METHODS
This study was approved by the Human Genome, Gene Analysis Ethics Committee of the University of Tokyo (approval number G0948). Bone marrow (BM) samples collected were analyzed after obtaining written informed consent from legal guardians of the patient.
Total RNA was extracted from recurrent BM and checked for integrity and concentration using the Agilent Tapestation (Agilent, Santa Clara, CA, USA). RNA-seq libraries were constructed using the NEBNext Ultra RNA library prep kit compatible with the Illumina platform (New England BioLabs, Ipswich, MA, USA). Next-generation sequencing was performed using the Illumina NovaSeq 6000 platform with a standard 101 bp paired-end read protocol according to the manufacturer’s instructions. Reference genome (hg19) alignment and fusion gene detection were performed using Genomon v2.6.2. For expression analysis, fragments per kilobase of exon per million reads mapped (FPKM) were used as normalized count data. T-distributed stochastic neighbor embedding (t-SNE) was performed using R (v3.6.1) package Rtsne to reduce dimensions. For t-SNE, RNA-seq data of DLBCL and KMT2A -rearranged ALL were obtained from published studies (accession number GSE147986 from Gene Expression Omnibus [ncbi.nlm.nih.gov/geo] for DLBCL and a personal communication for KMT2A -rearranged ALL).11,12
Primary and recurrent BM samples were subjected to reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing ofKMT2A -MLLT3 and internal tandem duplication (ITD) and variants in N676 and D835 of FLT3 . ITD and the variants ofFLT3 are additional gene alterations in KMT2A -rearranged leukemia.13,14 Supplemental Table S1 lists the primer sequences.