Abstract
Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, comprising multiple subtypes characterized by aneuploidy, recurring submicroscopic gains and losses of DNA, and inter- and intra-chromosomal rearrangements. A common feature in ALL with 44-45 chromosomes (from here on referred to as near-diploid ALL) is the presence of a dicentric chromosome, which may lead to the formation of gene fusions and the expression of chimeric proteins. At present, however, we have a limited understanding of the nature of these chimeric proteins, but some are known or predicted to dysregulate the function of oncoproteins or hematopoietic growth factors.
We recently performed genome-wide profiling of over 120 pediatric cases with hypodiploid ALL, including 68 near-haploid cases (harboring 24-31 chromosomes), 34 low-hypodiploid cases (32-39 chromosomes) and 22 near-diploid cases, 20 of which harbored a dicentric chromosome. Near-haploid ALL is characterized by a high frequency of activating mutations of Ras signaling (mainly NF1 alterations) and IKZF3 deletions, and low-hypodiploid ALL by IKZF2, TP53 and RB1alterations. In contrast, these alterations are infrequent in dicentric ALL. To gain insight into the genetic basis of near-diploid ALL with dicentric chromosomes, we examined the 20 cases including 6 with dic(9;20), 3 with dic(7;12) and 2 with dic(9;17), while the remaining 9 cases had dicentric chromosomes involving various chromosomal arms. Affymetrix SNP 6.0 microarrays, gene expression profiling and candidate gene resequencing for 19 genes were performed for all cases, and transcriptome sequencing (mRNA Seq) on tumor and whole exome sequencing on tumor and matched normal genomic DNA have to date been carried out for 10 cases.
The most common lesion was focal deletion of CDKN2A/CDKN2B, encoding the tumor suppressors INK4/ARF (77%). Approximately one third of near-diploid cases harbored mutations targeting Ras and receptor tyrosine kinase signaling, including mutations in NRAS (18%), KRAS (9%), PTPN11 (9%) and NF1 (5%). The B-lymphoid transcription factor PAX5 was altered by deletion, sequence mutation or amplification in 59% of near-diploid ALL. PAX5 has previously been reported to be involved in gene fusions created by dicentric chromosomes in ALL, including PAX5-ETV6 in some cases with dic(9;12)(p13;p13). PAX5 has previously also been shown to be targeted by dic(9;20)(p11-13;q11), which creates several non-productive fusion transcripts.
The burden of non-synonymous single nucleotide variations (SNVs) and insertion/deletion (indel) mutations in coding- and splice regions in mRNA- and exome sequenced dicentric ALL varied with 8-25 SNVs and 4-15 indels per case. Except for the recurrent lesions stated above, the only recurrently sequence mutated gene identified in these 10 cases was NOTCH1, with mutations found in two cases, both of T-lineage. Singleton mutations were identified in e.g. IL7R, JAK3, STAT3, MYCN, PTEN and DOT1L. Fusion detection in mRNA Seq data identified 1-4 head-to-tail gene fusions per case, including the previously characterized fusions DDX3X-MLLT10, PICALM-MLLT10, MLL-AFF1 and P2RY8-CRLF2, none of which are involved in the dicentric chromosomes. The P2RY8-CRLF2 fusion was by RT-PCR shown to be present in two additional dicentric ALL cases. Further, novel in-frame fusions including ETV6-AMPH and PAX5-FBRSL1 created by dic(7;12)(p11.2;p11.2) and dic(9;12), respectively, were identified, the function of which in tumorigenesis is still unknown.
An extended mRNA Seq study is currently ongoing, investigating an additional 30 near-diploid ALL cases with a focus on the dicentric chromosomes dic(7;9), dic(9;12) and dic(9;20). Further, recurrence screening will be performed on a cohort including another 45 cases, and functional evaluation is on an ongoing basis carried out on identified fusions.
Altogether, this study is anticipated to provide critical new insights into the genetic basis of near-diploid ALL harboring a dicentric chromosome, and to shed more light on the putative chimeric genes produced by the formation of dicentric chromosomes. Further, we hope to identify new targets for therapy.
Disclosures:
No relevant conflicts of interest to declare.
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