The Effect of Unique Molecular Identifier Family Size Using Tumor-Informed Circulating Tumor DNA Analysis in Childhood Cancers.

Analysis of circulating tumor DNA (ctDNA) provides a molecular assessment that can complement routine imaging in childhood cancer management. Detailed monitoring of ctDNA levels may provide information on treatment efficacy and resistance and minimal residual disease and allows for early detection of relapse. Here, tumor-informed ctDNA analysis was applied to 90 blood plasma samples collected from eight children with malignant tumors. A total of 4 to 10 tumor-specific mutations per patient were assessed using SiMSen-Seq, a digital sequencing approach using unique molecular identifiers (UMIs). The effects of individual SiMSen-Seq assays and plasma samples were evaluated in relation to their impact on background error rate, number of detected target molecules, and mutant calling using different UMI family size cutoff settings. The use of at least two sequencing reads per UMI provided the best overall performance by generating the highest number of detected target molecules and hence the optimal chance to detect low-frequency mutations. Data were consistent between SiMSen-Seq assays and plasma samples, providing robust ctDNA profiling over time for all patients. In conclusion, the results show that optimal use of UMIs in tumor-informed ctDNA analysis enables sensitive molecular readout that can assist in management of childhood cancers.