Deletions of the Transcription Factor Ikaros in Myeloproliferative Neoplasms at Transformation to Acute Myeloid Leukemia.

Abstract 435

Transformation to acute myeloid leukemia (AML) is a major complication of myeloproliferative neoplasms (MPNs). Although clinical prognostic factors have been defined such as age, severe anemia, elevated white blood cell count with a high proportion of immature myeloid cells and a high number of circulating CD34+ cells, there is little knowledge of the genetic changes leading to leukemic transformation in MPN. To gain insight into these genetic changes, we studied 12 patients with post-MPN leukemia using Affymetrix SNP 6.0 microarrays. Acquired genetic lesions were detected in 10 patients. Beyond well described frequent events like 9pUDP, chromosome 7p deletions (del7p) emerged as a novel recurrent defect in this patient group. Mapping of the minimal deleted region of del7p by combining microarray, copy number and loss of heterozygosity analysis resulted in a minimal deleted region of 0.25 Mb in size, restricted to the IKZF1 gene. IKZF1 is encoding the transcription factor Ikaros, which is known to have a pleiotropic function in the regulation of hematopoiesis, as well as crucial involvement in hematopoietic malignancies, primarily of the lymphoid lineages. Based on the observation of 16.7% (2 out of 12) of post-MPN leukemia patients carrying an IKZF1 deletion, we further screened a chronic phase MPN patient cohort from Vienna for the defect and found 1 out of 235 informative patients carrying an IKZF1 deletion. Thus, deletion of IKZF1 exhibited statistically significant clustering with post-MPN leukemia when compared to chronic phase MPN (P=0.0063). To validate these results in an independent patient cohort, we screened 20 post-MPN leukemic and 156 chronic phase MPN patients from Pavia for loss of IKZF1, and found 4 and 1 positive patients, respectively (P=0.0006). Taking these two cohorts together, IKZF1 deletions could be found in 0.5% of chronic MPN patients and 18.8% of post-MPN leukemic patients, showing statistically significant association with post-MPN leukemia (P<0.0001). We could define haploinsufficiency as mechanism of IKZF1 tumor suppressor inactivation, since IKZF1 exon sequencing of del7p patients did not show any mutations on the non-deleted allele of IKZF1 and IKZF1 mRNA could be detected in del7p patients. In order to characterize the role of IKZF1 deletion in clonal expansion, we monitored oncogenic mutation and deletion burdens in granulocytes of two del7p patients in serial samples over time. In both patients, one being positive for JAK2-V617F and the other for MPL-W515L, increased IKZF1 deletion burden was detected as a late event after the acquisition of oncogenic mutations. We also examined the hematopoietic progenitors of a del7p patient by genotyping individual BFU-E and CFU-GM colonies for defects detected in granulocytes. IKZF1 deletion was found to be a late defect in the clonal evolution of MPN, occurring after oncogenic mutations such as JAK2-V617F and chromosomal aberrations such as del13q. In murine hematopoietic progenitor cells, sh-RNA induced deficiency for Ikzf1 resulted in cytokine hypersensitivity in vitro. Based on these data we can conclude that IKZF1 deletion does not induce arrest in myeloid differentiation, it is a late genetic event in the clonal evolution of MPN and represents an important step in the leukemic transformation of a subpopulation of MPN patients.