Chromosomal Instability Causes Genetic and Clonal Heterogeneity in Myeloproliferative Neoplasms and Is Not Restricted to JAK2-V617F Positive Cells

A number of oncogenic mutations have been identified in myeloproliferative neoplasms (MPN) in the past few years. Among these JAK2-V617F is most frequent, followed by mutations of the thrombopoietin receptor MPL and JAK2 exon 12. In addition, cytogenetic lesions occur frequently in MPN detected either at diagnosis or later in the course of the disease. To explore the genetic aberrations present in MPN patients, we performed microarray genotyping using Affymetrix SNP 6.0 arrays in a series of 71 MPN patients with variable presence of JAK2 and MPL mutations. More than half of the analyzed patients exhibited loss of heterozygosity (LOH) in at least one chromosomal region. Complex karyotypes with two and more regions with LOH were detected in 18 patients. Uniparental disomy (UPD) on chromosomes 9p, 1p, 11q, 14q and 17q represented the largest proportion of LOH detected followed by deletions on chromosome 13q, 20q, and 12p. All patients with UPD on chromosome 1p were homozygous for the MPL-W515L mutation. We observed frequent aberrations of chromosome 7 including monosomy, deletions on 7p and 7q, and UPD of 7q. Using microsatellite PCR, we validated the microarray findings and further determined the frequency of these aberrations in a total of 367 MPN patients. Multiple occurrences of individual chromosomal lesions allowed us to define the minimal genomic regions involved in deletions or UPDs. The sizes of the common deleted regions (CDRs) were variable ranging from 9 mega base pairs (Mb) to 0.5 Mb. The CDR on chromosome 7p included only the IKZF1 and FIGNL1 genes previously shown to associate with leukemic transformation. To determine the clonal composition of the hematopoietic progenitor pool of patients with complex karyotypes we genotyped individual BFU-E and CFU-GM colonies in a series of 27 patients. We observed a remarkable clonal heterogeneity at the progenitor cell level. Using four clonal markers we defined 9 different types of clonal structures. In a set of patients, JAK2-V617F or MPL-W515L mutations occurred before the acquisition of chromosomal deletions. Other patients acquired deletions before the acquisition of JAK2-V617F. In summary, our results show that somatic mutations in MPN are not acquired in a predetermined order as seen in other malignancies, but occur randomly. The chromosomal instability in MPN is not caused by JAK2-V617F exclusively, since many patients show aberrations outside of the JAK2-V617F positive clone. Heterogeneity of somatic mutations in MPN leads to high clonal variability within the progenitor pool potentially affecting therapeutic outcome. Thus, targeting JAK2-V617F alone may not lead to restoration of polyclonal hematopoiesis. An individualized therapeutic approach and/or combination therapy might be necessary to achieve clonal remission in MPN.