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

Abstract

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 DUX4rearranged 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 (55Á7%) 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 97Á2%, 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.

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

“…Cytogenetics, FISH and Multiplex Ligation-dependent Probe Ampli cation (MLPA) (MRC Holland, The Netherlands) were performed, as previously described. [23][24][25] WTS was performed on RNA samples, extracted from diagnostic bone marrow using the RNeasy Extraction kit (Qiagen, Manchester, UK), from 85 patients within the same B-ALL cohort. Sequencing reads were processed and aligned to Human Reference Genome GRCh38.…”

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

“…Neither DUX4 nor MEF2D were included in the t-NGS kit, as they were unknown at the time of design, thus highlighting the importance of exibility when choosing tools for diagnostic testing. We were able to screen for MEF2D rearrangements by FISH, 12 however, accurate DUX4-r identi cation was only possible using WGS with a bespoke analysis pipeline. 22 It remains to be determined whether standard PCR testing or t-NGS with a similar customised pipeline can reliably identify DUX4-r.…”

“…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).…”

“…These remained unclassi ed, except for four patients who presented with a subtype-de ning chromosomal abnormality by cytogenetic analysis: PAX5alt with dic(9;20)(p11 ~ 13;q11) (n = 3) and t(6;14)(p22;q32)/IGH::ID4 (n = 1). Additionally, 304 patients, not tested by NGS, were screened using FISH and/or MLPA, with 93 classi ed as previously described 12 (Fig. 1).…”

“…1 Among approximately 30% of B-ALL patients (B-other-ALL) lacking these subtypede ning abnormalities, distinct genetic entities have emerged. [2][3][4][5][6][7][8][9][10] For example, DUX4-rearranged (DUX4r) and patients with ABL-class fusions have been shown to have good and poor outcomes, respectively, 4,[10][11][12][13][14][15][16][17][18][19][20][21] while the clinical relevance of other subtypes, including alterations of PAX5 (PAX5alt) and ETV6::RUNX1-like, remain unclear. Although these subtypes display characteristic gene expression signatures, their underlying genetic pro les are heterogeneous.…”