ETV6::RUNX1-like ALL is defined by a gene expression signature similar to that of ETV6::RUNX1-positive ALL and absence of all genetic subtype-defining aberrations, including the ETV6::RUNX1 fusion. Within the International BFM Study Group, we assembled and analyzed a cohort of 100 patients (including 97 children) with ETV6::RUNX1-like ALL. We describe their diverse genetic landscape, centered around ETV6 aberrations with frequent IKZF1 disruptions, as previously shown, but including various rare non-ETV6/non-IKZF1 gene fusions, and rearrangements of CRLF2 (CRLF2r). We show that ETV6 and IKZF1 aberrations do not occur exclusively in this subtype, which hampers its classification based solely on genomic data. We confirm our previous observation of a strong association of the CD27-positive/CD44low-negative immunophenotype with ETV6::RUNX1(-like) subtype. Compared to ETV6::RUNX1-positive ALL, patients with ETV6::RUNX1-like ALL are younger, have higher white blood cell counts at diagnosis, and have an inferior early treatment response. While overall survival is comparable, event-free survival is significantly lower in patients with ETV6::RUNX1-like ALL, with NCI risk, early treatment response, IKZF1 deletions, CRLF2r, and JAK2 mutations having prognostic relevance. Notably, Down syndrome is highly prevalent and associated with a worse outcome in ETV6::RUNX1-like ALL. In conclusion, we provide biological, demographic, and clinical characteristics of the largest ETV6::RUNX1-like cohort presented to date.

Pediatric regimens improve outcomes in adolescent and young adult (AYA) acute lymphoblastic leukemia (ALL) patients. End-consolidation (time point 2 [TP2]) minimal residual disease negativity (MRDneg) is associated with improved survival. In this study, standard consolidation chemotherapy was replaced with blinatumomab to improve TP2 MRDneg-a key survival surrogate in B-lineage ALL. From 2019 to 2022, 55 patients constituted the intention-to-treat (ITT) cohort, median age 25 (range, 16-39) years. Using a Simon's 2-stage design, blinatumomab replaced standard consolidation chemotherapy cycles with TP2 MRDneg as the primary endpoint. Blinatumomab was associated with an improved TP2 MRDneg rate of 70.8% (95% CI, 55.9%-83.0%) versus the null hypothesis of 60% (P = 0.037). When compared to our previous ALL06 study, median time from protocol I commencement to next treatment phase was 84 versus 97 days (P = 0.0001), with 82.7% versus 45.1% (P < 0.0001), commencing protocol M or high-risk block therapy by day 94. Induction mortality was 1.8%. Blinatumomab was well tolerated. Median follow-up was 42.9 (range, 1.9-54.7) months, with 3-year disease-free survival (DFS) 88.6% (95% CI, 76.3%-94.7%) and 3-year overall survival (OS) 90.5% (95% CI, 78.6%-95.9%) in the ITT cohort. Higher than medium-risk patients had poorer DFS but not OS. Standard genomic risk patients had 100% 3-year DFS and OS. Adverse genomic risk stratified by TP2 MRDpos predicted poorer DFS but not OS. Blinatumomab consolidation for de novo B-lineage AYA ALL was associated with high MRDneg rates and excellent survival, particularly in standard-risk disease. Genomics may assist in predicting response to blinatumomab in de novo ALL (ACTRN12618001734257).

Background: The IGH locus is susceptible to translocations or insertions that contribute to B-cell precursor acute lymphoblastic leukemia (ALL) by ectopic or enhanced expression of a gene relocated to the IGH enhancer. The frequency of IGH rearrangements is relatively high in Down syndrome (DS) ALL. IGH rearrangements can be cryptic and might not be detected as a chimeric transcript, hence, their frequency, partner genes and prognostic value are largely unknown. Methods: We performed RNA-sequencing and IGH break-apart fluorescent in-situ hybridization (FISH) to determine the genetic and clinical characteristics of IGH rearrangements in 50 DS ALL patients. Results: We identified 10 patients with a chimeric IGH transcript and another 22 IGH-rearranged patients solely by FISH. The IGH rearrangement was clonal (>= 50 % of leukemic cells) in 11 cases and subclonal (10-50 % of cells) in 21 cases. Almost one-third of the subclonal IGH rearrangements co-occurred with known oncogenic driver aberration. The partner gene was identified in 16 cases and the most frequent partners were CEBPD (n = 6) and CRLF2 (n = 4). A trend towards a worse event-free survival was seen for DS ALL patients with a clonal IGH rearrangement (clonal: HR 3.34, p = 0.053; subclonal: HR 1.80, p = 0.31) compared with DS ALL patients without an IGH rearrangement. Conclusion: By combining RNA-sequencing and FISH, we identified IGH rearrangements in 64 % (n = 32) of DS ALL. A clonal IGH rearrangement (22 %) may point to an unfavorable outcome in DS ALL.

Measurable residual disease (MRD) testing ​in acute lymphoblastic leukaemia (ALL) is considered standard of care as it is the strongest prognostic factor predicting disease outcome and is critical in therapeutic decision-making and assessing response to therapy. Across many centres, allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQPCR) has been the gold standard for assessing MRD in ALL patients for over 20 years. More recently, next-generation sequencing (NGS) technology has rapidly developed, with several platforms currently available for ALL MRD assessment. Comparative analysis between NGS and ASO-RQPCR is vital to ensure quality performance and to guide future use. Using longitudinal samples from 10 patients in a real-world clinical setting, we correlated the results of the Invivoscribe LymphoTrack system for NGS and ASO-RQPCR. Across the 10 patients, 63 timepoints were analysed showing nine discordant samples, six NGS+/PCR- ​and three PCR+/NGS-. Our findings are consistent with previous literature ​that the two methodologies are highly concordant in ALL MRD assessment (r=0.89). We also demonstrate that NGS has a greater quantitative range allowing for earlier detection of relapse in some cases. Furthermore, in our study, NGS was able to identify an MRD marker in more patients than ASO-RQPCR. Therefore, NGS MRD has potential advantages; however, clinical trials are required to evaluate clinical impact.

Measurable residual disease testing (MRD) in acute lymphoblastic leukaemia (ALL) is considered standard of care as it is the strongest prognostic factor predicting disease outcome and is critical in therapeutic decision making and assessing response to therapy. Across many centres, allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQPCR) has been the gold standard for assessing MRD in ALL patients for over 20 years1,2. More recently, next-generation sequencing (NGS) technology has rapidly developed, with several platforms currently available for ALL MRD assessment. Comparative analysis between NGS and ASO-RQPCR is vital to ensure quality performance and to guide future use. Using longitudinal samples from 10 patients in a real-world clinical setting, we correlated the results of the Invivoscribe LymphoTrack system for NGS and ASO-RQPCR. Across the 10 patients, 63 timepoints were analysed showing nine discordant samples, six NGS+/PCR–, and three PCR+/NGS–. Our findings are consistent with previous literature, that the two methodologies are highly concordant in ALL MRD assessment (r=0.89). We also demonstrate that NGS has greater quantitative range allowing for earlier detection of relapse in some cases. Further, in our study NGS was able to identify an MRD marker in more patients than ASO-RQPCR. NGS MRD therefore has potential advantages, however clinical trials are required to evaluate clinical impact.

Acute leukemia continues to be a major cause of death from disease worldwide and current chemotherapeutic agents are associated with significant morbidity in survivors. While better and safer treatments for acute leukemia are urgently needed, standard drug development pipelines are lengthy and drug repurposing therefore provides a promising approach. Our previous evaluation of FDA-approved drugs for their antileukemic activity identified disulfiram, used for the treatment of alcoholism, as a candidate hit compound. This study assessed the biological effects of disulfiram on leukemia cells and evaluated its potential as a treatment strategy. We found that disulfiram inhibits the viability of a diverse panel of acute lymphoblastic and myeloid leukemia cell lines ( n = 16) and patient-derived xenograft cells from patients with poor outcome and treatment-resistant disease ( n = 15). The drug induced oxidative stress and apoptosis in leukemia cells within hours of treatment and was able to potentiate the effects of daunorubicin, etoposide, topotecan, cytarabine, and mitoxantrone chemotherapy. Upon combining disulfiram with auranofin, a drug approved for the treatment of rheumatoid arthritis that was previously shown to exert antileukemic effects, strong and consistent synergy was observed across a diverse panel of acute leukemia cell lines, the mechanism of which was based on enhanced ROS induction. Acute leukemia cells were more sensitive to the cytotoxic activity of disulfiram than solid cancer cell lines and non-malignant cells. While disulfiram is currently under investigation in clinical trials for solid cancers, this study provides evidence for the potential of disulfiram for acute leukemia treatment. Key messages Disulfiram induces rapid apoptosis in leukemia cells by boosting oxidative stress. Disulfiram inhibits leukemia cell growth more potently than solid cancer cell growth. Disulfiram can enhance the antileukemic efficacy of chemotherapies. Disulfiram strongly synergises with auranofin in killing acute leukemia cells by ROS induction. We propose testing of disulfiram in clinical trial for patients with acute leukemia.

Background: Chromosome 21 is affected in ∼60% of paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients and includes somatic and constitutional gains, intrachromosomal amplification of chromosome 21 (iAMP21), and the translocation t(12;21) resulting in the ETV6::RUNX1 gene fusion. Methods: Since these numeric and structural chromosome 21 alterations are not targetable, we studied the type and frequency of yet-proven targetable events co-occurring with chromosome 21 alterations. Results: Among 307 primary paediatric BCP-ALL cases, JAK/STAT pathway lesions were most frequent in patients with constitutional gain of chromosome 21 (Down syndrome ALL; 35/71, 49%) and iAMP21 (9/22, 41%). RAS pathway lesions were most frequent in high hyperdiploidy (62/108, 57%) and FLT3 lesions were most frequent in iAMP21 (7/22, 32%). Virtually all cases expressed CD19 and CD22 at the cell surface. Positivity for CD20 surface expression ranged from 67% in iAMP21 (8/12) to 20% in ETV6::RUNX1 (26/129). Conclusion: Activated JAK/STAT, RAS or FLT3 signalling, and CD marker surface expression may provide targetable treatment options for the majority of chromosome 21-altered BCP-ALL cases.

Pediatric B-cell precursor (BCP) lymphoblastic malignancies are neoplasms with manifestation either in bone marrow/blood (BCP acute lymphoblastic leukemia, BCP-ALL) or less common in extramedullary tissue (BCP lymphoblastic lymphoma, BCP-LBL). Although both presentations are similar in morphology and immunophenotype, molecular studies are virtually restricted to BCP-ALL so far. The lack of molecular studies on BCP-LBL is due to its rarity and the restriction to small, mostly formalin-fixed paraffin embedded (FFPE) tissues. Here we present the first comprehensive mutational and transcriptional analysis of what we consider the largest BCP-LBL cohort described to date (n=97). Whole exome sequencing indicates a mutational spectrum of BCP-LBL strikingly similar to that found in BCP-ALL. However, epigenetic modifiers were more frequently mutated in BCP-LBL, whereas BCP-ALL was more frequently affected by mutation in genes involved in B-cell development. Integrating copy number alterations, somatic mutations and gene expression by RNA-sequencing revealed that virtually all molecular subtypes originally defined in BCP-ALL are present in BCP-LBL too, with only 7% of lymphomas that were not assigned to a subtype. Similar to BCP-ALL, the most frequent subtypes of BCP-LBL were high hyperdiploidy and ETV6::RUNX1. Tyrosine kinase/cytokine-receptor rearrangements were detected in 7% of BCP-LBL. These results indicate that genetic subtypes can be identified in BCP-LBL using next-generation sequencing, even on FFPE tissue, and may be relevant to guide treatment.

Background The BCR::ABL1 is a hallmark of chronic myeloid leukemia (CML) and is also found in acute lymphoblastic leukemia (ALL). Most genomic breaks on the BCR side occur in two regions - Major and minor - leading to p210 and p190 fusion proteins, respectively. Methods By multiplex long-distance PCR or next-generation sequencing technology we characterized the BCR::ABL1 genomic fusion in 971 patients (adults and children, with CML and ALL: pediatric ALL: n = 353; pediatric CML: n = 197; adult ALL: n = 166; adult CML: n = 255 patients) and designed "Break-App" web tool to allow visualization and various analyses of the breakpoints. Pearson's Chi-Squared test, Kolmogorov-Smirnov test and logistic regression were used for statistical analyses. Results Detailed analysis showed a non-random distribution of breaks in both BCR regions, whereas ABL1 breaks were distributed more evenly. However, we found a significant difference in the distribution of breaks between CML and ALL. We found no association of breakpoints with any type of interspersed repeats or DNA motifs. With a few exceptions, the primary structure of the fusions suggests non-homologous end joining being responsible for the BCR and ABL1 gene fusions. Analysis of reciprocal ABL1::BCR fusions in 453 patients showed mostly balanced translocations without major deletions or duplications. Conclusions Taken together, our data suggest that physical colocalization and chromatin accessibility, which change with the developmental stage of the cell (hence the difference between ALL and CML), are more critical factors influencing breakpoint localization than presence of specific DNA motifs.

Acute lymphoblastic leukemia (ALL) is a cancer of precursor lymphoid cells characterised by clonal proliferations of cells committed to the B- or T-cell lineage. Measurable residual disease (MRD) is the strongest prognostic factor for ALL,1 and is also used to access blinatumomab therapy in B-cell disease. Patients with low or undetectable MRD have an excellent prognosis whereas persistent MRD is a high-risk for relapse.1 Allele-specific oligonucleotide quantitative PCR (ASO qPCR) has been the gold standard for assessing MRD in ALL patients for over 20 years,2 but methods incorporating massively parallel sequencing (MPS) have been increasingly deployed internationally in recent years.

Over the last decade, next generation sequencing has enabled classification of multiple new recurrent genomic drivers of acute lymphoblastic leukemia (ALL). The aim of this study was to describe the genomic drivers of ALL in an adolescent and young adult (AYA) cohort (ALL06 target age 15-39 years, recruited age 16.6-39 years), treated uniformly on a pediatric inspired protocol (the Australasian Leukaemia and Lymphoma Group (ALLG) ALL06 study). ALL06 assessed the safety and efficacy of adapting a pediatric chemotherapy protocol in older patients. Genomic risk classification of B- and T-ALL patients enrolled to the study was based on the use of multiple assays: mRNA-Sequencing, Multiplex Ligation-dependent Probe Amplification (MLPA), immunophenotyping and cytogenetics. Using this approach, 36/40 (90%) B- and 13/17 (76.5%) T-ALL patients were classified according to genomic risk. A strong correlation existed between adverse genomic risk and minimal residual disease (MRD) at the end of consolidation, translating to inferior overall and relapse free survival. Patients with adverse risk genomics who achieved negative MRD status had improved responses compared to those with persistent MRD. Patients with standard risk genomics had excellent responses regardless of MRD status. This is the first report of the impact of genomics in an individual cohort of AYA patients treated on a single protocol. These data argue strongly for incorporation of a genomic risk classification into future ALL treatment paradigms at the time of diagnosis, and also for the rigorous assessment of risk assignments in a group of patients who are not children and not older adults. ACTRN12611000814976 https://anzctr.org.au/

Average intensities of all light (PDX) and heavy (SILAC) Class I phosphosites p(STY) identified in 16 PDXs with Tyrosine kinase downstream targets highlighted in yellow.

Expression of the exon 10 fusion transcript in patient 17 and patient 32. Standard curve was established using patient 32 sample in a range from 25ng/µl to 0.1 ng/µl in 1 in 2 serial dilutions.

Background: The advent of pediatric inspired regimens has improved the outcome for younger adults with Acute Lymphoblastic Leukaemia (ALL), however this comes at a considerable toxicity burden limiting its applicability in older adults. The Australasian Leukaemia and Lymphoma Group (ALLG) undertook a phase 2 proof-of-concept (POC) study of Blinatumomab with RI chemotherapy in adults with newly diagnosed (newDx) Ph neg BCP-ALL. Aims: To assess response, the effectiveness and tolerability of the combination of Blinatumomab with RI chemotherapy following a debulking steroids and chemotherapy for newDx Ph- B-ALL in older adults. Methods: The ALLG ALL8 study (ACTRN12617000084381) is a phase II POC study for patients fit for treatment with a Hyper-CVAD-like regimen (between 40-65 years) with newly diagnosed Ph neg B-lineage ALL. Patients with CNS involvement were excluded. A steroid pre-phase (Prednisolone 100mg daily for 7 days) was followed by a disease debulking phase of cyclophosphamide 150mg/m2 BD day 1-3, vincristine 2mg day 1 & 11 and dexamethasone 10mg/m2 day 1-4 and 11-14. Patients then received alternating cycles of Blinatumomab (at 9mcg/d for the first 7 days of cycle 1 followed by 28mcg/d until day 28) with B-cycles of Hyper-CVAD (Methotrexate 1g/m2 day 1, Cytarabine 3g/m2 BD day 2,3, Methylprednisolone 50mg BD day 1-3) (figure 1) for a total of 4 cycles. All received intrathecal prophylaxis with methotrexate, cytarabine and hydrocortisone prior to blinatumomab treatment blocks and day 1 and 8 of each B-cycle, total of 8 doses. High-risk patients (MLL translocations, hypodiploid, complex karyotype or MRD positive at TP3) were recommended for allogeneic stem cell transplant while others continued to receive 24 months of POMP maintenance. Minimal residual disease testing was performed at a centralised EuroMRD accredited laboratory. MRD positivity was defined as a detectable level of ≥ 1 x 10-4. This is the analysis of the final endpoint as at 14th February 2023. POC criteria were defined on an observed event free rate of ≥ 63%. Results: 30 subjects were enrolled (21 (70%) Male), with a median age of 51.7 years (39.5 – 66.5 years) with 14 (47%) ECOG 0, 12 (40%) ECOG 1, and 4 (13%) ECOG 2 at time of study entry. 5 (17%) subjects had high-risk cytogenetics. 100% of subjects attained composite CR (CR/CRi) with 28 (93%) attaining this by the end of 1B, and a further 2 by the end of cycle 2B. MRD response was ≤10-4 in 19/27 (70%) of subjects at the end of 1B and 20/24 (83%) of subjects at the end of 2B. The estimated EFS at 24 months was 60.4% (median EFS 36.1 mo) with OS at 24 months of 78.6% (median OS NR). This was insufficient to meet the pre-specified proof-of-concept criteria. The regimen was well tolerated with the major toxicity being infective (53 episodes of infection). There were 2 episodes of cytokine release syndrome (1 grade 3), and 7 episodes of grade 3 neurological toxicity (1 myelopathy and 4 neuropathy related to chemotherapy and 2 episodes of neurotoxicity related to blinatumomab). 4 patients proceeded to alloHSCT. Summary/Conclusion: Blinatumomab with chemotherapy was well tolerated with a high rate of remission and deep MRD responses in the majority of patients. Responses appeared durable with this lower intensity treatment approach despite a low rate of allogeneic stem cell transplantation and the exclusion of anthracyclines and asparaginase from treatment. Despite failing to meet proof-of-concept criteria the EFS and OS were encouraging for older ALL patients and demonstrates the feasibility of combining low-intensity chemotherapy with blinatumomab in older adults with BCP-ALL.Keywords: B cell acute lymphoblastic leukemia, Acute lymphoblastic leukemia

Legend for Supplementary Figures S1-S9.

Abstract IKZF1 deletions are associated with an increased risk of relapse in B-cell precursor acute lymphoblastic leukemia (B-ALL), and their accurate detection has great clinical impact. Here, we included four international cohorts of pediatric and adult patients with B-ALL, and reviewed literature to illustrate the recombination map of IKZF1 deletions, with a focus at non-recurrent deletions. We provide a substantial basis for the improvement of diagnostic methods based on MLPA and multiplex PCR for the identification of IKZF1 deletions, and also demonstrate that rare IKZF1 deletions increase the incidence of relapse in these patients. Of note, non-recurrent deletions comprised a wide range of alterations, but the majority were Δ1 and Δ1–3. They were often associated with reciprocal IKZF1 fusions. So far, a total of 23 IKZF1 gene fusions were identified in B-ALL. We also verified the occurrence of the heptamer sequence (E-value: 9.9 x 10− 9) and an enrichment of GC nucleotides (71% versus 56%; P value = 4.9 x 10− 3) exclusively within breakpoint clusters, suggesting that RAG recombination and TdT activity may promote the majority of IKZF1 deletions, although rare types of alterations may be associated with other molecular mechanism of leukemogenesis, such as microhomology-mediated end joining.

Supplementary Figure Legends 1-3 from ODC1 Is a Critical Determinant of MYCN Oncogenesis and a Therapeutic Target in Neuroblastoma

Average normalized ratio of phosphorylated tyrosine (pY) sites of tyrosine kinases identified in 16 PDXs

AbstractPediatric regimens have improved outcomes in adolescent and young adult (AYA) acute lymphoblastic leukemia (ALL). However, results remain inferior to children with ALL. The Australasian Leukaemia and Lymphoma Group (ALLG) ALL06 study (anzctr.org.au/ACTRN12611000814976) was designed to assess whether a pediatric ALL regimen (Australian and New Zealand Children's Haematology and Oncology Group [ANZCHOG] Study 8) could be administered to patients aged 15 to 39 years in a comparable time frame to children as assessed by the proportion of patients completing induction/consolidation and commencing the next phase of therapy (protocol M or high-risk [HR] treatment) by day 94. Minimal residual disease (MRD) response stratified patients to HR treatment and transplantation. From 2012 to 2018, a total of 86 patients were enrolled; 82 were eligible. Median age was 22 years (range, 16-38 years). Induction/consolidation was equally deliverable in ALL06 as in Study 8. In ALL06, 41.5% (95% confidence interval [CI], 30.7-52.9) commenced protocol M or HR therapy by day 94 vs 39.3% in Study 8 (P = .77). Median time to protocol M/HR treatment was 96 days (interquartile range, 87.5-103 days) in ALL06 vs 98 days in Study 8 (P = .80). Induction mortality was 3.6%. With a median follow-up of 44 months (1-96 months), estimated 3-year disease-free survival was 72.8% (95% CI, 62.8-82.7), and estimated 3-year overall survival was 74.9% (95% CI, 65.3-84.5). End induction/consolidation MRD negativity rate was 58.6%. Body mass index ≥30 kg/m2 and day 79 MRD positivity were associated with poorer disease-free survival and overall survival. Pediatric therapy was safe and as deliverable in AYA patients as in children with ALL. Intolerance of pediatric ALL induction/consolidation is not a major contributor to inferior outcomes in AYA ALL.