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The contribution of genetic predisposing factors to the development of pediatric acute lymphoblastic leukemia (ALL), the most frequently diagnosed cancer in childhood, has not been fully elucidated. Children presenting with multiple de novo leukemias are more likely to suffer from genetic predisposition. Here, we selected five of these patients and analyzed the mutational spectrum of normal and malignant tissues. In two patients, we identified germline mutations in TYK2, a member of the JAK tyrosine kinase family. These mutations were located in two adjacent codons of the pseudokinase domain (p.Pro760Leu and p.Gly761Val). In silico modeling revealed that both mutations affect the conformation of this autoregulatory domain. Consistent with this notion, both germline mutations promote TYK2 autophosphorylation and activate downstream STAT family members, which could be blocked with the JAK kinase inhibitor I. These data indicate that germline activating TYK2 mutations predispose to the development of ALL.

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.

Response to therapy as determined by minimal residual disease (MRD) is currently used for stratification in treatment protocols for pediatric acute lymphoblastic leukemia (ALL). However, the large MRD-based medium risk group (MRD-M; 50-60% of the patients) harbors many relapses. We analyzed MRD in 131 uniformly treated precursor-B-ALL patients and evaluated whether combined MRD and IKZF1 (Ikaros zinc finger-1) alteration status can improve risk stratification. We confirmed the strong prognostic significance of MRD classification, which was independent of IKZF1 alterations. Notably, 8 of the 11 relapsed cases in the large MRD-M group (n=81; 62%) harbored an IKZF1 alteration. Integration of both MRD and IKZF1 status resulted in a favorable outcome group (n=104; 5 relapses) and a poor outcome group (n=27; 19 relapses), and showed a stronger prognostic value than each of the established risk factors alone (hazard ratio (95%CI): 24.98 (8.29-75.31)). Importantly, whereas MRD and IKZF1 status alone identified only 46 and 54% of the relapses, respectively, their integrated use allowed prediction of 79% of all the relapses with 93% specificity. Because of the unprecedented sensitivity in upfront relapse prediction, the combined parameters have high potential for future risk stratification, particularly for patients originally classified as non-high risk, such as the large group of MRD-M patients.

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.

Episomes with the NUP214-ABL1 fusion gene have been observed in 6% of T-ALL. In this multicentric study we collected 27 cases of NUP214-ABL1-positive T-ALL. Median age was 15 years with male predominance. Outcome was poor in 12 patients. An associated abnormality involving TLX1 or TLX3 was found in all investigated cases. Fluorescent in situ hybridization revealed a heterogeneous pattern of NUP214-ABL1 amplification. Multiple episomes carrying the fusion were detected in 24 patients. Episomes were observed in a significant number of nuclei in 18 cases, but in only 1-5% of nuclei in 6. In addition, intrachromosomal amplification (small hsr) was identified either as the only change or in association with episomes in four cases and two T-ALL cell lines (PEER and ALL-SIL). One case showed insertion of apparently non-amplified NUP214-ABL1 sequences at 14q12. The amplified sequences were analyzed using array-based CGH.These findings confirm that the NUP214-ABL1 gene requires amplification for oncogenicity; it is part of a multistep process of leukemogenesis; and it can be a late event present only in subpopulations. Data also provide in vivo evidence for a model of episome formation, amplification and optional reintegration into the genome. Implications for the use of kinase inhibitors are discussed.

Gross cytogenetic anomalies are traditionally being used as diagnostic, prognostic and therapeutic markers in the clinical management of cancer, including childhood acute lymphoblastic leukemia (ALL). Recently, it has become increasingly clear that genetic lesions driving tumorigenesis frequently occur at the submicroscopic level and, consequently, escape standard cytogenetic observations. Therefore, we profiled the genomes of 40 childhood ALLs at high resolution. We detected multiple de novo genetic lesions, including gross aneuploidies and segmental gains and losses, some of which were subtle and affected single genes. Many of these lesions involved recurrent (partially) overlapping deletions and duplications, containing various established leukemia-associated genes, such as ETV6, RUNX1 and MLL. Importantly, the most frequently affected genes were those controlling G1/S cell cycle progression (e.g. CDKN2A, CDKN1B and RB1), followed by genes associated with B-cell development. The latter group includes microdeletions of the B-lineage transcription factors PAX5, EBF, E2-2 and IKZF1 (Ikaros), as well as genes with other established roles in B-cell development, that is RAG1 and RAG2, FYN, PBEF1 or CBP/PAG. The fact that we frequently encountered multiple lesions affecting genes involved in cell cycle regulation and B-cell differentiation strongly suggests that both these processes need to be targeted independently and simultaneously to trigger ALL development.