RESULTS
After obtaining informed consent from his parents, DNA was extracted
from the peripheral blood and biopsy samples of the tumors at initial
diagnosis (D), each relapse (second relapse; R2, third relapse; R3,
fourth relapse; R4) and autopsy (abdominal lymph node; LN, liver; Liver)
except for the sample at first relapse. Direct sequencing identifiedBCOR -ITD (c.5526_5621dup) at all the analyzed samples
(Supplementary Fig. S2). SNP array analysis did not reveal any copy
number alterations (CNAs) at D whereas consistent CNAs on chromosomes
1q, 7q, and 15q were detected (Supplementary Fig. S3). Each sample
possessed several sample specific CNAs. Moreover, we detected a focal
loss at chromosome 9q32-33 in the samples of R2 through autopsy, where
only the tumor suppressor gene BRINP1 was located (Fig. 2A). We
further analyzed other genetic alterations using targeted-capture
sequencing (TCS) for 381 genes relating to pediatric solid
tumors12. Candidate mutations were filtered according
to our previous report12. Detected 13 mutations were
classified into 7 clones based on the detected samples and variant
allele frequency. Only the BCOR -ITD was included in clone 1,
whereas 3 clade mutations (FLT1 , JAK2 , and MYH7 )
were additionally detected in relapsed tumor samples (clone 2, 3). The
other mutations (PRKRIR , KMT2D , TSHR , CDK6 ,IL6ST , NF1 , KDM6A , OBSCN , and SYNE1 )
were clone specific (clone 4–7) (Supplementary Table S1). The
number of detected mutations increased during each tumor recurrence. All
R3 mutations (clone 1–3) were detected upon metastatic samples from
autopsy, suggesting the accumulation of metastasis site specific
mutations during tumor progression. Importantly, the newly acquired
mutations at each relapse were not detected even as a minor clone in the
previous samples. We analyzed the clonal evolution of the present case
by using a software: clonevol13,14. Multiregional
sequencing analysis at different time points allowed us to delineate the
clonal history of tumor cells, suggesting that each relapse developed
based on the preexisting tumors (Fig. 2C).