Detection of Novel Pathogenic Gene Rearrangements in Pediatric Acute Myeloid Leukemia By RNA Sequencing
Authors: Shiba Norio , Yoshida Kenichi , Shiraishi Yuichi , Hara Yusuke , Yamato Genki , Kaburaki Taeko , Sanada Masashi , Oki Kentaro , Tomizawa Daisuke , Sotomatsu Manabu , Arakawa Hirokazu , Horibe Keizo , PARK Myoung-Ja , Taga Takashi , Adachi Souichi , Tawa Akio , Miyano Satoru , Ogawa Seishi , Hayashi Yasuhide 
Published: October 2019

Abstract
Background
Pediatric acute myeloid leukemia (AML) comprises approximately 20% of pediatric leukemia cases. AML is a major therapeutic challenge in pediatric oncology, and the current overall survival rate is <70%. The pathogenesis of AML is heterogeneous, and causes include various chromosomal aberrations, gene mutations/epigenetic modifications, and deregulated/overregulated gene expression, resulting in increased proliferation and decreased hematopoietic progenitor cell differentiation. Recurrent chromosomal structural aberrations such as t(8;21), inv(16), and MLL -rearrangements are well established as diagnostic and prognostic markers in AML. Furthermore, recurrent mutations in FLT3, KIT, and RAS have been reported in both adult and pediatric AML. Recently, massively parallel sequencing has facilitated the discovery of recurrent mutations in DNMT3A, TET2, and IDH, which are clinically useful for predicting the prognosis. However, these mutations are rare in pediatric AML, thereby suggesting that other genetic alterations may exist in pediatric AML.
In addition, recent studies have reported that the NUP98-NSD1 fusion is an adverse AML prognostic marker and that PRDM16 (also termed MEL1) is a representative gene that is overexpressed in patients who have the NUP98-NSD1 fusion. PRDM16 overexpression occurs in nearly a quarter of pediatric AML patients who are NUP98-NSD1 negative, and this overexpression is increased in specimens with other high-risk lesions (e.g., FLT3-ITD, NUP98-NSD1,and MLL-PTD).
Patients and Methods
To obtain a complete overview of gene rearrangements and other genetic lesions, we performed RNA sequencing of samples from 47 de novo pediatric AML patients using Illumina HiSeq 2000, including 39 patients with normal karyotypes and 6 patients with Trisomy 8. Among these 47 patients, 35 patients overexpressed PRDM16, which was strongly associated with a poor prognosis in our previous studies. As a control, we selected 12 patients with low PRDM16 expression levels. All patients were enrolled and treated as part of the AML-05 study conducted by the Japan Pediatric Leukemia/Lymphoma Study Group. We determined the known gene mutations present in these patients using the RNA sequencing data.
Results
Approximately 300 candidate gene rearrangements were identified in 46/47 samples, including 26 in-frame and 78 out-of-frame gene rearrangements. Several recurrent gene rearrangements identified in this study involved previously reported targets in AML, such as FUS-ERG, NUP98-NSD1,and MLL-MLLT3. However, several novel gene rearrangements were identified in the current study, including HOXA10-HOXA-AS3, PRDM16-XXX, CUL1-YYY, and DAZAP1-ZZZ. At present, we are validating these novel gene rearrangements using Sanger sequencing. Known gene alterations, such as FLT3-ITD, MLL-PTD, and mutations of RAS, KIT, CEBPA, WT1, and NPM1 genes, were detected by RNA sequencing.
Conclusion
In the present study, RNA sequencing was employed to elucidate the complexity of gene rearrangements/mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that can be discriminated from adult AML in terms of the spectrum of gene rearrangements/mutations. We identified at least one potential gene rearrangement or driver mutation in nearly all AML samples, including various novel fusion genes. Thus, our results suggest that gene rearrangements and mutations play essential roles in pediatric AML.

Disclosures
No relevant conflicts of interest to declare.