Genetic characterisation of childhood B-other-acute lymphoblastic leukaemia in UK patients by fluorescence in situ hybridisation and Multiplex Ligation-dependent Probe Amplification.

While next-generation sequencing technologies provide excellent strategies to screen for newly defined genetic abnormalities of prognostic or therapeutic significance in patients with B-other-acute lymphoblastic leukaemia (ALL), they are not widely available. We used a dual screening approach, incorporating fluorescence in situ hybridisation (FISH) and Multiplex Ligation-dependent Probe Amplification (MLPA), to establish the frequency and long-term outcome of a representative cohort of specific subgroups of B-other-ALL recruited to the childhood ALL trial, UKALL2003. We focussed on abnormalities of known prognostic significance, including ABL-class fusions and ERG deletions, as a surrogate marker for DUX4-rearranged ALL. ABL-class fusions accounted for ~4% of B-other-ALL and were associated with high levels of minimal residual disease (MRD; 14/23 with MRD >5%) and a high relapse rate (55·7%) following treatment without tyrosine kinase inhibitor (TKI), confirming the importance of prospective screening with a view to incorporating TKI into therapy. Patients with deletions of ERG (~10% of B-other-ALL) had a 10-year event-free-survival of 97·2%, validating previous reports of their excellent outcome. Rearrangements of ZNF384, MEF2D and NUTM1 were observed at low frequencies. Here, we estimate that approximately one third of B-other-ALL patients can be reliably classified into one of the known genetic subgroups using our dual screening method. This approach is rapid, accurate and readily incorporated into routine testing.

Genetic and genomic analysis of acute lymphoblastic leukemia in older adults reveals a distinct profile of abnormalities: analysis of 210 patients from the UKALL14 and UKALL60+ clinical trials.

Despite being predominantly a childhood disease, the incidence of acute lymphoblastic leukemia (ALL) has a second peak in adults aged 60 years and over. These older adults fare extremely poorly with existing treatment strategies and very few studies have undertaken a comprehensive genetic and genomic characterization to improve prognosis in this age group. We performed cytogenetic, single nucleotide polymorphism (SNP) array and next-generation sequencing (NGS) analyses on samples from 210 patients aged ≥60 years from the UKALL14 and UKALL60+ clinical trials. BCR-ABL1-positive disease was present in 26% (55/210) of patients, followed by low hypodiploidy/near triploidy in 13% (28/210). Cytogenetically cryptic rearrangements in CRLF2, ZNF384 and MEF2D were detected in 5%, 1% and <1% of patients, respectively. Copy number abnormalities were common and deletions in ALL driver genes were seen in 77% of cases. IKZF1 deletion was present in 51% (40/78) of samples tested and the IKZF1plus profile was identified in over a third (28/77) of cases of B-cell precursor ALL. The genetic good-risk abnormalities high hyperdiploidy (n=2), ETV6-RUNX1 (no cases) and ERG deletion (no cases) were exceptionally rare in this cohort. RAS pathway mutations were seen in 17% (4/23) of screened samples. KDM6A abnormalities, including biallelic deletions, were discovered in 5% (4/78) of SNP arrays and 9% (2/23) of NGS samples, and represent novel, potentially therapeutically actionable lesions using EZH2 inhibitors. Outcome remained poor with 5-year event-free and overall survival rates of 17% and 24%, respectively, across the cohort, indicating a need for novel therapeutic strategies.

In vivo synaptic scaling is mediated by GluA2-lacking AMPA receptors in the embryonic spinal cord.

When spiking activity within a network is perturbed for hours to days, compensatory changes in synaptic strength are triggered that are thought to be important for the homeostatic maintenance of network or cellular spiking activity. In one form of this homeostatic plasticity, called synaptic scaling, all of a cell's AMPAergic miniature postsynaptic currents (mEPSCs) are increased or decreased by some scaling factor. Although synaptic scaling has been observed in a variety of systems, the mechanisms that underlie AMPAergic scaling have been controversial. Certain studies find that synaptic scaling is mediated by GluA2-lacking calcium receptors (CP-AMPARs), whereas others have found that scaling is mediated by GluA2-containing calcium-impermeable receptors (CI-AMPARs). Spontaneous network activity is observed in most developing circuits, and in the spinal cord this activity drives embryonic movements. Blocking spontaneous network activity in the chick embryo by infusing lidocaine in vivo triggers synaptic scaling in spinal motoneurons; here we show that AMPAergic scaling occurs through increases in mEPSC conductance that appear to be mediated by the insertion of GluA2-lacking AMPA receptors at the expense of GluA2-containing receptors. We have previously reported that in vivo blockade of GABAA transmission, at a developmental stage when GABA is excitatory, also triggered AMPAergic synaptic scaling. Here, we show that this form of AMPAergic scaling is also mediated by CP-AMPARs. These findings suggest that AMPAergic scaling triggered by blocking spiking activity or GABAA receptor transmission represents similar phenomena, supporting the idea that activity blockade triggers scaling by reducing GABAA transmission.

A robust and validated integrated prognostic index for defining risk groups in adult acute lymphoblastic leukemia: an EWALL collaborative study.

ABSTRACT: Risk stratification is crucial to the successful treatment of acute lymphoblastic leukemia (ALL). Although numerous risk factors have been identified, an optimal prognostic model for integrating variables has not been developed. We used individual patient data from 4 contemporary academic national clinical trials, UKALL14, NILG-ALL10/07, GIMEMA-LAL1913, and PETHEMA-ALL-HR2011, to generate and validate the European Working Group for Adult ALL prognostic index (EWALL-PI), which is based on white blood cell count, genetics, and end of induction minimal residual disease (MRD). Individual patient risk scores were calculated for 778 patients aged 15 to 67 years in complete remission using the validated UKALL-PI formula, applying minor modifications to reflect differences between pediatric and adult ALL. Per-trial analysis revealed that EWALL-PI correlated with relapse and death. Regression analysis revealed that each unit increase in EWALL-PI increased the risk of relapse or death by ∼30% with no evidence of heterogeneity across trials or patient subgroups. EWALL-PI-defined risk models outperformed the stratification algorithms used by each trial. Threshold analysis revealed an EWALL-PI threshold that divided patients with B cell and T cell into standard (EWALL-PI <2.50) and high (EWALL-PI ≥2.50) risk groups, respectively. Per-trial analysis showed that patients at high risk had a significantly increased relapse rate and inferior survival compared with patients with standard risk (subdistribution hazard ratio for relapse, ranged from 1.85 to 3.28; hazard ratio for death, 1.73 to 3.03). Subgroup analysis confirmed the robustness of these risk groups by sex, age, white blood cell count, and lineage. In conclusion, we validated an integrated risk model across 4 independent adult ALL clinical trials, demonstrating its utility defining clinically relevant risk groups.

Integrative genomic analysis of childhood acute lymphoblastic leukaemia lacking a genetic biomarker in the UKALL2003 clinical trial.

Incorporating genetics into risk-stratification for treatment of childhood B-progenitor acute lymphoblastic leukaemia (B-ALL) has contributed significantly to improved survival. In about 30% B-ALL (B-other-ALL) without well-established chromosomal changes, new genetic subtypes have recently emerged, yet their true prognostic relevance largely remains unclear. We integrated next generation sequencing (NGS): whole genome sequencing (WGS) (n = 157) and bespoke targeted NGS (t-NGS) (n = 175) (overlap n = 36), with existing genetic annotation in a representative cohort of 351 B-other-ALL patients from the childhood ALL trail, UKALL2003. PAX5alt was most frequently observed (n = 91), whereas PAX5 P80R mutations (n = 11) defined a distinct PAX5 subtype. DUX4-r subtype (n = 80) was defined by DUX4 rearrangements and/or ERG deletions. These patients had a low relapse rate and excellent survival. ETV6::RUNX1-like subtype (n = 21) was characterised by multiple abnormalities of ETV6 and IKZF1, with no reported relapses or deaths, indicating their excellent prognosis in this trial. An inferior outcome for patients with ABL-class fusions (n = 25) was confirmed. Integration of NGS into genomic profiling of B-other-ALL within a single childhood ALL trial, UKALL2003, has shown the added clinical value of NGS-based approaches, through improved accuracy in detection and classification into the range of risk stratifying genetic subtypes, while validating their prognostic significance.

Time to Cure for Childhood and Young Adult Acute Lymphoblastic Leukemia Is Independent of Early Risk Factors: Long-Term Follow-Up of the UKALL2003 Trial.

PURPOSE: The aim of the randomized trial, UKALL2003, was to adjust treatment intensity on the basis of minimal residual disease (MRD) stratification for children and young adults with acute lymphoblastic leukemia. We analyzed the 10-year randomized outcomes and the time for patients to be considered cured (ClinicalTrials.gov identifier: NCT00222612). METHODS: A total of 3,113 patients were analyzed including 1,054 patients who underwent random assignment (521 MRD low-risk and 533 MRD high-risk patients). Time to cure was defined as the point at which the chance of relapse was < 1%. The median follow-up time was 10.98 (interquartile range, 9.19-13.02) years, and survival rates are quoted at 10 years. RESULTS: In the low-risk group, the event-free survival was 91.7% (95% CI, 87.4 to 94.6) with one course of delayed intensification versus 93.7% (95% CI, 89.9 to 96.1) with two delayed intensifications (adjusted hazard ratio, 0.73; 95% CI, 0.38 to 1.40; P = .3). In the high-risk group, the event-free survival was 82.1% (95% CI, 76.9 to 86.2) with standard therapy versus 87.1% (95% CI, 82.4 to 90.6) with augmented therapy (adjusted hazard ratio, 0.68; 95% CI, 0.44 to 1.06; P = .09). Cytogenetic high-risk patients treated on augmented therapy had a lower relapse risk (22.1%; 95% CI, 15.1 to 31.6) versus standard therapy (52.4%; 95% CI, 28.9 to 80.1; P = .016). The initial risk of relapse differed significantly by sex, age, MRD, and genetics, but the risk of relapse for all subgroups quickly coalesced at around 6 years after diagnosis. CONCLUSION: Long-term outcomes of the UKALL2003 trial confirm that low-risk patients can safely de-escalate therapy, while intensified therapy benefits patients with high-risk cytogenetics. Regardless of prognosis, the time to cure is similar across risk groups. This will facilitate communication to patients and families who pose the question "When am I/is my child cured?"

EVITA Dengue: a cluster-randomized controlled trial to EValuate the efficacy of Wolbachia-InfecTed Aedes aegypti mosquitoes in reducing the incidence of Arboviral infection in Brazil.

BACKGROUND: Arboviruses transmitted by Aedes aegypti including dengue, Zika, and chikungunya are a major global health problem, with over 2.5 billion at risk for dengue alone. There are no licensed antivirals for these infections, and safe and effective vaccines are not yet widely available. Thus, prevention of arbovirus transmission by vector modification is a novel approach being pursued by multiple researchers. However, the field needs high-quality evidence derived from randomized, controlled trials upon which to base the implementation and maintenance of vector control programs. Here, we report the EVITA Dengue trial design (DMID 17-0111), which assesses the efficacy in decreasing arbovirus transmission of an innovative approach developed by the World Mosquito Program for vector modification of Aedes mosquitoes by Wolbachia pipientis. METHODS: DMID 17-0111 is a cluster-randomized trial in Belo Horizonte, Brazil, with clusters defined by primary school catchment areas. Clusters (n = 58) will be randomized 1:1 to intervention (release of Wolbachia-infected Aedes aegypti mosquitoes) vs. control (no release). Standard vector control activities (i.e., insecticides and education campaigns for reduction of mosquito breeding sites) will continue as per current practice in the municipality. Participants (n = 3480, 60 per cluster) are children aged 6-11 years enrolled in the cluster-defining school and living within the cluster boundaries who will undergo annual serologic surveillance for arboviral infection. The primary objective is to compare sero-incidence of arboviral infection between arms. DISCUSSION: DMID 17-0111 aims to determine the efficacy of Wolbachia-infected mosquito releases in reducing human infections by arboviruses transmitted by Aedes aegypti and will complement the mounting evidence for this method from large-scale field releases and ongoing trials. The trial also represents a critical step towards robustness and rigor for how vector control methods are assessed, including the simultaneous measurement and correlation of entomologic and epidemiologic outcomes. Data from this trial will inform further the development of novel vector control methods. TRIAL REGISTRATION: ClinicalTrials.gov NCT04514107 . Registered on 17 August 2020 Primary sponsor: National Institute of Health, National Institute of Allergy and Infectious Diseases.

Prognostic impact of chromosomal abnormalities and copy number alterations in adult B-cell precursor acute lymphoblastic leukaemia: a UKALL14 study.

Chromosomal abnormalities are established prognostic markers in adult ALL. We assessed the prognostic impact of established chromosomal abnormalities and key copy number alterations (CNA) among 652 patients with B-cell precursor ALL treated on a modern MRD driven protocol. Patients with KMT2A-AFF1, complex karyotype (CK) and low hypodiploidy/near-triploidy (HoTr) had high relapse rates 50%, 60% & 53% and correspondingly poor survival. Patients with BCR-ABL1 had an outcome similar to other patients. JAK-STAT abnormalities (CRLF2, JAK2) occurred in 6% patients and were associated with a high relapse rate (56%). Patients with ABL-class fusions were rare (1%). A small group of patients with ZNF384 fusions (n = 12) had very good survival. CNA affecting IKZF1, CDKN2A/B, PAX5, BTG1, ETV6, EBF1, RB1 and PAR1 were assessed in 436 patients. None of the individual deletions or profiles were associated with survival, either in the cohort overall or within key subgroups. Collectively these data indicate that primary genetic abnormalities are stronger prognostic markers than secondary deletions. We propose a revised UKALL genetic risk classification based on key established chromosomal abnormalities: (1) very high risk: CK, HoTr or JAK-STAT abnormalities; (2) high risk: KMT2A fusions; (3) Tyrosine kinase activating: BCR-ABL1 and ABL-class fusions; (4) standard risk: all other patients.