Error-Corrected Sequencing of Cord Bloods Identifies Pediatric AML-Associated Clonal Hematopoiesis

Background:

Recently, several studies have used next-generation sequencing (NGS) to characterize an age-specific increase in the variant allele frequencies (VAF) and number of somatic mutations in AML-associated genes in adults without hematological malignancies. Termed Clonal Hematopoiesis of Indeterminate Significance (CHIP), this phenomenon is defined as being >2% VAF and associated with an increased risk of hematologic malignancy later in life. The threshold of 2% VAF is arbitrarily defined because mutations below the threshold could not be reliably called due to the inherent error rate of sequencing. In light of this, the lab has previously developed error-corrected sequencing (ECS) to identify point mutations and small indels from adult blood and marrow samples at VAF as low as 0.01%. Moving on to pediatric samples, the most commonly mutated genes in pediatric AML vary substantially from adult AML, and the prevalence of clonal hematopoiesis in pediatric AML-specific genes in early childhood is unknown. Using ECS, we investigated the prevalence of clonal hematopoiesis in 94 adult and pediatric AML-associated genes in 31 random human umbilical cord blood (UCB) samples. We hypothesized clonal mutations in cord blood would be rare because of the age-related linear acquisition of mutations observed in older individuals, but the genes in question had not been previously assessed.

Methods:

Random cord blood samples were obtained from the St. Louis Cord Blood Bank (http://www.slcbb.org/). ECS libraries from extracted genomic DNA were prepared according to established protocol. A total of 1602 amplicons were targeted (total queried genome space ~270kb) using an Illumina panel designed to enrich 94 genes frequently mutated in both adult and pediatric AML. The libraries were sequenced on the Illumina HiSeq 3000 platform and raw sequence data was analyzed using a validated bioinformatic pipeline established in the lab. A subset of called mutations was secondarily validated using the QX200 droplet digital PCR platform (Bio-Rad) in the Druley lab.

Results and Conclusions:

We found that 18.2% of cord blood samples harbor validated somatic mutations with VAFs ranging from 0.002 - 0.006. With ECS, we showed that the prevalence of clonal hematopoiesis in younger population is much higher than previously expected. These results suggest that clonal hematopoiesis is likely a stochastic process that happens in everyone as early as fetal development, yet the incidence of pediatric leukemia is estimated to be 7.7 cases per million children.

Future work:

In order to rule out the possibility of maternal blood contamination of the UCB, we are flow sorting CD34+ cells from the cord blood samples and screening for the mutations.