Novel biological subtypes and clinically important genetic aberrations (druggable lesions, prognostic factors) have been described in B-other acute lymphoblastic leukemia during the last decade; however, due to a lack of studies on unselected cohorts, their population frequency and mutual associations remain to be established. We studied 110 consecutively diagnosed and uniformly treated childhood B-other patients using single nucleotide polymorphism arrays and whole exome/transcriptome sequencing. The frequency of DUX4-rearranged, BCR-ABL1-like, ZNF384-rearranged, ETV6-RUNX1-like, iAMP21 and MEF2D-rearranged subtypes was 27%, 15%, 5%, 5%, 4% and 2%, respectively; 43% of cases were not classified into any of these subtypes (B-rest). We found worse early response to treatment in DUX4-rearranged leukemia and a strong association of ZNF384-rearranged leukemia with B-myeloid immunophenotype. Of druggable lesions, JAK/STAT-class and RAS/RAF/MAPK-class aberrations were found in 21% and 43% of patients, respectively; an ABL-class aberration was found in a single patient. A recently described negative prognostic factor, IKZF1plus, was found in 14% of patients and was enriched in (but not exclusive for) BCR-ABL1-like subtype. PAX5 fusions (including 4 novel), intragenic amplifications and P80R mutations were mutually exclusive and occurred exclusively in B-rest subset, altogether accounting for 20% of B-other group. PAX5 P80R was associated with a specific gene expression signature potentially defining a novel leukemia subtype. Our study shows unbiased European population-based frequencies of novel ALL subtypes, recurrent (cyto)genetic aberrations and their mutual associations; this study also strengthens and widens the current knowledge of B-other acute lymphoblastic leukemia and provides an objective basis for optimization of current genetic diagnostics.

Despite attempts to improve the definitions of ambiguous leukemia (ALAL) during the last two decades, general therapy recommendations are missing. Herein, we report a large cohort of children with ALAL and propose a treatment strategy. A retrospective multinational study iBFM-AMBI2012 on 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (5yEFS) of patients with acute lymphoblastic leukemia (ALL)-type primary therapy (80±4%) was superior to that of children who received acute myeloid leukemia (AML)-type or combined-type treatments (36±7.2% and 50±12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5yEFS of CD19^pos leukemia was 83±5.3% on ALL-type primary treatment compared to 0±0% and 28±14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using WHO and/or EGIL definitions), and in the bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19^pos ALAL. AML-type of treatment is preferred in a minority of ALAL cases with CD19^neg and no other lymphoid features ALALs. No overall benefit of transplantation was documented and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change to the treatment type, this is to be considered only in cases with 5% blast or greater after remission induction. The results provide a basis for a prospective trial. Key points The largest cohort of ambiguous leukemias to date reveals a better prognosis of children who started on lymphoid-directed treatment. Myeloid type of primary treatment correlated with dismal outcomes in CD19pos leukemias.

To characterize the incidence, clinical features and genetics of ETV6-ABL1 leukemias, representing targetable kinase-activating lesions, we analyzed 44 new and published cases of ETV6-ABL1-positive hematologic malignancies [22 cases of acute lymphoblastic leukemia (13 children, 9 adults) and 22 myeloid malignancies (18 myeloproliferative neoplasms, 4 acute myeloid leukemias)]. The presence of the ETV6-ABL1 fusion was ascertained by cytogenetics, fluorescence in-situ hybridization, reverse transcriptase-polymerase chain reaction and RNA sequencing. Genomic and gene expression profiling was performed by single nucleotide polymorphism and expression arrays. Systematic screening of more than 4,500 cases revealed that in acute lymphoblastic leukemia ETV6-ABL1 is rare in childhood (0.17% cases) and slightly more common in adults (0.38%). There is no systematic screening of myeloproliferative neoplasms; however, the number of ETV6-ABL1-positive cases and the relative incidence of acute lymphoblastic leukemia and myeloproliferative neoplasms suggest that in adulthood ETV6-ABL1 is more common in BCR-ABL1-negative chronic myeloid leukemia-like myeloproliferations than in acute lymphoblastic leukemia. The genomic profile of ETV6-ABL1 acute lymphoblastic leukemia resembled that of BCR-ABL1 and BCR-ABL1-like cases with 80% of patients having concurrent CDKN2A/B and IKZF1 deletions. In the gene expression profiling all the ETV6-ABL1-positive samples clustered in close vicinity to BCR-ABL1 cases. All but one of the cases of ETV6-ABL1 acute lymphoblastic leukemia were classified as BCR-ABL1-like by a standardized assay. Over 60% of patients died, irrespectively of the disease or age subgroup examined. In conclusion, ETV6-ABL1 fusion occurs in both lymphoid and myeloid leukemias; the genomic profile and clinical behavior resemble BCR-ABL1-positive malignancies, including the unfavorable prognosis, particularly of acute leukemias. The poor outcome suggests that treatment with tyrosine kinase inhibitors should be considered for patients with this fusion.