NGS-Based Tumor-Informed Analysis of Circulating Tumor DNA

Accurate circulating tumor DNA (ctDNA) detection has an immense biomarker potential in all phases of the cancer disease course. Presence of ctDNA in the blood has been shown to have prognostic value in various cancer types as it may reflect the actual tumor burden. There are two main methods to consider, a tumor-informed and a tumor-agnostic analysis of ctDNA. Both techniques exploit the short half-life of circulating cell-free DNA (cfDNA)/ctDNA for disease monitoring and ultimately future clinical treatment intervention. Urothelial carcinoma is characterized by a high mutation spectrum but very few hotspot mutations. This limits tumor agnostic usability of hotspot mutation or fixed sets of genes for ctDNA detection. Here we focus on a tumor-informed analysis for ultrasensitive patient- and tumor-specific ctDNA detection using personalized mutation panels, probes that bind to specific genomic sequences to enrich for the region of interest. In this chapter, we describe methods for purification of high-quality cfDNA and guidelines for designing tumor-informed customized capture panels for sensitive detection of ctDNA. Furthermore, a detailed protocol for library preparation and panel capture utilizing a double enrichment strategy with low amplification is described.