Non-Functional ("haploinsufficient"), but Not Dominant Negative Clonal IKZF1 Deletions Confer an Adverse Prognosis in Adult BCR-ABL-Negative Acute Lymphoblastic Leukemia
Authors: Kobitzsch Benjamin , Gökbuget Nicola , Schwartz Stefan , Reinhardt Richard , Brueggemann Monika , Viardot Andreas , Wäsch Ralph , Starck Michael , Thiel Eckhard , Hoelzer Dieter , Burmeister Thomas 
Published: October 2019

Background: IKAROS (IKZF1) is a key transcription factor for B-cell development. The IKZF1 protein exerts its functions as a dimer (homodimer or heterodimer with other IKAROS family members). Various IKZF1 intragenic deletions have been identified as recurrent aberrations in acute lymphoblastic leukemia: those resulting in the loss of transcription of one allele or at least loss of the dimerization domain ("haploinsufficient", "non-functional") and those involving only the DNA-binding domain but retaining the dimerization domain of the transcription factor, leading to so-called "dominant negative" isoforms.

Several studies in pediatric ALL have confirmed the negative prognostic impact of IKZF1 alterations although there is still no consensus if they are independent risk factors in a multivariate analysis. The situation in adult ALL is less clear and few larger studies have addressed this issue, in particular the prognostic impact of different types of IKZF1 alterations.

Objectives: We investigated archived patient samples obtained in the context of the German Multicenter ALL Study Group (GMALL) therapy studies for different IKZF1 alterations to assess their prognostic implications and their molecular pattern.

Methods: We used RT-PCR to determine the presence of aberrant IKZF1 transcript variants and additionally investigated all patients by DNA-based PCRs for the IKZF1 aberrations D4-7, D2-7, D4-8, D2-8. Rare transcript variants, such as D2-3, D2, D5-7 were molecularly characterized on the DNA level by specific PCRs. IKZF1 aberrations were quantified using gel densitometry and/or quantitative real time PCR such that clonal and subclonal aberrations could be distinguished. All chromosomal breakpoints were molecularly characterized.

Results: In total, 482 B cell precursor ALL patients aged between 15 and 65 years were included in the analysis. Samples were obtained at primary diagnosis and tested BCR-ABL -negative. Other molecular aberrations were tested according to the GMALL standards: 39 MLL-AF4-positive, 4 MLL-ENL, 1 MLL-AF9, 30 TCF3-PBX1, 3 ETV6-RUNX1. The immunophenotypes were the following: 424 pre B/common, 58 pro B.

One hundred and twenty-eight out of 482 (27%) patients carried at least one IKZF1 deletion, with 37 patients carrying more than one deletion. Altogether there were 175 IKZF1 deletions, 71 cases of D4-7, 47 with D2-7, 26 with D4-8, 19 with D2-3, 10 with D2-8, and 1 with D5-7 and D2 each. Taken together 56 patients (12%) carried only non-functional mutations while 50 (10%) had dominant-negative mutations only. There was a group of 22 patients with both types of mutations (5%).

Evaluating the prognostic impact, we considered the effect of non-functional and dominant-negative mutations separately. Patients with a non-functional IKZF1 mutation had a reduced overall survival (OS at 5 ys 37% vs 59% in IKZF1-unmutated patients, N=78/404, p=0.001) while dominant-negative mutations had no effect on OS (OS at 5 ys 54% compared to 56%, N=72/410, p=0.95). However, solely present subclonal non-functional mutations did not affect survival (OS at 5 ys 59% compared to 59%, N=23/404). In the dominant-negative group, clonality had no influence on OS (p=0.56, N=45/73/351). Taken together, patients with clonal non-functional IKZF1 had a significantly worse OS compared to those without (0.28 vs 0.59, N=53/427, p<.0001). This was true for patients with standard risk as defined by GMALL risk stratification (0.37 vs 0.68, N=24/243, p.0002). High risk patients also tended to do worse, which however did not reach significance (0.26 vs 0.46, N=30/184, p=.06).

With regard to molecular analysis 193 chromosomal fusion sites were characterized. In the great majority (>95%) putative cryptic recombination sites were identified in the near vicinity thus underlining the causal role of the VDJ recombination machinery in the generation of these aberrations.

Conclusions: We conducted a detailed and large-scale molecular analysis of intragenic IKZF1 deletions and identified clonal non-functional IKZF1 deletions as adverse prognostic factor in adult and adolescent B cell precursor ALL patients treated according to the GMALL protocols. Subclonal, or dominant negative IKZF1 mutations had no prognostic effect. It remains open for further analysis whether clonal non-functional IKZF1 deletions represent an independent prognostic factor in MRD-based risk stratification.

Disclosures

Wäsch: German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.