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).