Pediatric acute myeloid leukemia (AML) comprises ∼20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology. Nearly 40% of patients still relapse after present first-line therapies and once the relapse occurs, the long-term survival rates decrease, ranging from 21% to 34%. As for the pathogenesis of AML relapse, the recent development of massively parallel sequencing technologies has provided a new opportunity to investigate comprehensive genetic alterations that are involved in tumor recurrence of adult AML. However, little is known about the molecular details of relapsed pediatric AML.


In order to reveal the clonal origin and the major mutational events in relapsed pediatric AML, we performed whole exome sequencing of 4 trio samples from diagnostic, relapsed and complete remission phases using Illumina HiSeq 2000. Copy number abnormalities were also detected using whole exome sequencing. Subsequently, deep sequencing of identified mutations was performed to evaluate intra-tumor heterogeneity and the clonolocal history of relapsed clones.


Whole-exome sequencing of 12 samples from 4 patients were analyzed with a mean coverage of more than x100, and 95 % of the targeted sequences were analyzed at more than x20 depth on average. A total of 98 somatic mutations were identified, where mean number of non-silent mutations was higher at relapsed phase than at the time of diagnosis (14.0/case vs 10.5/case) (p=0.270). Assessment of clonality using variant allele frequencies of individual mutations suggested that some mutations were subclonal mutations, consisting of intra-tumor heterogeneity both at the time of diagnosis and at relapse. In all 4 patients, relapsed AML evolved from one of the subclones at the initial phase, which was accompanied by many additional mutations including common driver mutations that were absent or existed only with lower allele frequency in the diagnostic samples, indicating a multistep process of leukemia recurrence. Forty-six out of the 98 mutations were specific either at the time of diagnosis (n = 16) or at relapse (n = 30). Relapse-specific mutations and copy number changes were found in several genes including known drivers such as NRAS and CREBBP. These mutations were further investigated in an extended cohort of relapsed pediatric AML samples using targeted sequencing to evaluate their prevalence. In some cases, AML relapse may accompany a dynamic clonal change. For example, some bona fide driver mutations, such as KRAS mutations, that were predominant at the time of diagnosis disappeared in relapsed samples.


Whole exome sequencing unmasked clonal structure of primary and relapsed pediatric AML, which helped to understand the underlying mechanism of relapse in pediatric AML. Our results suggested that pediatric AML has intra-tumor heterogeneity and subclonal mutations such as NRAS and CREBBP occurring in one of the subclones could drive the AML relapse.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.