Transcriptome Analysis Revealed the Entire Genetic Understanding of Pediatric Acute Myeloid Leukemia with a Normal Karyotype

Background: Pediatric acute myeloid leukemia (AML) comprises approximately 20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with a current overall survival rate of less than 70%. The pathogenesis of AML is heterogeneous and can be caused by various chromosomal aberrations, gene mutations/epigenetic modifications, and deregulated/overregulated gene expressions, leading to increased proliferation and decreased hematopoietic progenitor cell differentiation. Recurrent chromosomal structural aberrations [e.g., t(8;21), inv(16), and MLL-rearrangements] have been well established as diagnostic and prognostic markers of AML. Furthermore, recurrent mutations in FLT3, KIT, NPM1, and CEBPA have been reported in both adult and pediatric AML. Recently, massively parallel sequencing enabled the discovery of recurrent mutations in DNMT3A, TET2, and IDH, which are clinically useful for the prediction of the prognosis. However, these mutations are rare in pediatric AML, suggesting that other genetic alterations exist in pediatric AML.

In contrast, recent reports have described NUP98-NSD1 fusion as an adverse AML prognostic marker and PRDM16 (also known as MEL1) as the representative overexpressed gene in patients harboring NUP98-NSD1 fusion. Intriguingly, PRDM16 overexpression occurs in nearly one-quarter of all children, with AML involving NUP98-NSD1-negative patients. Moreover, this overexpression is enriched in specimens with other high-risk lesions (e.g., FLT3-ITD, NUP98-NSD1, and MLL-PTD).

Patients and Methods: To reveal a complete registry of gene rearrangements and other genetic lesions in pediatric AML with a normal karyotype, we performed transcriptome analysis (RNA sequencing) of 61 of 70 de novo pediatric AML patients with a normal karyotype using Illumina HiSeq 2000. We could not perform RNA sequencing in nine patients because of a lack of RNA quantity or quality. Among the 70 AML patients with a normal karyotype, 33 patients overexpressed PRDM16, which was found to be strongly associated with a poor prognosis in our previous studies. All patients were enrolled and treated with AML-05 in the study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG). We also analyzed the known genetic mutations associated with these patients using the data derived from RNA sequencing.

Results: A total of 144 candidate gene rearrangements, which were not observed in normal samples, were identified in 51 of 61 samples. Many of the recurrent gene rearrangements identified in this study involved previously reported targets in AML, including NUP98-NSD1, NUP98-JARID1A, CBFA2T3-GLIS2, MLL-MLLT10, and MLL-MLLT3. However, several gene rearrangements were newly identified in the current study, including MLL-SEPT6, HOXA10-HOXA-AS3, PRDM16-SKI, and CUL1-EZH2. We have also performed the validation of these novel gene rearrangements using Sanger sequencing. Most of these gene rearrangements were found in patients with a high expressionof PRDM16. In contrast, CEBPA mutations were frequently observed in patients with a low expression of PRDM16. Known gene alterations, such as FLT3-ITD and MLL-PTD, and mutations of the RAS, KIT, CEBPA, WT1, and NPM1genes were also detected using RNA sequencing.

Conclusion: RNA sequencing unmasked a complexity of gene rearrangements and mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that could be discriminated from adult counterparts, regarding the spectrum of gene rearrangements and mutations. In the present study, we identified at least one potential gene rearrangement or driver mutation in nearly all AML samples, including some novel fusion genes. These findings suggest that gene rearrangements in conjunction with mutations also play essential roles in pediatric AML.