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Among the recently described subtypes in childhood B-lymphoblastic leukemia (B-ALL) were DUX4-and PAX5-altered (PAX5alt). By using whole transcriptome RNA sequencing in 377 children with B-ALL from the Malaysia-Singapore ALL 2003 (MS2003) and Malaysia-Singapore ALL 2010 (MS2010) studies, we found that, after hyperdiploid and ETV6-RUNX1, the third and fourth most common subtypes were DUX4(n = 51; 14%) and PAX5alt (n = 36; 10%). DUX4also formed the largest genetic subtype among patients with poor day-33 minimal residual disease (MRD; n = 12 of 44). But despite the poor MRD, outcome of DUX4B-ALL was excellent (5-year cumulative risk of relapse [CIR], 8.9%; 95% confidence interval [CI], 2.8%-19.5% and 5-year overall survival, 97.8%; 95% CI, 85.3%-99.7%). In MS2003, 21% of patients with DUX4B-ALL had poor peripheral blood response to prednisolone at day 8, higher than other subtypes (8%; P= .03). In MS2010, with vincristine at day 1, no day-8 poor peripheral blood response was observed in the DUX4subtype (P= .03). The PAX5alt group had an intermediate risk of relapse (5-year CIR, 18.1%) but when IKZF1was not deleted, outcome was excellent with no relapse among 23 patients. Compared with MS2003, outcome of PAX5alt B-ALL with IKZF1codeletion was improved by treatment intensification in MS2010 (5-year CIR, 80.0% vs 0%; P= .05). In conclusion, despite its poor initial response, DUX4B-ALL had a favorable overall outcome, and the prognosis of PAX5alt was strongly dependent on IKZF1codeletion.

Identifying patient-specific clonal IGH/TCR junctional sequences is critical for minimal residual disease (MRD) monitoring. Conventionally these junctional sequences are identified using laborious Sanger sequencing of excised heteroduplex bands. We found that the IGH is highly expressed in our diagnostic B-cell acute lymphoblastic leukemia (B-ALL) samples using RNA-Seq. Therefore, we used RNA-Seq to identify IGH disease clone sequences in 258 childhood B-ALL samples for MRD monitoring. The amount of background IGH rearrangements uncovered by RNA-Seq followed the Zipf’s law with IGH disease clones easily identified as outliers. Four hundred and ninety-seven IGH disease clones (median 2, range 0–7 clones/patient) are identified in 90.3% of patients. High hyperdiploid patients have the most IGH disease clones (median 3) while DUX4 subtype has the least (median 1) due to the rearrangements involving the IGH locus. In all, 90.8% of IGH disease clones found by Sanger sequencing are also identified by RNA-Seq. In addition, RNA-Seq identified 43% more IGH disease clones. In 69 patients lacking sensitive IGH targets, targeted NGS IGH MRD showed high correlation (R= 0.93; P= 1.3 × 10−14), better relapse prediction than conventional RQ-PCR MRD using non-IGH targets. In conclusion, RNA-Seq can identify patient-specific clonal IGH junctional sequences for MRD monitoring, adding to its usefulness for molecular diagnosis in childhood B-ALL.

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No relevant conflicts of interest to declare.

While acute lymphoblastic leukemia (ALL) is a prototype of cancer that can be cured by chemotherapy alone, current ALL treatment regimens rely primarily on conventional cytotoxic agents with significant acute and long-term side effects. Better understanding of genomic landscape of ALL is critical for developing molecularly targeted therapy and implementing genomics-based precision medicine in this cancer. In particularly, sentinel chromosomal translocations are common in ALL and often involve key transcription factors important for hematopoiesis. Epigenetic regulator genes are also frequently targeted by somatic genomic alterations such as sequence mutations (e.g., CREBBP) and gene fusions (e.g., MLL, EP300). To comprehensively define transcriptomic abnormalities in childhood ALL, we performed RNA-seq of an unselected cohort of 231 children enrolled on the MaSpore frontline ALL protocols in Singapore or Malaysia. In total, we identified 58 putatively functional and predominant fusion genes in 125 patients (54.1%), the majority of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature driven by chromosomal rearrangements of the ZNF384gene with different partners (i.e., histone acetyl-transferases EP300and CREBBP, TAF15,and TCF3). In 9 of 11 ALL cases with ZNF384rearrangements, the breakpoint in this gene was invariably between exon 2 and exon 3, resulting in deletion of the 5'-UTR and then in-frame fusion of the entire ZNF384coding sequence with the partner genes. The top two most significantly up-regulated genes in the ZNF384-rearranged group were CLCF1and BTLA, whose expression levels were 15.5- and 15.0-fold higher than in ALL cases with wildtype ZNF384, respectively. In fact, ZNF384 binding was identified within the CLCF1and BTLAloci (particularly the promoter regions) by chromatin immunoprecipitation sequencing in B lymphoblasoid cells. Using luciferase transcription driven by CLCF1promoter in HEK293T cells as a model system, we observed significantly greater transcription activity with EP300-ZNF384fusion compared to cells expressing wildtype ZNF384, suggesting that this chimeric gene resulted in gain of ZNF384 function. Similar results were obtained with luciferase transcription assay driven by the BTLApromoter. In human ALL cells, CLCF1and BTLApromoter activities were consistently and significantly higher in ZNF384-rearranged ALL than in ALL cell line with wildtype ZNF384. To examine the effects of ZNF384 fusion on hematopoietic stem and progenitor cell (HSPCs) function, we also evaluated colony forming potential of HSPC in vitroupon ectopic expression of ZNF384fusions. While there was marked suppression of colonies from myeloid and erythoid lineages, expression of EP300-ZNF384or CREBBP-ZNF384significantly stimulated preB cell colony formation. However, neither EP300-nor CREBBP-ZNF384fusion was able to transform mouse hematopoietic precursor cell Ba/f3 in vitro, but instead increased the transforming potential of other oncogenic mutations (NRASG12D). EP300-ZNF384 and CREBBP-ZNF384 fusion proteins lacked the histone acetyltransferase (HAT) domain, and showed only 25% and 10% of HAT activity of full-length EP300 and CREBBP, respectively, with dominant-negative effects. Also, expression of EP300-ZNF384led to significant decrease in global H3 acetylation in Ba/f3 cells in vitro. Finally, in NRASG12D-transformed Ba/f3 cells, co-expression of EP300-ZNF384or CREBBP-ZNF384substantially potentiated cytotoxic effects of histone deacetylase inhibitor vorinostat. Similarly, in a panel of human ALL cell lines, ZNF384-rearrangement was also associated with increased sensitivity to vorinostat, suggesting that some ZNF384-rearranged ALL may benefit from therapeutic agents targeting histone acetylation regulation. In conclusion, our results indicate that gene fusion is the major class of genomic abnormalities in childhood ALL and chromosomal rearrangements involving EP300and CREBBPmay cause global epigenetic deregulation in ALL with potentials for therapeutic targeting.