Background: Incorporating genetics into risk stratification for the treatment of childhood acute lymphoblastic leukaemia (ALL) has contributed to increased survival rates. About 30% of patients (B-other-ALL) harbour none of the known major chromosomal changes. Recently, we estimated that up to two thirds of B-other-ALL can be classified into novel subtypes using FISH and MLPA alone. We showed that ABL-class fusions and ERG deletions were linked to prognosis (Schwab et al, BJHaem, 2022). Aims: To characterise B-other-ALL in the UKALL2003 trial using next generation sequencing (NGS), to evaluate the added benefits of these techniques and validate the prognostic significance of B-other-ALL subtypes. Methods: B-other-ALL patients were tested using whole genome sequencing (WGS) (n=158) and bespoke targeted NGS (t-NGS) for the detection of abnormalities in 64 genes commonly mutated or rearranged in B-ALL (n=180), 42 patients had both WGS and t-NGS. Data were integrated with results from FISH, MLPA and cytogenetics. Results: A representative cohort of 347 B-other-ALL was classified into subgroups, including six with ≥20 cases (Figure). Among patients tested by WGS, 92% (n=146/158) were classified compared to 75% (n=104/138) tested by t-NGS. NGS-based approaches allowed for the detection of PAX5 P80R, IKZF1 N169Y and ZEB2 H1038R and fusion partner genes. Identification of DUX4-r was not possible using t-NGS. PAX5alt was the most frequently observed (n=90), including dic(9;20) (n=27), dic(9;12) (n=11), PAX5 fusions (n=21), PAX5 mutation (n=11) and PAX5-ITD (n=12). Additionally, 11 patients were observed with PAX5 P80R mutations, classified as a separate subgroup due to the associated gene expression signature. PAX5alt had an outcome similar to B-other-ALL overall (overall survival (OS) at 10yrs 82% vs 86%). There was no significant difference in outcome between PAX5 abnormalities, although an association with age was observed: dic(9;20) was more commonly observed in children aged 1-4 (p< 0.001), while both dic(9;12) and PAX5 P80R were seen in older children aged 10-15 years (p=0.005). One subgroup with DUX4 rearrangements and/or ERG deletions (DUX4-r/ERG-d) (n=78) had DUX4 involvement confirmed by WGS in 57 patients. In the remaining 21 patients ERG-d were identified by t-NGS and/or MLPA. ERG-d is exclusive to DUX4-r patients, thus can be used as a surrogate marker. In line with other studies, patients with DUX4-r had a lower relapse rate (RR) (5%) and improved OS (96%) compared to other subgroups. Patients classified as ETV6-RUNX1-like (n=21) were characterised by multiples abnormalities of ETV6, including rearrangements (n=17) and/or deletion (n=12) as well as rearrangements (n=7) and/or deletions (n=6) of IKZF1. An ETV6-RUNX1-like gene expression signature was confirmed by RNA-Seq (n=6). No relapses or deaths were reported within this subgroup. As previously reported, ABL-class fusions (n=25) were associated with an inferior outcome compared to other subgroups (RR=63% and OS=50%). Patients with rearrangements of CRLF2 (n=52) and ZNF384 (n=37) had similar outcomes to the cohort overall. Image:Summary/Conclusion: A combination of techniques has classified a representative cohort of B-other-ALL patients from UKALL2003 into clinically relevant subgroups. The identification of DUX4-r, subgroup defining mutations and fusion partner genes demonstrates the value of NGS-based approaches. We have confirmed the good and poor prognostic associations of DUX4-r and ABL-class fusions, respectively, and identified ETV6-RUNX1-like subgroup to have a good prognosis.