What is the relevance of Ikaros gene deletions as a prognostic marker in pediatric Philadelphia-negative B-cell precursor acute lymphoblastic leukemia?

Abstract

New prognostic markers are needed for upfront identification of patients with acute lymphocytic leukemia with a high risk of relapse or who are not likely to respond to the most aggressive chemotherapy. We focused our analysis on Ikaros (IKZF1) gene deletions in a homogeneous cohort of 410 pediatric patients with Philadelphia chromosome-negative, B-cell precursor acute lymphoblastic leukemia enrolled in Italy into the AIEOP-BFM ALL2000 study. We confirm their reported poor prognostic value, although the associated event-free survival was relatively high (approximately 70%). The difference in the cumulative incidence of relapse between patients positive or not for IKZF1 deletions was not marked: 24.2% (5.9) versus 13.1% (1.8) overall and 23.9% (6.6) versus 16.5% (2.5) in the intermediate-risk subgroup. In line with this, IKZF1 deletions were not an independent prognostic factor for the hazard of relapse. Most IKZF1-deleted cases stratified in the high-risk group relapsed, suggesting that once identified, patients with these deletions require an alternative treatment. In conclusion, the need of and benefit from introducing IKZF1 deletions as an additional stratification marker for patients with Philadelphia-negative B-cell precursor acute lymphoblastic leukemia remain questionable.

Introduction

In the AIEOP-BFM ALL2000 study of patients with acute lymphocytic leukemia (ALL), risk-adapted treatment, with the risk group stratification largely based on minimal residual disease monitoring as a measure of early response to therapy, led to a cure rate of over 80%. However, relapse is still the most frequent adverse event, occurring mainly in the largest and heterogeneous subgroup of non-high-risk patients.1 This emphasizes the need for new prognostic markers for upfront identification of patients with a high risk of relapse or of patients who are likely not to respond to the most aggressive chemotherapy.

Recently, genomic abnormalities of Cytokine receptor like factor 2 (CRLF2) and Ikaros (IKZF1) genes have been reported, not only in patients with Down syndrome or Philadelphia chromosome-positive disease, but also in patients without known chromosomal aberrations, although with different incidences.2–6 Indeed, IKZF1 deletions are rare in T-ALL (about 5%),7 very frequent in Philadelphia chromosome-positive ALL (about 80%)8 and frequent in patients with Down syndrome and ALL (reported incidence, 35%).9 The most frequent IKZF1 alterations identified in ALL patients were deletions encompassing the whole gene or involving only some exons.5,7–15 All these deletions cause the loss of IKZF1 activity.16

IKZF1 deletions were shown to be related to poor outcome in pediatric ALL patients,5,7–15 but their prognostic impact could be different in specific subgroups.

The potential benefit of the early identification of a new prognostic marker should be assessed within the subgroup of patients who are not at high risk due to other features and evaluated in a homogeneous cohort of cases. A recent study by Dorge et al.7 showed that patients with an IKZF1 deletion had an inferior outcome compared to those who did not have a deletion and it was, therefore, concluded that IKZF1 deletions may be a strong candidate for changing the stratification strategy. However, although the outcome of the patients with deletions was inferior to that of patients without deletions, it was still relatively favorable, since patients with deletions had a 5-year event-free survival of about 70%. Thus, IKZF1 deletions, although potentially useful for stratification, are not associated with a really poor prognosis. The aim of our study was to assess the prognostic value of IKZF1 deletions in a cohort of patients whose stratification and treatment17 were very similar to those in the study by Dorge et al.7

Our investigation was focused on a subgroup of ALL patients for whom a change in risk stratification could be most relevant in clinical practice. We, therefore, screened a cohort of 410 non-Down syndrome patients with non-T, Philadelphia chromosome-negative, B-cell precursor ALL enrolled in the AIEOP-BFM ALL2000 study in Italy and recently analyzed for CRLF2 alterations for evaluation of the prognostic role of IKZF1.18

Design and Methods

Patients

The study cohort was constituted by 410 non-Down syndrome, non-T, Philadelphia chromosome-negative, B-cell precursor ALL patients consecutively enrolled in the AIEOP-BFM ALL2000 study in AIEOP Centers from February 2003 to July 2005, who were included in the previous study on CRLF2 alterations and for whom DNA was still available.18 Data on recurrent genomic aberrations were available for most patients.19P2RY8-CRLF2 rearrangement was tested by reverse transcriptase polymerase chain reaction analysis in 372 (90.7%) patients.18

As shown in Online Supplementary Table S1, there was an unbalance toward more unfavorable features with respect to treatment response (prednisone-poor response and high minimal residual disease levels) in the non-investigated group. Despite this difference, the event-free survival curve of the analyzed patients was not different from that of the not analyzed patients diagnosed in AIEOP centers in the study period (2003–2005) (Online Supplementary Figure S1).

The project was approved by the AIEOP ALL Scientific Committee.

Risk group definitions and treatment outlines have already been reported17 and are summarized in the Online Supplementary Material.

DNA copy number variations

IKZF1 deletions, together with deletions in other genes (CDKN2A/B, PAX5, ETV6, BTG1, RB1 and EBF1) were investigated by multiplex ligation-dependent probe amplification (MLPA) using the Salsa MLPA P335-A3 ALL-IKZF1 kit (MRC-Holland, Amsterdam, the Netherlands) according to the manufacturer’s instructions.7,18,20 Patients positive for IKZF1 deletions were further analyzed by the more specific Salsa MLPA P202-A1 IKZF1 kit (MRC-Holland, Amsterdam, the Netherlands) to confirm and better define the extension of the alteration.

Samples from pediatric ALL patients in complete remission were used as wild-type controls.

Statistical analysis

Event-free survival time was calculated from the date of diagnosis to the date of an event, which was resistance, relapse, death or second neoplasm, whichever occurred first. Patients were censored at last follow-up if no events occurred. Event-free survival was estimated according to Kaplan-Meier, and compared using the log-rank test. The cumulative incidence of relapse at 5 years was estimated by adjusting for competing risks of other events and compared using Gray’s test.18 Multivariate Cox models for event-free survival and cause-specific hazard of relapse were applied to assess, with the Wald test, the impact of IKZF1 deletions, after accounting for the risk group, age and white blood cell count at diagnosis, and the presence of P2RY8-CRLF2 aberration. The Cox model was also applied for each variable separately (univariate analysis).

Results

IKZF1 deletions at diagnosis

IKZF1 deletions were detected in 54/410 cases (13.2%), in keeping with incidence data reported in the literature.3,13 In 25 cases (6.1%) the deletion was intra-genic, involving only some exons of the IKZF1 gene, while in 29 cases (7.1%) the deletion encompassed the whole IKZF1 gene. In detail, we identified nine cases with lack of exons 4–7 (Δ4–7), three cases with Δ2–8, two cases for each of the following deletions: Δ2–7, Δ4–8, Δ1–3, Δ2–3 and single cases for: Δ1–4, Δ4–5, Δ4–6, Δ6–8 and Δ2 (exon numbering according to Iacobucci et al.6) (Online Supplementary Table S2).

Table 1.Clinical features of the study cohort patients positive or negative for an IKZF1 deletion.

The clinical characteristics of the patients are presented in Table 1. The major difference regards treatment response, with lower proportions of patients with a prednisone-poor response among the IKFZ1-deleted group and of patients with high minimal residual disease levels among the non-IKFZ1-deleted group. The percentages of patients allocated to the high-risk group were, however, the same (7%) for patients with or without the IKFZ1 deletion. The relative incidence of major deletion subgroups did not vary according to final risk group assignment (Online Supplementary Table S2). Among the patients with an IKZF1 deletion, one was positive for the chromosomal translocation t(12;21) and none was positive for t(4;11). Only three IKZF1-deleted cases also carried a P2RY8-CRLF2 fusion (Table 1). The differences in the incidence of double-deleted cases reported in this and other studies2,3,5,7,11 is probably due to the relatively low number of patients in all studies.

Using the MLPA technique we further analyzed the presence of copy number variations of other genes frequently deleted in B-cell precursor ALL and known to be involved in lymphoid development (PAX5, ETV6, EBF1) or in cell cycle regulation (CDKN2A/B, BTG1, RB1).10,20–22 We did not find statistically significant differences in the incidence of these genetic alterations in children positive or negative for IKZF1 deletions (Online Supplementary Table S3). Most of these genetic alterations occurred simultaneously in the same patients and are described in detail in Online Supplementary Table S2. Twenty-five of the 54 patients with an IKZF1 deletion, and in particular seven of the nine carrying a Δ4–7 deletion, were negative for any additional tested alterations (Online Supplementary Table S2), although copy number variations of exons not detected by MLPA assays cannot be excluded. Moreover, aberrations present in less than 20–50% of cells would not have been detected because of the limited sensitivity of the MLPA assay.

Prognostic impact of IKZF1 deletions

Compared to patients without a deletion of IKZF1, those with a deletion of part or all of this gene had an inferior event-free survival [70.2% (6.2) versus 85.2% (1.9) at 5 years, P=0.007] and a significantly higher cumulative incidence of relapse [24.2% (5.9) versus 13.1% (1.8) at 5 years, P= 0.049] (Figure 1A-B). The corresponding survival figures at 5 years were 87.0%(4.6) versus 93.0%(1.4) (P-value=0.10).

These data are in accordance with those from other studies reported in the literature, in particular with those recently published by Dorge et al. who analyzed ALL patients enrolled in the AIEOP-BFM ALL2000 study in Germany [event-free survival 69% (5) versus 85% (1), P=<0.001 and cumulative incidence of relapse 21% (4) versus 10% (1), P=0.001].7

Figure 1.Association of IKZF1 deletions and treatment outcome. (A) Event-free survival and (B) cumulative incidence of relapse of study cohort patients according to the presence or absence of IKZF1 deletions. (C) Event-free survival and (D) cumulative incidence of relapse of intermediate risk patients according to the presence or absence of IKZF1 deletions.

The negative prognostic impact of IKZF1 deletions was retained, although without statistical significance, when the favorable factor t(12;21) was excluded from the analysis (Online Supplementary Figure S2A,B) and also when patients with hyperdiploidy were excluded (Online Supplementary Figure S2C,D). Patients positive for the Δ4–7 deletion, predicted to encode a dominant-negative IKZF1 isoform, did not show a worse outcome (3/9 relapsed) (Online Supplementary Table S2). We also analyzed the impact of IKZF1 deletions alone or in combination with additional copy number abnormalities. Interestingly, only three out of 25 patients positive for the IKZF1 deletion only relapsed compared with 10/28 who had additional alterations, pointing to a poor outcome when a major genetic instability was observed. Specifically, all three cases positive for both IKZF1 deletions and the P2RY8-CRLF2 fusion relapsed (Online Supplementary Table S2), but the limited numbers do not allow any conclusions to be drawn on a possible synergic effect of IKZF1 alterations with other coexistent abnormalities.

Table 2.Results of the univariate and multivariate analyses. Cox model on event-free survival (hazard of first event among resistance, relapse, death, second malignant neoplasm) and the hazard of relapse in 410 patients.

The Cox model analysis was performed on the 410 patients to assess whether, after adjusting for other relevant risk factors, IKZF1 deletions retained a prognostic impact on event-free survival (Table 2A) or on the specific hazard of relapse (Table 2B). IKZF1 alterations were significantly related to a higher rate of events (hazard ratio for event-free survival of 1.87; 95% CI 1.05–3.32, P=0.03) and, although not statistically significantly, to a higher rate of relapse (hazard ratio 1.7; 95% CI 0.9–3.18, P=0.1). More precisely, two deaths in induction and one death in complete clinical remission (total n=3) occurred in the 54 IKFZ1-deleted patients compared to three deaths in induction and two in complete clinical remission and one second malignant neoplasm (total n=6) in the 356 non-IKFZ1-deleted patients. These events contributed to the statistical significance of the difference in the event-free survival.

In both Cox model analyses, the P2RY8-CRLF2 aberration and risk group were significantly associated with outcome. Of note, when individually analyzed, the IKFZ1 deletion had a statistically significant effect on event-free survival and relapse, in keeping with results in Figure 1A,B.

We further analyzed the prognostic value of IKZF1 deletions within the subgroups according to protocol stratification. IKZF1 deletions were less frequent within the standard-risk group, being found in 8 out 117 standard-risk patients (6.8%), 42 out of 264 intermediate-risk patients (15.9%) and 4 out of 29 high-risk patients (13.8%) (Table 1). Interestingly, none of the eight IKZF1 deletion-positive standard-risk patients relapsed whereas 10/42 cases (23.8%) stratified in the intermediate-risk group and three out of four cases in the high-risk group did so. In particular, in the largest risk subgroup, which was the intermediate risk subgroup, IKZF1 deletion-positive patients had an inferior event-free survival and a higher cumulative incidence of relapse compared to the IKZF1 deletion-negative patients, but the differences did not reach statistical significance [event-free survival: 69.0%(7.2) versus 82.2%(2.6), P=0.052; cumulative incidence of relapse: 23.9%(6.6) versus 16.5%(2.5), P=0.30] (Figure 1C-D).

Discussion

Previous studies12–14 showed that the presence of IKZF1 deletions is a risk factor in childhood ALL and this finding was recently confirmed by Dorge et al.7 in the framework of a BFM treatment strategy also for patients at so-called intermediate risk. Our findings are substantially in keeping with those reported by Dorge et al.7, and yet the value of including IKZF1 deletions as a new marker for risk stratification is challenged by our results.

Indeed, overall event-free survival for patients with IKZF1 deletions, after excluding the confounding effects of Down syndrome, T-immunophenotype and Philadelphia chromosome-positive patients, was around 70% at 5 years also in our experience. The three patients with IKZF1 deletions who were at high risk and relapsed had poor response to treatment (high minimal residual disease levels) and accordingly were all eligible for transplantation, thus identification of IKZF1 deletions would not have contributed to a better stratification. In the intermediate risk group, with a 5-year event-free survival of 70%, treatment intensification could be justified to improve results. In our context, the recent AIEOP-BFM ALL 2009 study, with a more intensive use of L-asparaginase, might already provide a benefit that reduces the impact of an IKZF1 deletion. This is especially true if we consider that in our study cohort the difference in the cumulative incidence of relapse was not so marked, being approximately 7% in the intermediate risk subgroup and 11% overall. This, as well as the lower number of events in the multivariate analysis, may explain why the presence of IKZF1 deletions is an independent prognostic factor for event-free survival but not for the hazard of relapse alone.

In conclusion, based on our data, the suitability of IKZF1 deletions as an additional stratification marker for Philadelphia chromosome-negative, B-cell precursor ALL patients remains questionable, at least until new target therapy becomes available.

Acknowledgments

The authors would like to thank Simona Songia, Lilia Corral, Eugenia Mella, Tiziana Villa (Monza), Elena Seganfreddo and Katia Polato (Padova) for AIEOP minimal residual disease monitoring and all medical doctors of the AIEOP centers. This work was supported by grants from: Fondazione Tettamanti (Monza), Fondazione Città della Speranza (Padova), Associazione Italiana Ricerca sul Cancro (AIRC) (to GB, AB, MGV, GteK and GC), MIUR (to GB and AB), Fondazione Cariplo (to AB, GC and GteK), and CARIPARO project of excellence (to GteK). This work was (partly) funded by the European Commission (FP7) under the contract ENCCA (NoE-2011-261474).

Footnotes

• The online version of this article has a Supplementary Appendix.
• Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.

• Received October 10, 2012.
• Accepted March 25, 2013.