A field guide for cancer diagnostics using cell‐free DNA: From principles to practice and clinical applications

Abstract

Recently, many genome-wide profiling studies provided insights into the molecular make-up of major cancer types. The deeper understanding of these genetic alterations and their functional consequences led to 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 invasive sampling procedures carrying the risk of complications and may not represent the entire tumor due to underlying genetic heterogeneity. In this context, complementary characterization of genetic information in the blood of cancer patients can serve as minimal-invasive 'liquid biopsy'. Fragments of circulating cell-free DNA (cfDNA) are released from tissues of healthy individuals as well as cancer patients. The fraction of cfDNA that is released from primary tumors or metastases (i.e. circulating tumor DNA, ctDNA) represents genetic aberrations in cancer cells, which are a potential source for diagnostic, prognostic, and predictive biomarkers. Recent studies have demonstrated technical feasibility and clinical applications including detection of drug targets and resistance mutations as well as longitudinal monitoring of tumors under therapy. To this end, a variety of pre-analytical procedures for blood processing, isolation and quantification of cfDNA are being employed and several analytical methods and technologies ranging from PCR-based single locus assays to genome-wide approaches are available, which considerably differ in sensitivity, specificity, and throughput. However, broad implementation of ctDNA analysis in daily clinical practice requires a thorough understanding of theoretical, technical, and biological concepts and necessitates standardization and validation of pre-analytical and analytical procedures across different technologies. Here, we review the pertinent literature and discuss the advantages and limitations of available methodologies and their potential applications in molecular diagnostics.

Next generation sequencing of the cellular and liquid fraction of pancreatic cyst fluid supports discrimination of IPMN from pseudocysts and reveals cases with multiple mutated driver clones: First findings from the prospective ZYSTEUS biomarker study

“…In line with previous studies, 16,43-45 many of our samples harbored mutations with mutation allele frequencies below 10%. Sanger sequencing would thus be insufficient for preoperative mutation testing 46 and the prevalence of genetic changes in several previous studies on this topic is likely to be inaccurate as stated by Singhi et al 16 NGS, especially when analyzing liquid biopsies, may reveal a broader spectrum of mutations and hence allow for better prediction of future cyst development. Regarding separation of the samples into liquid and cellular fraction (sediment after centrifugation) of the liquid biopsy, analyzing the cellular fraction revealed more diverse sets of mutations ( Table 2).…”

Integrated clonal analysis reveals circulating tumor DNA in urine and plasma of glioma patients

“…Primary brain tumors, which are diagnosed in over 260,000 patients annually (1), have a poor prognosis and lack effective treatments. Better methods for early detection and identification of tumor recurrence may enable the development of novel treatment strategies including the use of immunotherapy (2,3). The development of new treatments would also benefit from minimally invasive methods that characterise the glioma genome (4)(5)(6).…”

A High-Throughput Protocol for Isolating Cell-Free Circulating Tumor DNA from Peripheral Blood

“…Purifications of ctDNA, tumor-derived cfDNA (cell-free DNA), can yield DNA fragments that vary in size. Smaller fragments can be produced by cells undergoing apoptosis, while larger fragments can be produced from necrotic cells [2]. A study by Raymond and colleagues reported that the majority of cfDNA is found in nucleosomal fragments and the expected yield per milliliter of blood is usually below 10ng, averages 6.6ng, and depends on type and stage of tumor [2].…”

“…Smaller fragments can be produced by cells undergoing apoptosis, while larger fragments can be produced from necrotic cells [2]. A study by Raymond and colleagues reported that the majority of cfDNA is found in nucleosomal fragments and the expected yield per milliliter of blood is usually below 10ng, averages 6.6ng, and depends on type and stage of tumor [2]. Other factors such as pregnancy, intense exercise, cardiovascular disease and post-radiation cellular damage can affect the abundance of cfDNA yield [2].…”

“…These tests have been introduced in research and clinical labs at scale, and the need for rapid and high-throughput purification of cfDNA has become apparent. Several methods for isolating cfDNA have been described in the literature [2]. A recent comprehensive review published by Gorgannezhad etal .…”