A. Kohlmann

Severe congenital neutropenia (CN) is a rare inherited disorder of hematopoiesis with a approx. 20% risk to evolve to acute myeloid leukemia. We investigated the patterns of acquisition of leukemia-associated-mutations in 31 CN patients developing leukemia using DNA deep sequencing. We found that 20 of the 31 patients (64.5%) demonstrated mutations within RUNX1. Of the 20 patients with RUNX1 mutations, 19 harboured CN-associated mutations: ELANE (11), ELANE+GFI1 (1), WAS (3), HAX1 (3) and G6PT (SLC37A4) (1). The majority of patients with RUNX1 mutations (75%) harboured cooperating acquired CSF3R mutations. Other leukemia-associated mutations in the patients with RUNX1 mutations were found infrequently: FLT3-ITD (2), EP300 (2), CBL (1) and SUZ12 (1). Ten patients with RUNX1 mutations revealed monosomy 7, 6 trisomy 21, 2 monosomy 7 plus trisomy 21. Sequential analysis of the time prior to overt leukemia revealed that RUNX1 mutation is a late event whereas CSF3R mutation is rather a earlier event in leukemogenic transformation. To evaluate whether RUNX1 and CSF3R mutations are co-segregated in the same leukemia-initiating clone, we performed CFU assay of AML blasts and bone marrow cells isolated 6 years before onset of leukemia. We analysed affected genes in each CFU by Sanger sequencing and ARMS-PCR. We found that in 38 out of 44 colonies mutations in both RUNX1 and CSF3R were presented. The high frequency of RUNX1 and CSF3R mutations in CN patients who developed leukemia suggests a unique molecular pathway of leukemia development. The detection of RUNX1 mutations in combination with CSF3R mutations could be a useful marker for identifying CN patients progressing to leukemia or MDS.

Rearrangements of the MLL gene occur in both acute lymphoblastic and acute myeloid leukemias (ALL, AML). This study addressed the global gene expression pattern of these two leukemia subtypes with respect to common deregulated pathways and lineage-associated differences. We analyzed 73 t(11q23)/MLL leukemias in comparison to 290 other acute leukemias and demonstrate that 11q23 leukemias combined are characterized by a common specific gene expression signature. Additionally, in unsupervised and supervised data analysis algorithms, ALL and AML cases with t(11q23) segregate according to the lineage they are derived from, that is, myeloid or lymphoid, respectively. This segregation can be explained by a highly differing transcriptional program. Through the use of novel biological network analyses, essential regulators of early B cell development, PAX5 and EBF, were shown to be associated with a clear B-lineage commitment in lymphoblastic t(11q23)/MLL leukemias. Also, the influence of the different MLL translocation partners on the transcriptional program was directly assessed. Interestingly, gene expression profiling did not reveal a clear distinct pattern associated with one of the analyzed partner genes. Taken together, the identified molecular expression pattern of MLL fusion gene samples and biological networks revealed new insights into the aberrant transcriptional program in 11q23/MLL leukemias.

Acute myeloid leukemia (AML) is a heterogeneous group of disorders that can be discriminated by morphology, immunophenotyping or more recently by cytogenetic and molecular techniques. By cytogenetics two major groups of AML can be discriminated: One group with detectable chromosomal aberrations accounting for approximately 52 % of all de novo AML and the second group without cytogenetically detectable karyotype abnormalities. In the first group two major subtypes can be further distinguished. The first group comprises AML with balanced aberrations mainly consisting in t(8;21), t(15;17) and inv(16). The second group comprizes cases with unbalanced aberrations including particularly 5q-, 17q- -5 and AML with complex karyotypes. AMLs with balanced aberrations have a good prognosis with long term survival being achieved in approximately 60 %-80 % of cases. AMLs with non-balanced aberrations on the other hand have a poor prognosis with only 10 %-15 % long-term survivors. AMLs with no detectable abnormalities or other cytogenetic aberrations comprise a group with an intermediate prognosis in which long term survival is achieved in approximately 25 %-30 % of cases. Biologically, AMLs with balanced aberrations regularly involve the deregulation of transcription factors resulting in an impairment of cell differentiation and proliferation. AMLs with unbalanced aberrations are mostly characterized by a loss of genetic material resulting in an alteration of cell cycle control and DNA repair. A new view on the biology of AML has recently been made possible through the analysis of gene expression profiles. This technique is still under investigation. First results, however, already show that gene expression patterns have a high diagnostic potential and allow to detect biology subgroups with a high accuracy. Furthermore, by this technique pathways can be identified that are altered in the leukemic process. Gene expression profiling therefore opens a new and exciting perspective in leukemia biology and therapy that may have substantial impact on the improvement of diagnosis and more importantly may guide therapeutic strategies.