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 can be challenging, due to the rapidly expanding list of key genetic abnormalities. Whole genome sequencing (WGS) has the potential to improve 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. WGS results were concordant 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): a good-risk B-ALL subtype with high survival rates. Overall, we have validated that WGS provides a standalone, reliable genetic test to detect all subtype-defining genetic abnormalities in B-ALL, accurately classifying patients for the risk-directed treatment stratification, while simultaneously performing as a research tool to identify novel disease biomarkers.

Fig. 1. The landscape of subtype-defining genetic alterations.

Oncoplot showing the subtype definition of each case and the associated subtype-defining genetic abnormalities observed by WGS. Colours define the subtype of each patient and the type of rearrangement for each genetic abnormality.

Fig. 2. Molecular subtyping of B-other-ALL.

A Genetic subtypes as defined by WGS of 173 B-ALL patients, including 172 B-other-ALL patients (cohort 2) and one patient with iAMP21-ALL (cohort 1) in which a DUX4-r was observed. Eight patients had no subtype-defining genetic abnormalities (termed ‘other’). B t-distributed stochastic neighbour embedding (tSNE) plot of 85 B-other-ALL patients from this study (red triangles) and 1452 B-ALL patients from our previous study, demonstrating the subtype groupings (colour coded) of each patient based on WTS data [7]. This analysis validated the identification of recent subtype-defining genetic abnormalities that were identified within the WGS data.

Fig. 3. Novel subtype-defining abnormalities discovered by WGS.

A Genetic abnormalities (CNA, SV, missense/frameshift/splice site mutations and small insertions (<500 bp)) involving individual exons of PAX5, CDKN2A, CDKN2B and MTAP in PAX5alt cases (n = 29). ‘CN profile’ describes a group of PAX5alt cases identified by WGS with PAX5 loss, biallelic CDKN2A and CDKN2B loss, often with MTAP abnormalities. PAX5alt subtyping was validated in all patients with matched WTS data (n = 17). B, C The mutational load in ETV6::RUNX1-like patients is shown to be elevated (median 2.91, range 0.63–6.5) (B), andthe AID/APOBEC family of cytidine deaminases represents >5% of the mutational signature profile in 10/12 ETV6::RUNX1-like cases (green) (C). Enrichment of the AID/APOBEC mutational signature is also evident in ETV6::RUNX1 patients (pink), as previously reported [30].

Fig. 4. Key genetic abnormalities in eight “other” patients subtyped by WGS.

The oncoplot provides details of genetic abnormalities that were clonal, recurrent or within ALL-associated genes detected by WGS. The primary subtype (black) defined by WTS is shown for four patients with matched WTS data. The subtype definition of each case based on Prediction Analysis of Microarrays (PAM) or two-dimensional t-distributed stochastic neighbour embedding (tSNE) analyses is shown. The presence of a subtype-defining fusion transcript in each sample is given; apart from patient 22980 with a ZNF618-NUTM1 fusion by WTS only. No fusion transcript was detected in the remaining patients.

Fig. 5. Characterisation of DUX4-r patients.

A Spanning reads per billion (SRPB) between the IGH and DUX4 loci. IGH::DUX4 patients (n = 57) were found to have 11.1-157.3 SRPB. ALL samples from other subtypes (and matched germline samples) show lower values, ranging from 0–9.6 SPRB. A threshold of >10 SRPB was applied to define patients with IGH::DUX4 abnormalities. Three DUX4-r patients did not show >10 SRPB as the rearrangement involved alternative genomic regions. B Breakpoint mapping of IGH::DUX4 breakpoint within the IGH locus of 53 patients. Breakpoints mapping to the forward (red) or reverse (brown) strand are shown. A cluster breakpoint region (CBR) within the IGH J (joining) segment is present, in which 47/53 cases harbour a breakpoint (chr14:105860602-105865246). C ERG abnormalities are seen in 68.3% (41/60) of DUX4-r patients. The exon structure of ERG is depicted in NM_001136154.1 and NM_182918.4 (not to scale); exons are numbered and represented with purple rectangles. The type of abnormalities range from deletion (pink), inversion (yellow), mutation (lollipop stick) and translocation (lollipop stick labelled ‘BND’). The width of the ribbon represents the number of cases with the abnormality.