Loss-of-function but not dominant-negative intragenic IKZF1 deletions are associated with an adverse prognosis in adult BCR-ABL-negative acute lymphoblastic leukemia

Abstract

Genetic alterations of the transcription factor IKZF1 ("IKAROS") are detected in around 15-30% of cases of BCR-ABL-negative B-cell precursor acute lymphoblastic leukemia. Different types of intragenic deletions have been observed, resulting in a functionally inactivated allele ("loss-of-function") or in "dominant-negative" isoforms. The prognostic impact of these alterations especially in adult acute lymphoblastic leukemia is not well defined. We analyzed 482 well-characterized cases of adult BCR-ABL-negative B-precursor acute lymphoblastic leukemia uniformly treated in the framework of the GMALL studies and detected IKZF1 alterations in 128 cases (27%). In 20%, the IKZF1 alteration was present in a large fraction of leukemic cells ("high deletion load") while in 7% it was detected only in small subclones ("low deletion load"). Some patients showed more than one IKZF1 alteration (8%). Patients exhibiting a loss-of-function isoform with high deletion load had a shorter overall survival (OS at 5 years 28% vs. 59%; P<0.0001), also significant in a subgroup analysis of standard risk patients according to GMALL classification (OS at 5 years 37% vs. 68%; P=0.0002). Low deletion load or dominant-negative IKZF1 alterations had no prognostic impact. The results thus suggest that there is a clear distinction between loss-of-function and dominant-negative IKZF1 deletions. Affected patients should thus be monitored for minimal residual disease carefully to detect incipient relapses at an early stage and they are potential candidates for alternative or intensified treatment regimes. (clinicaltrials.gov identifiers: 00199056 and 00198991).

Trial registration: ClinicalTrials.gov NCT00199056 NCT00198991.

Copyright© 2017 Ferrata Storti Foundation.

Figures

Figure 1

Flowchart of the analysis.

Figure 2

Detection of IKZF1 deletions by RT-PCR and PCR screening. (A–C) RT-PCR ex1/8, PCR Δ4–7 and PCR Δ2–7 of the same 9 patients. (A) RT-PCR with primers in exon 1/8. Increased Ik6 expression in lanes 4–6 and increased Ik10 expression in lanes 6–8. Reduced full length isoform expression in lanes 1 and 7 is attributed to an additional deletion Δ2–3 in these 2 patients detected by another RT-PCR (see Online Supplementary Figure S2). (B) PCR Δ4–7. In lanes 1–3, Δ4–7 is present with a low deletion load; in lanes 4–6, the deletion is present with a high deletion load. Corresponding qPCR results are given below. Control band of 1257bp. (C) PCR Δ2–7 with low deletion load in lanes 3–4 and high deletion load in lanes 6–8. Control band of 1257bp. (D) Structure of the IKZF1 transcript isoforms Ik1 (full-length), Ik6 (loss of exons 4–7) and Ik10 (loss of exons 2–7). (E and F) PCR Δ4–8 and PCR Δ2–8 of the identical patients in lanes 10–17. Control band of 1229 bp. (E) PCR Δ4–8. See double bands in lanes 10 and 11. (F) PCR Δ2–8. See variant breakpoint in lane 17.

Figure 3

Prevalence of IKZF1 deletions at the time of diagnosis. (A) Frequency of all deletions as detected by PCR (Δ2–7, Δ2–8, Δ4–7, Δ4–8) and RT-PCR (exon 1/4, exon 1/8). (B) Only deletions classified as high deletion load by quantitative PCR and densitometry.

Figure 4

Overall survival (OS) depending on IKZF1 deletions. (A) OS of patients with loss-of-function IKZF1 deletions. (B) OS of patients with dominant-negative deletions. (C) OS of patients with high or low deletion load loss-of-function IKZF1 deletions. (D) OS of patients with high or low deletion load dominant-negative IKZF1 deletions.

Figure 5

Distribution of IKZF1 breakpoints and clonality of deletions. (A) Chromatogram of patient #189 showing two distinguishable clones (sequenced sense and antisense reverse complement). (B) Chromatogram of patient #395 showing oligoclonality at the breakpoint junction in both sequencing directions. (C) Distribution of breakpoints in the IKZF1 gene locus. Proximal breakpoints are shown in black, distal breakpoints in blue. There are four major breakpoint clusters within intron 1, 3, 7 and 3′UTR of IKZF1.