IKZF1 and CRLF2 Gene Alterations Correlate with Poor Prognosis in BCR-ABL1-Negative High Risk B-Cell Precursor Acute Lymphoblastic Leukemia

Despite improved outcome of current therapy, up to 20% of pediatric patients with B-cell precursor Acute Lymphoblastic Luekemia (BCP-ALL) still relapse. Recent genome-wide analysis showed that IKZF1, JAK2 and CRLF2 gene alterations correlate with poor prognosis in these patients. We analyzed theses gene alterations in Japanese patient cohort.

Two hundred patients with BCR-ABL1-negative BCP-ALL between 1 and 18 years old, who were enrolled in Japanese Children's Cancer & Leukemia Study Group (JCCLSG)-2004 study from 2004 to 2008, were analyzed in this study. DNA in 179 patients and RNA in 147 patients, respectively, were extracted from the initial diagnostic bone marrow or peripheral blood samples. Using DNA samples, JAK2R683 point mutation was analyzed by direct sequencing, and the copy number alterations of IKZF1 by multiplex ligation-dependent probe amplification (MLPA). CRLF2 expression in RNA samples were measured by real-time quantitative polymerase chain reaction (RQ-PCR). At the same time, we detected IKZF1 transcript of Ik6 isoform (del. exon 4 to 7) and P2RY8-CRLF2 fusion gene expression by reverse transcribed (RT) - PCR. Event-free survival (EFS) and relapse-free interval (RFI) was calculated by Kaplan-Meier method. Event was defined with induction failure, relapse, death of any cause in EFS, and with relapse in RFI.

Twenty-two (12%) of 179 patients had some exon deletions in IKZF1. Seven (32%) of 22 patients had only one copy of all exon (exon 1 to 8) and 6 of 22 (27%) patients had deletion from exon 4 to 7, which expressed Ik6 isoform. The older the age was, the higher the frequency of IKZF1 deletion was. The frequency of IKZF1 deletions (12/59) in the patients of NCI-HR group was significantly higher than that (10/120) of the patients of NCI-SR group (p=0.029). In outcome, EFS for the patients with IKZF1 deletions was significantly worse than that for without IKZF1 deletions (EFS; 66.4%±12.1% vs. 89.2%±2.7% respectively, p=0.007). And RFI for the patients with IKZF1 deletions was also worse than that for without it, (RFI; 73.1%±12.4% vs. 90.9%±2.6%, respectively, p=0.047). IKZF1 deletions seemed to be poor prognostic factor in NCI-HR patients, while they are not in NCI-SR, as shown in Table.

High CRLF2 expression (10 times higher than median value, 14.4 copies) was detected in 15 (10%) of 147 patients. Only 2 of 15 CRLF2-high patients had P2RY8-CRLF2 fusion gene by RT-PCR. We also detected 3 of 135 CRLF2-low patients had this fusion. EFS for CRLF2-high expression patients had also significantly worse than that for CRLF2-low patients (EFS; 62.2%±13.7% vs. 90.6%±2.7%, p=0.003). And RFI for CRLF2-high patients was 73.1%±12.4%, while RFI for CRLF2-low patients was 90.9%±2.6% (p=0.069). EFS for CRLF2-high showed significantly worse than that for CRLF2-low in NCI-HR patients (53.3%±17.3% vs. 87.0%±6.1%, p=0.028), but not significantly in NCI-SR (p=0.445). Regardless of patients with or without CRLF2-high expression, they showed similar RFI in NCI-HR and SR. Five patients had IKZF1 deletions and CRLF2-high expression, simultaneously, and showed extremely poor prognosis (4 of 5 showed induction failure or relapse). None of 179 patients had JAK2R683 mutation in this study.

While IKZF1 and CRLF2 alterations were detected in similar frequency to previous reports, P2RY8-CRLF2 fusion was found only 13% of CRLF2-high patients. Moreover, JAK2R683 mutation was not detected in any patients in this study. These findings suggest that there might be racial difference in genetic mechanism of leukemogenesis. IKZF1 and CRLF2 alterations might be a poor prognostic factor in NCI-HR patients, but not in NCI-SR. These independency would be disclosed in next large-scale national study.

No relevant conflicts of interest to declare.