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Chromosomal rearrangements of the human KMT2A/MLLgene are associated with de novoas well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2Agene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2Agene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5’-KMT2A, two patients had a 5’-KMT2Adeletion, and one ETV6::RUNX1patient had an KMT2Ainsertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2Arecombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.

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 novoleukemias 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 silicomodeling 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 IKZF1are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1deletions affecting exons 4–7 and exons 1–8, but evidence for the remaining 33% of cases harboring other variants of IKZF1deletions 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 IKZF1wild-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 IKZF1deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1deletions 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 IKZF1deletion variants. We therefore conclude that all variants of rare IKZF1deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.