Complete or Partial Deletions of IKZF1 Occur In 28% of Blast Crisis CML and Are the Only Recurrent Submicroscopic Alteration Associated with Disease Progression: An Analysis of 43 Cases Using High-Resolution Genome-Wide Copy Number DNA Arrays and Molecular Assays
Authors: Grossmann Vera , Zenger Melanie , Kohlmann Alexander , Schnittger Susanne , Kern Wolfgang , Mueller Martin C , Hochhaus Andreas , Haferlach Torsten , Haferlach Claudia 
Published: October 2019

Abstract
Abstract 4172
In advanced CML, approximately 80% of patients develop additional non-random cytogenetic abnormalities in Ph+ cells. However, little is known about molecular mutations leading to disease progression. Only limited changes that occur in the clonal evolution of the chronic phase to blast crisis, both in gene expression patterns and DNA copy number alterations, have been described. Novel technologies allow comprehensive detection of additional molecular genomic aberrations. To identify recurrent submicroscopic gains and losses, as well as copy neutral loss of heterozygocity (CN-LOH), we employed whole-genome 2.7M arrays (Affymetrix, Santa Clara, CA) to study 22 cases with blast crisis CML (n=14 myeloid; n=6 lymphoblastic; n=2 not specified) and 18 cases of untreated patients in chronic phase, matched for age and gender. In four cases, the analyses were performed on paired samples. We first investigated the occurrence of CN-LOH and observed in two patients with blast crisis a recurrent CN-LOH for 1p (both ranging to the telomere as being typical for acquired CN-LOH). Secondly, all aberrations identified by chromosome banding analysis were also detected by microarray analysis. The microarray used provided copy number estimates for 2.7 million markers, thus, even very small gains and losses were identified. As such, 39 submicroscopic variations were found, which were not detectable by chromosome banding analysis. Aberrations occurred predominantly in blast crisis CML: number of alterations in chronic phase vs. blast crisis, n=4 vs. n=9 gains, n=7 vs. n=16 losses, and n=0 vs. n=3 CN-LOHs; e.g. gains for AKT2, MLLT4, ELN, and losses of CBFB, MLLT10 or MYC, respectively. Besides microdeletions in the breakpoint region of BCR (n=5) and ABL1 (n=5), the only recurrent submicroscopic alteration was a deletion confined to a subset of exons from the IKZF1 gene, located on the short arm of chromosome 7 (7p12.2), and was observed in three patients in the cohort of blast crisis. In addition, in three patients of blast crisis cohort a complete or partial monosomy 7p had been identified. IKZF1 encodes for a transcription factor which is an important regulator of lymphoid cell differentiation. To further investigate the role of IKZF1 deletions, additional 21 patients with blast crisis CML (n=14 myeloid; n=4 lymphoblastic; n=3 not specified) were screened. In this independent cohort, two patients showed monosomy 7 in chromosome banding analyses and in four patients deletions of internal IKZF1 exons were identified by PCR using specific primer pairs for the common deletions in the IKZF1 gene as described by Iacobucci et al. (Blood, 114:2159-67, 2009). In total, for both cohorts of blast crisis CML, in 28% (12/43) of cases either complete loss of IKZF1 due to cytogenetic aberrations leading to loss of 7p or intragenic IKZF1 deletions were observed. Of note, these aberrations never occurred concomitantly. In detail, a complete deletion of IKZF1 was detected in 5 patients due to a derivative chromosome 7 or a monosomy 7: 2/5 cases with lymphoblastic blast crisis and 2/5 cases with myeloid blast crisis (1/5 case not specified). A partial deletion of IKZF1 was found in 7 cases: 5/7 cases with partial IKZF1 deletions presented with lymphoblastic blast crisis and 1/7 case with myeloid blast crisis (1/7 case data not specified). In detail, in five cases the common partial deletion of exons 4–7 was detected, resulting in a dominant negative isoform, one case was harboring the exons 2–7 deletion, and another case was carrying both of the two mutations. In three of the seven cases with an intragenic IKZF1 deletion, matched DNA from the first diagnosis and chronic state was available. The respective IKZF1 deletions were not detectable in these specimens indicating that IKZF1 deletions developed during disease progression. Moreover, in one patient we performed a serial analysis of 8 time points across different disease states (interval from diagnosis to most recent investigation: 2.2 years). Here, exons 2–7 deletion was only detected at the stage of blast crisis. In addition, in 4 of 7 cases with a partial deletion of IKZF1 a point mutation in BCR-ABL1 (E279K, T315I, F317L, Q252H) was detected. In conclusion, these results underline a pathogenetic contribution of IKZF1 deletions to disease progression in CML and therefore constitute an important progression marker to blast crisis.

Disclosures:
Grossmann: MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding.