Recently, many genome-wide profiling studies provided insights into the molecular background of major cancer types. The deeper understanding of the genetic alterations and their functional consequences leveraged the discovery of novel therapeutic opportunities, improving clinical management of cancer patients. While tissue-based molecular patient stratification is the gold standard for precision medicine, it has certain limitations: Tissue biopsies are risky invasive sampling procedures and may not represent the entire tumor due to genetic heterogeneity. Complementary characterization of genetic information in the blood of cancer patients can serve as minimal-invasive 'liquid biopsy'. Released by different tissues in patients as well as healthy individuals, fragments of circulating cell-free DNA (cfDNA) are a potential source of diagnostic, predictive or prognostic biomarkers. cfDNA released from primary tumors or metastases (i.e. circulating tumor DNA, ctDNA) represents genomic aberrations in cancer cells. Recent studies have demonstrated technical feasibility and clinical applications of ctDNA including detection of drug targets and resistance mutations, longitudinal monitoring of tumors under therapy and detection of minimal residual disease. However, broad implementation of ctDNA analysis into daily clinical practice requires standardization and validation of pre-analytical and analytical procedures across different technologies. Currently, a variety of pre-analytical procedures for blood processing, isolation and quantification of cfDNA are used. Moreover, several analytical methods and technologies are available, ranging from PCR-based single locus assays to genome-wide approaches, which considerably differ in sensitivity, specificity, and throughput. In this review, we discuss the advantages and limitations of available methodologies and their potential applications in molecular diagnostics. This article is protected by copyright. All rights reserved.
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