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Deletions in IKZF1 are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1 deletions affecting exons 4-7 and exons 1-8, but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multicenter study we analyzed the prognostic value of these rare variants in a case-control design. Each IKZF1-deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1 deletions together revealed a poor prognosis (P<0.001) that was evident in each risk stratification arm. Rare variant types with the most unfavorable event-free survival were DEL 2-7 (P=0.03), DEL 2-8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4-7 and DEL 1-8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.

IKAROS family zinc finger 1/IKZF1 is a transcription factor important in lymphoid differentiation, and a known tumor suppressor in acute lymphoid leukemia. Recent studies suggest that IKZF1 is also involved in myeloid differentiation. To investigate whether IKZF1 deletions also play a role in pediatric acute myeloid leukemia, we screened a panel of pediatric acute myeloid leukemia samples for deletions of the IKZF1 locus using multiplex ligation-dependent probe amplification and for mutations using direct sequencing. Three patients were identified with a single amino acid variant without change of IKZF1 length. No frame-shift mutations were found. Out of 11 patients with an IKZF1 deletion, 8 samples revealed a complete loss of chromosome 7, and 3 cases a focal deletion of 0.1-0.9Mb. These deletions included the complete IKZF1 gene (n=2) or exons 1-4 (n=1), all leading to a loss of IKZF1 function. Interestingly, differentially expressed genes in monosomy 7 cases (n=8) when compared to non-deleted samples (n=247) significantly correlated with gene expression changes in focal IKZF1-deleted cases (n=3). Genes with increased expression included genes involved in myeloid cell self-renewal and cell cycle, and a significant portion of GATA target genes and GATA factors. Together, these results suggest that loss of IKZF1 is recurrent in pediatric acute myeloid leukemia and might be a determinant of oncogenesis in acute myeloid leukemia with monosomy 7., (Copyright© Ferrata Storti Foundation.)

Phosphatase and tensin homolog (PTEN)-inactivating mutations and/or deletions are an independent risk factor for relapse of T-cell acute lymphoblastic leukemia (T-ALL) patients treated on Dutch Childhood Oncology Group or German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia protocols. Some monoallelic mutated or PTEN wild-type patients lack PTEN protein, implying that additional PTEN inactivation mechanisms exist. We show that PTEN is inactivated by small deletions affecting a few exons in 8% of pediatric T-ALL patients. These microdeletions were clonal in 3% and subclonal in 5% of patients. Conserved deletion breakpoints are flanked by cryptic recombination signal sequences (cRSSs) and frequently have non-template-derived nucleotides inserted in between breakpoints, pointing to an illegitimate RAG recombination-driven activity. Identified cRSSs drive RAG-dependent recombination in a reporter system as efficiently as bona fide RSSs that flank gene segments of the T-cell receptor locus. Remarkably, equivalent microdeletions were detected in thymocytes of healthy individuals. Microdeletions strongly associate with the TALLMO subtype characterized by TAL1 or LMO2 rearrangements. Primary and secondary xenotransplantation of TAL1-rearranged leukemia allowed development of leukemic subclones with newly acquired PTEN microdeletions. Ongoing RAG activity may therefore actively contribute to the acquisition of preleukemic hits, clonal diversification, and disease progression., (© 2014 by The American Society of Hematology.)

Recurrent submicroscopic deletions in genes affecting key cellular pathways are a hallmark of pediatric acute lymphoblastic leukemia (ALL). To gain more insight into the mechanism underlying these deletions, we have studied the occurrence and nature of abnormalities in one of these genes, the B-cell translocation gene 1 (BTG1), in a large cohort of pediatric ALL cases. BTG1 was found to be exclusively affected by genomic deletions, which were detected in 65 out of 722 B-cell precursor ALL (BCP-ALL) patient samples (9%), but not in 109 T-ALL cases. Eight different deletion sizes were identified, which all clustered at the telomeric site in a hotspot region within the second (and last) exon of the BTG1 gene, resulting in the expression of truncated BTG1 read-through transcripts. The presence of V(D)J recombination signal sequences at both sites of virtually all deletions strongly suggests illegitimate RAG1/RAG2-mediated recombination as the responsible mechanism. Moreover, high levels of histone H3 lysine 4 trimethylation (H3K4me3), which is known to tether the RAG enzyme complex to DNA, were found within the BTG1 gene body in BCP-ALL cells, but not T-ALL cells. BTG1 deletions were rarely found in hyperdiploid BCP-ALLs, but were predominant in other cytogenetic subgroups, including the ETV6-RUNX1 and BCR-ABL1 positive BCP-ALL subgroups. Through sensitive PCR-based screening, we identified multiple additional BTG1 deletions at the subclonal level in BCP-ALL, with equal cytogenetic distribution which, in some cases, grew out into the major clone at relapse. Taken together, our results indicate that BTG1 deletions may act as "drivers" of leukemogenesis in specific BCP-ALL subgroups, in which they can arise independently in multiple subclones at sites that are prone to aberrant RAG1/RAG2-mediated recombination events. These findings provide further evidence for a complex and multiclonal evolution of ALL., Competing Interests: The authors have declared that no competing interests exist.

Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL) and is often difficult to predict. To explore the prognostic impact of recurrent DNA copy number abnormalities on relapse, we performed high-resolution genomic profiling of 34 paired diagnosis and relapse ALL samples. Recurrent lesions detected at diagnosis, including PAX5, CDKN2A and EBF1, were frequently absent at relapse, indicating that they represent secondary events that may be absent in the relapse-prone therapy-resistant progenitor cell. In contrast, deletions and nonsense mutations in IKZF1 (IKAROS) were highly enriched and consistently preserved at the time of relapse. A targeted copy number screen in an unselected cohort of 131 precursor B-ALL cases, enrolled in the dexamethasone-based Dutch Childhood Oncology Group treatment protocol ALL9, revealed that IKZF1 deletions are significantly associated with poor relapse-free and overall survival rates. Separate analysis of ALL9-treatment subgroups revealed that non-high-risk (NHR) patients with IKZF1 deletions exhibited a approximately 12-fold higher relative relapse rate than those without IKZF1 deletions. Consequently, IKZF1 deletion status allowed the prospective identification of 53% of the relapse-prone NHR-classified patients within this subgroup and, therefore, serves as one of the strongest predictors of relapse at the time of diagnosis with high potential for future risk stratification.