Detection by a simple and cheaper methodology of Ik6 and Ik10 isoforms of the IKZF1 gene is highly associated with a poor prognosis in B‐lineage paediatric acute lymphoblastic leukaemia

BCR-ABL1-like acute lymphoblastic leukaemia (ALL) is a subtype of high-risk lymphoblastic leukaemia identified as having a gene expression profile similar to that of Philadelphia chromosome (Ph)-positive ALL, and is characterized by rearrangements or mutations causing activation of the cytokine receptor and the signalling of tyrosine kinases, comprising approximately 15% of B-cell precursor (BCP-ALL) in children and adolescents. As is the case for Ph-positive ALL, deletions of the IKZF1 gene have been reported in approximately 70% of children and adults with Ph-like ALL, thus becoming a hallmark of this type of ALL due to their high frequency and being associated with a worse prognosis (Pui et al, 2017). The IKZF1 gene encodes the IKAROS protein that functions at the very early stage of haematopoiesis. It is known that IKAROS works as a suppressor of lymphoblastic neoplasms (Tokunaga et al, 2013; Marke et al, 2018). The most frequent alteration of IKZF1 found in patients with ALL is a deletion involving the entire gene or, more frequently, involving only some exons, which has been associated with a poor prognosis (Mullighan et al, 2009; Chen et al, 2012; Dörge et al, 2013). The IKZF1 gene consists of eight exons, seven of which are coders and allow multiple isoforms to be generated (Marke et al, 2018). The isoforms differ in the number of N-terminal zinc-fingers, and only the isoforms with at least three N-terminal zinc-fingers are able to bind to DNA. Therefore, shorter isoforms without some or all of the N-terminal zinc-finger attenuate DNA binding capacity and may act as dominant-negative isoforms (Zhou et al, 2011). The short isoforms (Ik4 to Ik10) lack two or more zinc-finger domains, thus being unable to bind to DNA, impairing the function of IKAROS proteins and behaving as a negative domain (Vshyukova et al, 2018). Ik6 is the most common form of shorter isoforms, being able to suppress DNA binding function and is the strongest transcription repressor in the Ikaros family. Studies have shown that overexpression of Ik6 has been identified as a marker of poor prognosis in children with ALL (Zhou et al, 2011). Classical methodologies for the detection of IKZF1 deletions, such as multiplex ligation-dependent probe amplification or single nucleotide polymorphism arrays are laborious, expensive and inaccessible to most treatment centres, especially in developing countries. A simplified and cheaper methodology for the assessment of IKZF1 isoforms may provide good predictive criteria of an unfavourable course in children with ALL and could be used to identify patients at high risk of relapse. We analysed 100 consecutive samples of bone marrow collected at diagnosis of BCP-ALL (3 were Ph+) and 35 samples of T-ALL. All children were classified and treated according to the Brazilian protocol GBTLI-99 (Scrideli et al, 2009; Brandalise et al, 2010) in order to detect the presence of IKZF1 deletions using an adapted and highly simplified methodology and conventional reverse transcription polymerase chain reaction (RT-PCR) as previously described (Tokunaga et al, 2013). The cDNA IKZF1 sequence from exons 1–8 was amplified by RT-PCR using the following primers: forward primer 5′-AAAGCGCGACGCACAAATCC-3′, reverse primer 5-ATGGCGTTGTTGATGGCTTGGTC-3′. RT-PCR was performed as described in Data S1. The relative amounts of isoforms Ik6 and Ik10 were assessed by gel band densitometry using ImageJ software (ImageJ, US National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/). The Ik6 or Ik10 isoforms were compared to full length isoforms of the IKZF1 gene by RT-PCR. The isoforms were considered to be present in a large fraction of leukaemic cells (high load) when they were unique or their relative amount was higher than that of the full-length isoforms of IKZF1. All samples showing a change in IKZF1 in RT-PCR were directly sequenced to confirm the isoform detected (Figure S1). Of the 97 patients with Ph-negative BCP ALL, 45 were classified as high risk and 9 had a high level of minimal residual disease (MRD) at the end of induction therapy (available in 69 patients) according to GBTLI-99 (Scrideli et al, 2009). High load negative IKZF1 isoforms were found in 12/99 patients (10 with the Ik6 isoform and 2 with Ik10). All 3 patients with Ph+ ALL had the high load Ik6 isoform. Event-free survival (EFS) and overall survival (OS) were assessed by Kaplan-Meier curves and the log-rank test according to white blood cell count at diagnosis, age, risk classification, morphological evaluation of bone marrow at the end of induction, MRD status at the end of induction therapy, and the presence of high load expression of the Ik6 or Ik10 isoforms, with a median follow-up of 160 months (Table 1, Fig 1). The variables age, MRD at day 28 and presence of Ik6/Ik10 high load expression were statistically associated with EFS and OS in univariate analysis. Multivariate analysis by Cox regression model using the variables that were significantly associated with event or death in univariate analysis showed that MRD at day 28 and Ik6/Ik10 high load expression were independent prognostic factors (Fig 1). Even in patients classified as low risk, the presence of Ik6/Ik10 high load expression occurred in 6/52 cases and was associated with lower 10-year EFS (81·5 ± 5·8% vs. 16·7% ± 15·2% P = 0·002) and 10-year OS (86·3 ± 5·2% vs. 16·7 ± 15·2%, P < 0·0001), demonstrating the importance of IKZF1 gene evaluation for the diagnosis of childhood ALL, even in patients initially fulfilling criteria for low risk stratification. Proportional differences between the presence of Ik6/Ik10 high load expression and clinical criteria were analysed by Fisher’s exact text. Association was found between the presence of Ik6/Ik10 high load expression and MRD at day 28 (P = 0·009), unfavourable event (P < 0·001) and death (P < 0·001). Of the 35 patients with T-ALL, only 2 showed IKZF1 changes with a high load of Ik6. In summary, the presence of Ik6/Ik10 high load expression in children with BCP-ALL, detected by a simple and cheaper methodology, was the most important independent prognostic factor in these patients. This fact may modify the initial stratification of risk and may improve the survival rates in cases with the presence of negative domain isoforms and stratify patients for future treatment with target therapies. This work was supported by grants from the Brazilian Public Financial support agencies Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP – grant number 05/02279-6), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – grant number 471885/2013-4) and FAEPA (Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo). LPS, LGT, CAS were responsible for overall design, data collection, analysis, interpretation and statistical analysis, manuscript preparation and writing of the manuscript; LPS, RPQ, VKS, EP, CAS provided and analysed PCR and DNA sequencing data; SRB, JAY, collected samples, analysed and provided clinical data. All authors read and approved the final manuscript. The authors declare no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.