Personalized circulating tumor DNA analysis for sensitive disease monitoring and detection of relapse in neuroblastoma.

Circulating tumor DNA (ctDNA) has shown potential as a non-invasive tumor biomarker in neuroblastoma. Previous studies used generic assays for detection of selected predefined oncogenic variants as markers of ctDNA, which limits the sensitivity and excludes a subset of patients from analysis. Here we assessed patient-specific ctDNA analysis for treatment evaluation and detection of relapse in neuroblastoma. We generated personalized sequencing panels targeting 10 tumor-specific single nucleotide variants (SNVs) for each patient and performed ctDNA analysis of 136 plasma samples collected longitudinally in 13 children with neuroblastoma. ctDNA was detected using ultra-deep next generation sequencing with unique molecular identifiers to eliminate polymerase-induced errors. We found that the levels of ctDNA at diagnosis correlated with risk group and decreased gradually during effective treatment. All samples collected during follow-up in patients without disease relapse were ctDNA-negative. All four relapses were associated with elevated ctDNA levels, and a majority of the analyzed SNVs were detected at time of relapse. In one case, ctDNA became positive 78 days before the relapse was diagnosed with routine assessment and increased by over a thousandfold before the start of additional treatment. Overall, ctDNA was more uniformly elevated at diagnosis, showed less putative false positive results, and was more sensitive for detection of relapse compared to five serum or urine tumor markers used in clinical routine. In conclusion, personalized ctDNA analysis is suitable for clinical monitoring of tumor burden and may be used in all neuroblastoma patients regardless of risk group or tumor genetics.