Background

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) represents the most common hematologic malignancy in pediatric populations.¹ Approximately 25% of patients with BCP-ALL express the ETV6-RUNX1 fusion gene (ER), arising from the chromosomal translocation t(12;21)(p13;q22). Conversely, in a small proportion of patients referred to as B-other BCP-ALL, no clear chromosomal or genetic anomalies can be identified, compromising diagnosis, prognostication, risk stratification, and treatment decisions (e.g., targeted therapies).

Recombination-activating genes (RAGs) have been demonstrated to have an essential role in the expression of membrane bound B-cell antigen receptors, with mutations contributing to the malignant processes leading to B-cell ALL.¹ While RAGs do not directly drive the ER fusion gene translocation, they have been identified as driving rearrangements in other distinct loci within the subtype.¹ Here, we describe the work of Dongfeng Chen and colleagues,¹ published in Cancer Medicine, exploring the RAG1 expression profiles among patients with BCP-ALL and whether any co-expressed genes, signifying novel subtypes, could be elucidated.

Study design

• Meta-analysis of published gene expression data from patients diagnosed with BCP-ALL
• 1,582 pediatric BCP-ALL patients from eight studies (Sweden, n = 3; US, n = 5):
  • six microarray studies
  • two RNA sequencing studies
• Eight controls from one study (South Korea; healthy fetal bone marrow)
• 289 replicant adult BCP-ALL patients from two studies (both from US)

Data handling and analysis

Gene expression microarray data were log2 transformed and normalized through the Robust Multichip Average (RMA) method.

Strand-specific RNA sequencing libraries were constructed from rRNA-depleted RNA and paired-end sequenced. Sequence reads were mapped to the Human 1000 Genomes, build 37. Feature counts were used to summarize gene expression at the exon-level per gene.

Results

• RAG1 and RAG2 were expressed in the ER BCP-ALL subtype in all the six microarray studies.
• RAG1 expression was consistently higher in the ER subtype compared with all other subtypes, except for a subset of samples with unclassified chromosomal abnormalities (B-other).
• In a genome-wide screen for genes expressed with RAG1 and RAG2 in BCP-ALL patients, 31 high-ranking coexpressed genes were identified with RAG1 (referred to as the RAG1-signature).
• The RAG1-signature was strongly associated with the ER subtype, alongside four B-other, and one CRLF2 sample.
• Using leukemia and pro-B gene expression profiling, B-other samples with the RAG1-signature clustered closely with ER samples, and were distinct from the other subtypes.
• In the RNA sequence analysis, RAG1 and RAG2 were expressed in higher levels in ER relative to other subtypes, with the RAG1-signature identifying all ER subtypes and four B-other samples.
• The ETV6-target genes WBP1L, CLIC5, BIRC7, and ANGPTL2 were expressed at high levels in both ER and B-other samples expressing the RAG1-sigature profiles.

Conclusion

This study demonstrates that within the B-other subset of BCP-ALL patients, who lack classifying chromosomal abnormalities, there is a small proportion of patients with increased RAG1 expression, and associated expression of 31 other genes—referred to as the RAG1-signature. B-other patients with the RAG1-signature clustered closely with ER BPC-ALL patients on both leukemia and pro-B expression profiles, and while deficient in the ER fusion gene, express ETV6-target genes at high levels, as seen in ER patients. Overall, the study supports that the RAG1-signature profile defines an ER-like BCP-ALL subtype in children, with potential utility for the diagnosis and identification of this subtype in the pediatric population