Abstract
Renal cell carcinoma (RCC) is known for its variable clinical behavior and outcome, including heterogeneity in developing relapse or metastasis. Recent data highlighted the potential of somatic mutations as promising biomarkers for risk stratification in RCC. Likewise, the analysis of circulating tumor DNA (ctDNA) for such informative somatic mutations (liquid biopsy) is considered an important advance for precision oncology in RCC, allowing to monitor molecular disease evolution in real time. However, our knowledge about the utility of ctDNA analysis in RCC is limited, in part due to the lack of RCC-appropriate assays for ctDNA analysis. Here, by interrogating different blood compartments in xenograft models, we identified plasma cell-free (cf) DNA and extracellular vesicles (ev) DNA enriched for RCC-associated ctDNA. Additionally, we developed sensitive targeted sequencing and bioinformatics workflows capable of detecting somatic mutations in RCC-relevant genes with allele frequencies ≥ 0.5%. Applying this assay to patient-matched tumor and liquid biopsies, we captured tumor mutations in cf- and ev-DNA fractions isolated from the blood, highlighting the potentials of both fractions for ctDNA analysis. Overall, our study presents an RCC-appropriate sequencing assay and workflow for ctDNA analysis and provides a proof of principle as to the feasibility of detecting tumor-specific mutations in liquid biopsy in RCC patients.