IKZF1 Deletion Is An Independent Predictor of Outcome In Pediatric Acute Lymphoblastic Leukemia Treated According to the ALL-BFM 2000 Protocol

Abstract 409

Alteration of the IKZF1 gene – encoding the transcription factor IKAROS, a key player in lymphoid development and tumor suppression – has been reported to be associated with a poor outcome in pediatric precursor B-cell ALL, especially in cases positive for the BCR-ABL1 fusion gene. In order to assess the prognostic value of IKZF1 deletions in a representative cohort of pediatric ALL patients treated on the German ALL-BFM 2000 study protocol, we screened 409 patients by applying a multiplex ligation-dependent probe amplification (MLPA) assay covering all eight IKZF1 exons (P335-A3 ALL-IKZF1 probemix; MRC-Holland, Amsterdam, The Netherlands). In ALL-BFM 2000, risk group stratification (standard, SR; intermediate, MR; high, HR) was based on minimal residual disease (MRD) analysis at two different time points (TP) and required two MRD targets with sensitivities of ≤10⁻⁴ (Flohr et al. Leukemia 2008). SR patients were MRD-negative on treatment days 33 (TP1) and 78 (TP2). HR patients had residual disease (≥10⁻³) at TP2. MRD MR patients had positive MRD detection at either one and or both time points but at a level of <10⁻³ at TP2. Although MRD-based stratification criteria were introduced in ALL-BFM 2000, established high-risk parameters were also retained: patients with prednisone poor-response or ≥5% leukemic blasts in the bone marrow on day 33 or positivity for a t(9;22) or t(4;11) or their molecular equivalents (BCR/ABL1 or MLL/AF4 fusion RNA) were stratified into the high-risk group independent of their MRD results. First results on MRD and outcome were published earlier (Conter et al. Blood 2010). Out of the 409 patients analyzed in our study, 46 (11%) displayed a deletion in at least one of the eight IKZF1 exons. Forty-three out of the 46 cases showed heterozygous deletions, while 3 patients displayed homozygous loss of IKZF1 exons. MLPA results of 11 patients were validated with results derived from copy number/LOH analyses using Affymetrix SNP 6.0 arrays. IKZF1 deletion was significantly more common in precursor B compared to T cell ALL (13% vs. 4%, P = 0.03) and less frequent in TEL/AML1-positive ALL (3% vs. 13%, P = 0.004). Out of 11 BCR/ABL1-positive samples, only two were characterized by an IKZF1 deletion. Forty-four patients with IKZF1-deleted ALL had results of MRD analyses available for both informative time points (day 33 after induction and day 78 after consolidation). Despite a trend towards increasing incidence of IKZF1 deletion in patients with slow response, the distribution of IKZF1-deleted ALL patients over the risk groups was not significantly different from non-deleted ALL (SR: 40.9 vs. 41.9; MR: 45.5 vs. 52.3; HR: 13.6 vs. 5.7%; P = 0.153). Regarding treatment outcome, patients with an IKZF1 deletion had a significantly lower 5-year event-free survival (EFS) compared to non-deleted patients (0.78±0.06 vs. 0.86±0.02; P = 0.015). This result was due to a higher cumulative incidence of relapses in IKZF1-deleted patients (0.16±0.05 vs. 0.10±0.02; P = 0.031). In multivariate Cox regression analyses including known prognostic variables (gender, immunophenotype, WBC count at diagnosis, TEL/AML1 status, risk group criteria of ALL-BFM 2000), IKZF1 deletion conferred a risk of 2.16 (95% confidence interval 1.14 – 4.10; P = 0.018) for an event when compared to non-deleted patients. We conclude that IKZF1 deletion is an independent predictor of treatment outcome for patients enrolled on the ALL-BFM 2000 protocol and represents a candidate marker to be integrated in future algorithms for early risk stratification in pediatric ALL.