We used the genomic breakpoint between and genes for the DNA-based monitoring of minimal residual disease (MRD) in 48 patients with childhood acute lymphoblastic leukemia (ALL). Comparing the results with standard MRD monitoring based on immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements and with quantification of deletion, we observed very good correlation for the methods in a majority of patients; however,>20% of children (25% [8/32] with minor and 12.5% [1/8] with major- variants in the consecutive cohorts) had significantly (>1 log) higher levels of fusion than Ig/TCR rearrangements and/or deletion. We performed cell sorting of the diagnostic material and assessed the frequency of -positive cells in various hematopoietic subpopulations; 12% to 83% of non-ALL B lymphocytes, T cells, and/or myeloid cells harbored the fusion in patients with discrepant MRD results. The multilineage involvement of the -positive clone demonstrates that in some patients diagnosed with-positive ALL, a multipotent hematopoietic progenitor is affected by the fusion. These patients have-positive clonal hematopoiesis resembling a chronic myeloid leukemia (CML)-like disease manifesting in "lymphoid blast crisis." The biological heterogeneity of -positive ALL may impact the patient outcomes and optimal treatment (early stem cell transplantation vs long-term administration of tyrosine-kinase inhibitors) as well as on MRD testing. Therefore, we recommend further investigations on CML-like-positive ALL.

Thiosemicarbazones (TSCs) are an interesting class of ligands that show a diverse range of biological activity, including anti-fungal, anti-viral and anti-cancer effects. Our previous studies have demonstrated the potent in vivo anti-tumor activity of novel TSCs and their ability to overcome resistance to clinically used chemotherapeutics. In the current study, 35 novel TSCs of 6 different classes were designed using a combination of retro-fragments that appear in other TSCs. Additionally, di-substitution at the terminal N4 atom, which was previously identified to be critical for potent anti-cancer activity, was preserved through the incorporation of an N4-based piperazine or morpholine ring. The anti-proliferative activity of the novel TSCs were examined in a variety of cancer and normal cell-types. In particular, compounds 1d and 3c demonstrated the greatest promise as anti-cancer agents with potent and selective anti-proliferative activity. Structure-activity relationship studies revealed that the chelators that utilized “soft” donor atoms, such as nitrogen and sulfur, resulted in potent anti-cancer activity. Indeed, the N,N,S donor atom set was crucial for the formation of redox active iron complexes that were able to mediate the oxidation of ascorbate. This further highlights the important role of reactive oxygen species generation in mediating potent anti-cancer activity. Significantly, this study identified the potent and selective anti-cancer activity of 1d and 3c that warrants further examination.

ERG -deletions occur recurrently in acute lymphoblastic leukemia, especially in the DUX4 -rearranged subtype. The ERG -deletion was shown to positively impact prognosis of patients with IKZF1 -deletion and its presence precludes assignment into IKZF1 plus group, a novel high-risk category on AIEOP-BFM ALL trials. We analyzed the impact of different methods on ERG -deletion detection rate, evaluated ERG -deletion as a potential marker for DUX4 -rearranged leukemia, studied its associations with molecular and clinical characteristics within this leukemia subtype, and analyzed its clonality. Using single-nucleotide-polymorphism array, genomic polymerase chain reaction (PCR) and amplicon-sequencing we found ERG -deletion in 34% (16 of 47), 66% (33 of 50) and 78% (39 of 50) of DUX4 -rearranged leukemia, respectively. False negativity of ERG -deletion by single-nucleotide-polymorphism array caused IKZF1 plus misclassification in 5 patients. No ERG -deletion was found outside the DUX4 -rearranged cases. Within DUX4 -rearranged leukemia, the ERG -deletion was associated with higher total number of copy-number aberrations, and, importantly, the ERG -deletion positivity by PCR was associated with better outcome [5-year event-free survival (EFS), ERG -deletion-positive 93% vs. ERG -deletion-negative 68%, P =0.022; 5-year overall survival (OS), ERG -deletion-positive 97% vs. ERG -deletion-negative 75%, P =0.029]. Ultra-deep amplicon-sequencing revealed distinct co-existing ERG -deletions in 22 of 24 patients. In conclusion, our data demonstrate inadequate sensitivity of single-nucleotide-polymorphism array for ERG -deletion detection, unacceptable for proper IKZF1 plus classification. Even using more sensitive methods (PCR/amplicon-sequencing) for its detection, ERG -deletion is absent in 22-34% of DUX4 -rearranged leukemia and does not represent an adequately sensitive marker of this leukemia subtype. Importantly, the ERG -deletion potentially stratifies the DUX4 -rearranged leukemia into biologically/clinically distinct subsets. Frequent polyclonal pattern of ERG -deletions shows that late origin of this lesion is more common than has been previously described.

Oxidative stress is a common denominator of numerous cardiovascular disorders. Free cellular iron catalyzes formation of highly toxic hydroxyl radicals and iron chelation may thus be an effective therapeutic approach. However, using classical iron chelators in diseases without iron overload poses risks that necessitate more advanced approaches, such as prochelators that are activated to chelate iron only under disease-specific oxidative stress conditions. In this study, three cell membrane-permeable iron chelators (clinically-used deferasirox and experimental SIH and HAPI) and five boronate-masked prochelator analogs were evaluated for their ability to protect cardiac cells against oxidative injury induced by hydrogen peroxide. Whereas the deferasirox-derived agents TIP and TRA-IMM displayed negligible protection and even considerable toxicity, aroylhydrazone prochelators BHAPI and BSIH-PD provided significant cytoprotection and displayed lower toxicity following prolonged cellular exposure compared to their parent chelators HAPI and SIH, respectively. Overall, the most favorable properties in terms of protective efficiency and low inherent cytotoxicity were observed with aroylhydrazone prochelator BSIH. BSIH efficiently protected both H9c2 rat cardiomyoblast-derived cells as well as isolated primary rat cardiomyocytes against hydrogen peroxide-induced mitochondrial and lysosomal dysregulation and cell death. At the same time, BSIH was non-toxic at concentrations up to its solubility limit (600 µM) and 72-hour incubation. Hence, BSIH merits further investigation for prevention and/or treatment of cardiovascular disorders associated with a known (or presumed) component of oxidative stress.