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Liquid biopsies, in particular the profiling of circulating tumor DNA (ctDNA), have long held promise as transformative tools in cancer precision medicine. Despite a prolonged incubation phase, ctDNA profiling has recently experienced a strong wave of development and innovation, indicating its imminent integration into the cancer management toolbox. Various advancements in mutation-based ctDNA analysis methodologies and technologies have greatly improved sensitivity and specificity of ctDNA assays, such as optimized preanalytics, size-based pre-enrichment strategies, targeted sequencing, enhanced library preparation methods, sequencing error suppression, integrated bioinformatics and machine learning. Moreover, research breakthroughs have expanded the scope of ctDNA analysis beyond hotspot mutational profiling of plasma-derived apoptotic, mono-nucleosomal ctDNA fragments. This broader perspective considers alternative genetic features of cancer, genome-wide characterization, classical and newly discovered epigenetic modifications, structural variations, diverse cellular and mechanistic ctDNA origins, and alternative biospecimen types. These developments have maximized the utility of ctDNA, facilitating landmark research, clinical trials, and the commercialization of ctDNA assays, technologies, and products. Consequently, ctDNA tests are increasingly recognized as an important part of patient guidance and are being implemented in clinical practice. Although reimbursement for ctDNA tests by healthcare providers still lags behind, it is gaining greater acceptance. In this work, we provide a comprehensive exploration of the extensive landscape of ctDNA profiling methodologies, considering the multitude of factors that influence its development and evolution. By illuminating the broader aspects of ctDNA profiling, the aim is to provide multiple entry points for understanding and navigating the vast and rapidly evolving landscape of ctDNA methodologies, applications, and technologies.

The changing face of circulating tumor DNA (ctDNA) profiling: Factors that shape the landscape of methodologies, technologies, and commercialization