Glucocorticoid (GC) resistance is a crucial determinant of inferior response to chemotherapy in pediatric acute lymphoblastic leukemia (ALL); however, molecular mechanisms underlying this phenomenon are poorly understood. Deregulated splicing is a common feature of many cancers, which impacts drug response and constitutes an attractive therapeutic target. Therefore, the aim of the current study was to characterize global splicing profiles associated with GC resistance and determine whether splicing modulation could serve as a novel therapeutic option for GC-resistant patients. To this end, 38 primary ALL samples were profiled using RNA-seq-based differential splicing analysis. The impact of splicing modulators was investigated in GC-resistant leukemia cell lines and primary leukemic specimens. Our findings revealed, for the first time, markedly distinct splicing landscapes in ALL samples of B-cell precursor (BCP)-ALL and T-ALL lineages. Differential splicing events associated with GC resistance were involved in RNA processing, a direct response to GCs, survival signaling, apoptosis, cell cycle regulation and energy metabolism. Furthermore, our analyses showed that GC-resistant ALL cell lines and primary samples are sensitive to splicing modulation, alone and in combination with GC. Together, these findings suggest that aberrant splicing is associated with GC resistance and splicing modulators deserve further interest as a novel treatment option for GC-resistant patients.

Objective interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients’ samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein.

Pediatric acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and is characterized by clonal heterogeneity. Genomic mutations can increase proliferative potential of leukemic cells and cause treatment resistance. However, mechanisms driving mutagenesis and clonal diversification in ALL are not fully understood. In this proof of principle study, we performed whole genome sequencing of two cases with multiple relapses in order to investigate whether groups of mutations separated in time show distinct mutational signatures. Based on mutation allele frequencies at diagnosis and subsequent relapses, we clustered mutations into groups and performed cluster-specific mutational profile analysis and de novo signature extraction. In patient 1, who experienced two relapses, the analysis unraveled a continuous interplay of aberrant activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) activity. The associated signatures SBS2 and SBS13 were present already at diagnosis, and although emerging mutations were lost in later relapses, the process remained active throughout disease evolution. Patient 2 had three relapses. We identified episodic mutational processes at diagnosis and first relapse leading to mutations resembling ultraviolet light-driven DNA damage, and thiopurine-associated damage at first relapse. In conclusion, our data shows that investigation of mutational processes in clusters separated in time may aid in understanding the mutational mechanisms and discovery of underlying causes.

Intrachromosomal amplification of chromosome 21 (iAMP21) B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) in children is a high‐risk subtype for which targeted drugs are lacking. In this study, we determined the frequency of secondary lesions in 28 iAMP21 BCP‐ALL patient samples and investigated cellular sensitivity for candidate‐targeted drugs. iAMP21 was enriched in FLT3 aberrations (10.7% vs. 50.0%, p = 0.003) and SH2B3 inactivation (7.14% vs. 46.4%, p = 0.002), compared with 28 B‐other cases, and these alterations co‐occurred in 21.4%. The occurrence of lesions in CRLF2 and IL7R was similar between iAMP21 and B‐other cases (25% vs. 17.9%, p = 0.746 and 7.14% vs. 0%, p = 0.491 respectively) as were mutations in JAK1 and JAK2 (3.57% vs. 0% and 10.7% vs. 10.7%, p = 1 for both). Sensitivity to the FLT3 inhibitor gilteritinib did not differ between iAMP21 and B‐other cases irrespective of FLT3 status. However, iAMP21 samples harboring both FLT3‐ITD and SH2B3 lesions showed the highest sensitivity. CRLF2‐rearranged iAMP21 samples were slightly more sensitive to JAK inhibitor ruxolitinib than those without, although a lack of sensitivity was present in 50% of iAMP21 cases. Trametinib sensitivity varied among iAMP21 samples with over half of iAMP21 samples being sensitive irrespective of RAS‐pathway mutation status or other secondary lesions. In summary, iAMP21 leukemias were enriched in FLT3 and in SH2B3 lesions, which when co‐occurring affected sensitivity to FLT3 inhibition by gilteritinib but not JAK inhibition by ruxolitinib. Together, our results suggest that FLT3 and RAS signaling inhibitors are of interest for further (pre)clinical evaluation in iAMP21 BCP‐ALL.