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Background: ABL-class fusions including NUP214-ABL1 and EBF1-PDGFRB occur in high risk acute lymphoblastic leukaemia (ALL) with gene expression patterns similar to BCR-ABL-positive ALL. Our aim was to evaluate new DNA-based measurable residual disease (MRD) tests detecting these fusions and IKZF1-deletions in comparison with conventional immunoglobulin/T-cell receptor (Ig/TCR) markers., Methods: Precise genomic breakpoints were defined from targeted or whole genome next generation sequencing for ABL-fusions and BCR-ABL1. Quantitative PCR assays were designed and used to re-measure MRD in remission bone marrow samples previously tested using Ig/TCR markers. All MRD testing complied with EuroMRD guidelines., Results: ABL-class patients had 46% 5year event-free survival and 79% 5year overall survival. All had sensitive fusion tests giving high concordance between Ig/TCR and ABL-class fusion results (21 patients, n = 257 samples, r2 = 0.9786, P < 0.0001) and Ig/TCR and IKZF1-deletion results (9 patients, n = 143 samples, r2 = 0.9661, P < 0.0001). In contrast, in BCR-ABL1 patients, Ig/TCR and BCR-ABL1 tests were discordant in 32% (40 patients, n = 346 samples, r2 = 0.4703, P < 0.0001) and IKZF1-deletion results were closer to Ig/TCR (25 patients, n = 176, r2 = 0.8631, P < 0.0001)., Conclusions: MRD monitoring based on patient-specific assays detecting gene fusions or recurrent assays for IKZF1-deletions is feasible and provides good alternatives to Ig/TCR tests to monitor MRD in ABL-class ALL., (© 2022. The Author(s).)

Background: Minimal residual disease (MRD) measurement is a cornerstone of contemporary acute lymphoblastic leukaemia (ALL) treatment. The presence of immunoglobulin (Ig) and T cell receptor (TCR) gene recombinations in leukaemic clones allows widespread use of patient-specific, DNA-based MRD assays. In contrast, paediatric solid tumour MRD remains experimental and has focussed on generic assays targeting tumour-specific messenger RNA, methylated DNA or microRNA., Methods: We examined the feasibility of using whole-genome sequencing (WGS) data to design tumour-specific polymerase chain reaction (PCR)-based MRD tests (WGS-MRD) in 18 children with high-risk relapsed cancer, including ALL, high-risk neuroblastoma (HR-NB) and Ewing sarcoma (EWS) (n = 6 each)., Results: Sensitive WGS-MRD assays were generated for each patient and allowed quantitation of 1 tumour cell per 10 -4 (0.01%)-10 -5 (0.001%) mononuclear cells. In ALL, WGS-MRD and Ig/TCR-MRD were highly concordant. WGS-MRD assays also showed good concordance between quantitative PCR and droplet digital PCR formats. In serial clinical samples, WGS-MRD correlated with disease course. In solid tumours, WGS-MRD assays were more sensitive than RNA-MRD assays., Conclusions: WGS facilitated the development of patient-specific MRD tests in ALL, HR-NB and EWS with potential clinical utility in monitoring treatment response. WGS data could be used to design patient-specific MRD assays in a broad range of tumours., (© 2021. The Author(s).)

Background: The prognosis for high-risk childhood acute leukaemias remains dismal and established treatment protocols often cause long-term side effects in survivors. This study aims to identify more effective and safer therapeutics for these patients., Methods: A high-throughput phenotypic screen of a library of 3707 approved drugs and pharmacologically active compounds was performed to identify compounds with selective cytotoxicity against leukaemia cells followed by further preclinical evaluation in patient-derived xenograft models., Results: Auranofin, an FDA-approved agent for the treatment of rheumatoid arthritis, was identified as exerting selective anti-cancer activity against leukaemia cells, including patient-derived xenograft cells from children with high-risk ALL, versus solid tumour and non-cancerous cells. It induced apoptosis in leukaemia cells by increasing reactive oxygen species (ROS) and potentiated the activity of the chemotherapeutic cytarabine against highly aggressive models of infant MLL-rearranged ALL by enhancing DNA damage accumulation. The enhanced sensitivity of leukaemia cells towards auranofin was associated with lower basal levels of the antioxidant glutathione and higher baseline ROS levels compared to solid tumour cells., Conclusions: Our study highlights auranofin as a well-tolerated drug candidate for high-risk paediatric leukaemias that warrants further preclinical investigation for application in high-risk paediatric and adult acute leukaemias.

Background: While current chemotherapy has increased cure rates for children with acute lymphoblastic leukaemia (ALL), the largest number of relapsing patients are still stratified as medium risk (MR) at diagnosis (50-60%). This highlights an opportunity to develop improved relapse-prediction models for MR patients. We hypothesised that bone marrow from MR patients who eventually relapsed would regrow faster in a patient-derived xenograft (PDX) model after induction chemotherapy than samples from patients in long-term remission., Methods: Diagnostic bone marrow aspirates from 30 paediatric MR-ALL patients (19 who relapsed, 11 who experienced remission) were inoculated into immune-deficient (NSG) mice and subsequently treated with either control or an induction-type regimen of vincristine, dexamethasone, and L-asparaginase (VXL). Engraftment was monitored by enumeration of the proportion of human CD45 + cells (%huCD45 + ) in the murine peripheral blood, and events were defined a priori as the time to reach 1% huCD45 + , 25% huCD45 + (TT25%) or clinical manifestations of leukaemia (TTL)., Results: The TT25% value significantly predicted MR patient relapse. Mutational profiles of PDXs matched their tumours of origin, with a clonal shift towards relapse observed in one set of VXL-treated PDXs., Conclusions: In conclusion, establishing PDXs at diagnosis and subsequently applying chemotherapy has the potential to improve relapse prediction in paediatric MR-ALL.

Background: Zinc-finger protein 384 (ZNF384) fusions are an emerging subtype of precursor B-cell acute lymphoblastic leukaemia (pre-B-ALL) and here we further characterised their prevalence, survival outcomes and transcriptome., Methods: Bone marrow mononuclear cells from 274 BCR-ABL1-negative pre-B-ALL patients were immunophenotyped and transcriptome molecularly characterised. Transcriptomic data was analysed by principal component analysis and gene-set enrichment analysis to identify gene and pathway expression changes., Results: We exclusively detect E1A-associated protein p300 (EP300)-ZNF384 in 5.7% of BCR-ABL1-negative adolescent/young adult (AYA)/adult pre-B-ALL patients. EP300-ZNF384 patients do not appear to be a high-risk subgroup. Transcriptomic analysis revealed that EP300-ZNF384 samples have a distinct gene expression profile that results in the up-regulation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) and cell adhesion pathways and down-regulation of cell cycle and DNA repair pathways., Conclusions: Importantly, this report contributes to a better overview of the incidence of EP300-ZNF384 patients and show that they have a distinct gene signature with concurrent up-regulation of JAK-STAT pathway, reduced expression of B-cell regulators and reduced DNA repair capacity.