Relative Expression of Ikaros Isoforms Has a Prognostic Impact in Phildelphia-Negative Childhood Acute Lymphoblastic Leukemia.

Abstract 1282

Poster Board I-304

Ikaros, encoded by the IKZF1 gene, is a zinc-finger transcription factor crucial for normal differentiation of the lymphoid lineage. Multiple isoforms of Ikaros are generated by alternative splicing in lymphoid progenitors. Recent studies based primarily on high-risk (HR) patients with ALL, including Philadelphia-positive cases, have shown an inferior prognosis of patients with Ikaros alterations, leading in most cases to the expression of short, non-DNA binding isoforms of IKZF1. There is only a limited information about the overall frequency and prognostic impact of IKZF1 alterations in non-selected, BCR/ABL-negative ALL cohorts. Using a simplified yet efficient approach based on expression assay described by Iaccobucci et al. together with Agilent-on-chip semi-quantitative electrophoresis, we examined the expression of IKZF1 transcript variants in diagnostic bone marow (BM) samples from 94 children with Ph- ALL. The patients were diagnosed between November 2002 and December 2004 and treated by ALL IC-BFM 2002 protocol. Based on the analysis of peripheral blood from healthy donors and remission BM samples, we determined physiological range for relative expression of IKZF1 isoforms. The ratio between non-DNA binding (IK4, IK4del, IK4A, IK8) and functional IK1 and IK2 isoforms was significantly elevated in 26 of 94 patients (28%). There were no associations between elevated short/long isoforms ratio and age, WBC, ALL IC risk group, TEL/AML1 or hyperdiploidy. Considering the key role of Ikaros in lymphoid lineage specification, we tested whether its altered expression was related to the expression of myeloid markers on leukemic blasts. Neither short/long isoforms ratio, nor single transcript variant expression had any relation to the level of myeloperoxidase (MPO), CD13, CD33, CD65, CD117, CD14 or CD15 expression estimated by flow cytometry. Patients having the short/long isoforms ratio more than 80% had a 5-year RFS 66.7±13.6% compared to 87.5±4.1% in other patients (p=0.04). The main difference between leukemic and normal samples was observed in the relative expression of IK6 dominant-negative isoform. Using a cut-off of 10%, 14 of 94 (15%) ALL samples had increased IK6 expression in relation to other isoforms. Only 2 of 94 patients (2%) expressed IK6 alone. Elevated relative IK6 expression in the range 10-20% (6 patients) had no prognostic impact. Using a cut-off of 20%, 5-year RFS survival was 90.2± 3.3% in the group with low IK6 expression compared to 37.5±17.1% in patients with the high expression (p<0.0001). With the cut-off of 50%, RFS was 20±17.9% in the IK6 high expression group compared to 89.4±3.3% in the group with low expression (p<0.0001). Of the 5 patients with IK6 expression higher than 50%, two were treated in ALL IC HR group (based on poor prednisone response), one in the intermediate risk and two in the standard risk group, based on WBC, age and BM status at day 15. Only 1 of 4 patients with available MRD would be classified as MRD-HR based on MRD higher than 10⁻³ at week 12. Surprisingly, elevated relative expression of IK4, IK4del, IK4A and IK8 (all non-DNA binding isoforms) had no prognostic impact. The absolute level of IKZF1 expression, which might have indicated IKZF1 haploinsufficiency, had no prognostic impact either. In conclusion, we showed that a substantial proportion of childhood Ph-negative ALL cases had an increased expression of short, non-DNA binding Ikaros transcripts. However, only elevated expression of the IK6 isoform had prognostic impact. Patients with IK6 expression higher than 50% (5% of all patients) had very poor prognosis. The method used in this study could serve as a rapid screening for a new subgroup of HR patients with ALL.

Supported by GA UK 7393/2007, MSMT NPV 2B06064, VZ MSM 0021620813 and MZ 000064203.