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.