Whole genome sequencing provides comprehensive genetic testing in childhood B-cell acute lymphoblastic leukaemia

Abstract

Childhood B-cell acute lymphoblastic leukaemia (B-ALL) is characterised by recurrent genetic abnormalities that drive risk-directed treatment strategies. Using current techniques, accurate detection of such aberrations is challenging, due to the rapidly expanding list of key genetic abnormalities. Whole genome sequencing (WGS) has the potential to revolutionise genetic testing, but requires comprehensive validation. We performed WGS on 210 childhood B-ALL samples annotated with clinical and genetic data. We devised a molecular classification system to subtype these patients based on identification of key genetic changes in tumour-normal and tumour-only analyses. This approach detected 294 subtype-defining genetic abnormalities in 96% (202/210) patients. Novel genetic variants, including fusions involving genes in the MAP kinase pathway, were identified. There was excellent concordance with standard-of-care methods and whole transcriptome sequencing (WTS). We expanded the catalogue of genetic profiles that reliably classify PAX5alt and ETV6::RUNX1-like subtypes. Our novel bioinformatic pipeline improved detection of DUX4 rearrangements (DUX4-r). We defined the excellent survival rates of DUX4-r and ETV6::RUNX1-like subtypes. Overall, we comprehensively validated that WGS provides a standalone, reliable genetic test to detect all subtype-defining genetic abnormalities in B-ALL, accurately classifying patients for risk-directed treatment stratification, while simultaneously performing as an excellent research tool to identify novel disease biomarkers.

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

“…WGS detected a higher number of focal copy number abnormalities (CNA) than MLPA, notably of ERG deletions, as we have previously reported. 22 Similarly, t-NGS identi ed ERG deletions in three patients that had been called normal by MLPA. These deletions were either focal, with evidence of a single probe deletion only by MLPA, which is insu cient to call a deletion (n = 2), or sub-clonal, where the MLPA ratio for the deleted probes was 0.76-0.9 but not below the required 0.75 cut-off level to call a deletion (n = 1).…”

“…32 Whole Genome Sequencing WGS was performed on matched diagnostic and remission DNA samples, as previously described. 22 Fluorescence in-situ hybridisation FISH results were available for rearrangements associated with B-other-ALL from our previously published studies, including: ABL-class genes: ABL1, ABL2, PDGFRB/CSF1R; JAK-STAT pathway genes: CRLF2, JAK2; other newly de ned subtypes: ZNF384, MEF2D and NUTM1; 12 as well as IGH and associated partner genes. 33 Additionally, FISH was performed to identify rearrangements of ETV6, PAX5 and IKZF1, using commercial or home-grown break-apart FISH probes 34 (Cytocell, UK; Leica Microsystems, UK).…”

“…Four cases tested by both techniques remained unclassi ed, although WGS identi ed novel abnormalities, including three in the MAP kinase pathway, as previously reported. 22 Although the t-NGS panel did not include DUX4, t-NGS identi ed ERG abnormalities in 8/11 DUX4-r cases analysed by WGS and t-NGS, including deletions (n = 7) and inversions (n = 1).…”

“…These cases were excluded from the ETV6::RUNX1-like subtype, as previous studies have shown that these fusions do not drive the distinctive gene expression signature associated with this subtype. 2,4,9 Other Subtypes Within the DUX4-r subtype (n = 80), WGS identi ed DUX4 rearrangements in 61 patients (see accompanying article 22 ), while ERG deletions were identi ed in a further 19 cases tested by t-NGS and/or MLPA (Supplementary Fig. 4).…”

“…2 We have demonstrated that whole genome sequencing (WGS) provides an excellent method for classifying B-ALL patients into clinically relevant genetic subtypes. 22 Here, we combine results from cytogenetics, uorescence in situ hybridisation (FISH) and Multiplex Ligation-dependent Probe Ampli cation (MLPA) with both WGS and targeted next generation sequencing (t-NGS) of a large cohort of B-other-ALL from a single highly successful UK childhood ALL clinical trial, UKALL2003. Using this integrated approach, we have accurately classi ed these patients into 15 distinct genetic subtypes, described the spectrum of underlying abnormalities, and clari ed their frequency and clinical signi cance.…”