publi­ca­ti­ons

Repre­sen­ta­tive publi­ca­ti­ons can be found in the entire list of publi­ca­ti­ons.

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