Ras Signalling Pathway and Novel Target Genes Related to Down Syndrome Contribute to the Development of B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) in iAMP21 Patients

Abstract 70

Intrachromosomal amplification of chromosome 21 (iAMP21) represents a distinct cytogenetic subgroup of BCP-ALL, in which patients experience a high-risk of relapse on standard treatment protocols. The abnormal chromosome 21 defining iAMP21 has a heterogeneous, complex profile at the genomic level. This complexity has made it difficult to elucidate target genes or the initiating mechanism giving rise to iAMP21 using standard genomic approaches. In this study, detailed genomic and mutational analysis has highlighted potential novel targets in the development of iAMP21 BCP-ALL.

DNA was available from 45 iAMP21 patient samples. Patient 1 was a 10 year old female; her diagnostic karyotype was 47,XX,+10,der(21)dup(21)(q?)r(21)(q?). Fluorescence in situ hybridization detected multiple copies of RUNX1, thus defining iAMP21. SNP 6.0 arrays indicated the characteristic genomic profile of chromosome 21, comprising a ∼30Mb (from 17–47Mb) region of copy number gain/amplification. Many of the breakpoints occurred within the Down Syndrome Critical Region (DSCR), specifically within the gene, DSCAM at 41.4Mb, and a telomeric deletion was identified with a breakpoint within the gene, TSPEAR at 45.9Mb. Chromosome 7 abnormalities were frequent, with one deletion including IKZF1 at 50.3Mb. The IKZF1, ETV6 and RB1 deletions seen by SNP 6.0 arrays were confirmed by Multiplex Ligation Probe-dependent Amplification (MLPA) and quantitative PCR.

Whole-exome sequencing of the diagnostic and remission DNA of patient 1 identified 44 somatic mutations; 21 were computationally predicted to be potentially damaging to the function of the protein. The variant frequency of the individual somatic mutations ranged from 1.1% − 65.2%, indicating heterogeneity within the iAMP21 genome. The majority of the variants were detected at a frequency of <10%, emphasising the importance of in depth coverage for the detection of mutations. A number (n=8) were confirmed by direct Sanger sequencing.

Novel somatic mutations were identified in the B-cell development (ENPEP) and histone modification (SMYD3, RPL24, SIN3B) pathways. Genomic abnormalities in components of these pathways have been previously associated with BCP-ALL development.

Six of the potentially-damaging mutations occurred within 3 genes: SIM2, SYNJ1, UMODL1, located within the DSCR, which have been implicated in the development of Down syndrome. We have previously demonstrated that Down syndrome-ALL and iAMP21 BCP-ALL share common genetic abnormalities: gain of an X chromosome and a high incidence of P2RY8-CRLF2 fusion.

Furthermore, we identified a mutation in NF1, a component of the Ras signalling pathway. The mutation (P1496S) occurred in the kinase-binding domain at a variant frequency of 27%. Activation of the Ras signalling pathway was confirmed by pERK expression shown by Western blot analysis. This is the first report of deregulation of the Ras signalling pathway in iAMP21 BCP-ALL. Additional studies using a combination of target-enrichment strategies and massively parallel deep-sequencing identified NRAS and KRAS mutations in 18 of the remaining 44 iAMP21 patients. These mutations were frequently identified in hotspot regions: NRAS G12D, KRAS G13D. Dual mutations were identified in both NRAS and KRAS in two patients, suggesting a cooperative effect of Ras signalling pathway defects.

In conclusion, genomic analysis and whole exome sequencing of a single patient has identified novel mutations: ENPEP and SMYD3, RPL24, SIN3B; in the B-cell development and histone modification pathways, respectively, indicating the involvement of pathways in iAMP21 patients that are common to other BCP-ALL subtypes. Although previously reported at a high level in high-risk childhood BCP-ALL, the involvement of the Ras signalling pathway was observed for the first time in iAMP21 BCP-ALL, through the identification of a NF1 mutation in patient 1. The involvement of the Ras signalling pathway in 41% of iAMP21 patients was confirmed by the finding of NRAS and KRAS mutations in a larger iAMP21 cohort. The novel identification of 3 mutated genes (SIM2, SYNJ1, UMODL1) and breakpoints within 2 others (DSCAM, TSPEAR) located within the DSCR reinforces common features between Down syndrome-ALL and iAMP21 BCP-ALL, which may further our understanding of the increased leukemia risk of Down syndrome individuals.