Molecular characterization and biomarker identification in paediatric B-cell acute lymphoblastic leukaemia

Abstract

B-cell acute lymphoblastic leukaemia (B-ALL) is the most prevalent hematologic malignancy in children and a leading cause of mortality. Managing B-ALL remains challenging due to its heterogeneity and relapse risk. This study aimed to delineate the molecular features of paediatric B-ALL and explore the clinical utility of circulating tumour DNA (ctDNA). We analysed 146 patients with paediatric B-ALL who received systemic chemotherapy. The mutational landscape was profiled in bone marrow (BM) and plasma samples using next-generation sequencing. Minimal residual disease (MRD) testing on day 19 of induction therapy evaluated treatment efficacy. RNA sequencing identified gene fusions in 61% of patients, including 37 novel fusions. Specifically, the KMT2A-TRIM29 novel fusion was validated in a boy who responded well to initial therapy but relapsed after 1 year. Elevated mutation counts and maximum variant allele frequency in baseline BM were associated with significantly poorer chemotherapy response (p = 0.0012 and 0.028, respectively). MRD-negative patients exhibited upregulation of immune-related pathways (p < 0.01) and increased CD8⁺ T cell infiltration (p = 0.047). Baseline plasma ctDNA exhibited high mutational concordance with the paired BM samples and was significantly associated with chemotherapy efficacy. These findings suggest that ctDNA and BM profiling offer promising prognostic insights for paediatric B-ALL management.

Keywords: ctDNA; immune microenvironment; next‐generation sequencing; paediatric B‐cell acute lymphoblastic leukaemia; predictive biomarker.

FIGURE 1

Study design.

FIGURE 2

Gene fusions in paediatric B‐ALL identified using RNA‐seq. (A) Gene fusions detected in patients. (B) Overview of the 37 novel fusions and proximal chromosomal locations of their corresponding fusion partners. (C) Representative illustration of novel KMT2A‐TRIM29 fusion in a patient. The fusion was validated using RT‐PCR and Sanger sequencing. A representative FISH graph depicts the partial loss of the KMT2A gene detected in the patient. B‐ALL, B‐cell acute lymphoblastic leukaemia; FISH, fluorescence in situ hybridization; RNA‐seq, RNA sequencing; RT‐PCR, reverse transcription–PCR.

FIGURE 3

Genomic landscapes and variation differences across B‐ALL subtypes. (A) Oncoprint depicting the patients' genetic variations. (B) The prevalence of top genetic and pathway alterations across different B‐ALL subtypes. B‐ALL, B‐cell acute lymphoblastic leukaemia.

FIGURE 4

Baseline mutational and immune features associated with therapeutic efficacy in B‐ALL. Therapeutic efficacy was evaluated using the MRD status on Day 19 of treatment. MRD− patients had significantly lower mutation counts (A) and maxVAF (B) compared to MRD+ patients. MRD− patients had lower trends in KRAS (C) and PTPN11 (D) mutation frequencies than MRD+ patients. Differences in pathway alterations were identified using KEGG (E), GSEA (F) and GO enrichment analysis (G, H). Infiltration of CD8⁺ T cells was significantly enriched in MRD− patients compared to MRD+ patients (I). B‐ALL, B‐cell acute lymphoblastic leukaemia; GO, Gene Ontology; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; maxVAF, maximum variant allele frequency; MRD, minimal residual disease; MRD−, MRD‐negative; MRD+, MRD‐positive.

FIGURE 5

Plasma ctDNA has high mutational concordance with paired BM and associates with therapeutic efficacy in B‐ALL. (A) The detection ratio of genetic variations indicated no significant difference between plasma and BM. (B, C) Plasma ctDNA had significantly lower mutation counts (B) and maxVAF (C) than BM. (D, E) BM and plasma ctDNA had high mutational concordance. (F) Plasma ctDNA positivity at baseline was significantly associated with MRD status at Day 19 (i.e., therapeutic efficacy). MRD− patients had significantly lower mutation counts in baseline plasma (G) and a lower trend of maxVAF (H) than MRD+ patients. B‐ALL, B‐cell acute lymphoblastic leukaemia; BM, bone marrow; ctDNA, circulating tumour DNA; maxVAF, maximum variant allele frequency; MRD, minimal residual disease; MRD−, MRD‐negative; MRD+, MRD‐positive.