While tissue biopsy remains the gold standard for tumor biomarker testing, assays using plasma-derived cfDNA, aka circulating-tumor DNA (ctDNA), have recently demonstrated validity in the setting of limited tissue or recurrent disease. Tumor-derived cfDNA is also present in nonplasma biofluids and supernatants procured through interventional procedures. Evaluation of cfDNA extracted from these fluids may have benefits at nearly every stage of cancer patient management, from diagnosis and prognosis to monitoring disease progression and predicting therapeutic response. This review will focus on preanalytical, analytical, and postanalytical variables that must be considered when analyzing “liquid biopsies” outside the plasma compartment.
While there are no uniform standards for cfDNA testing in oncology, recommendations and guidelines concerning the processing of plasma-derived ctDNA can inform molecular diagnostics performed on cfDNA derived from supernatants and nonplasma biofluids.
Preanalytical variables affecting cfDNA include biological considerations of the tumors and their communicating fluid compartment(s), as well as their collection, transport, storage, and extraction.
Assays for cfDNA evaluation cover a range of targets, from allele-specific to broad whole exome or genome sequencing; assay selection primarily depends on the testing indication and required limit of detection.
cfDNA acquisition results in fewer surgical complications, and assays using cfDNA demonstrate improved quality metrics, reduced turnaround time and expense, and higher sensitivity (potentially at the cost of specificity).
Nearly 75 years ago, Mandel and Metais described the presence of fragments of cell-free DNA (cfDNA) in human peripheral blood. While to date the mechanism of this phenomenon remains incompletely understood, cfDNA is thought to be the result of passive release of DNA during apoptosis, necrosis, NETosis, or physical cell damage and/or active secretion due to an underlying (patho)physiologic process, including pregnancy, organ transplant, autoimmune disease, trauma, infection, or neoplasia. The potential of cfDNA in evaluating neoplasia is substantial, with implications in cancer screening, diagnosis, prognosis, monitoring of disease progression, and the prediction of therapeutic response.
Cell-Free DNA in Peripheral Blood
The relative increase in serum cfDNA in patients with cancer compared with normal controls was first demonstrated in 1977. Stroun and colleagues would later report that at least some of this cfDNA was attributable to the cancer cells themselves, which was further supported by the identification of identical KRAS mutations in plasma-derived cfDNA of patients with pancreatic cancer and their corresponding tumor tissue. This potential of tumor-derived cfDNA in peripheral blood, also known as circulating tumor DNA (ctDNA), to act as a surrogate for tissue led many to theorize that a “liquid biopsy” evaluating ctDNA might become the gold standard for tumor testing, at least in certain contexts. Advances in technology in the last decade have allowed the approval of multiple ctDNA liquid biopsy assays by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA).
Cell-Free DNA in Biofluids and Supernatants
cfDNA, however, is not limited to plasma, having been reported in bodily fluids (biofluids), including but not limited to cerebrospinal fluid (CSF), aqueous humor, tears, saliva, sputum, effusions, ascites, stool, urine, and organ-cyst fluid, as well as within interventional byproducts of lavage and fine-needle aspiration (FNA) ( Fig. 1 ). Rather than an exhaustive survey of the historic contributions and institutional experiences for each of these analytes, this review will instead focus on the preanalytical, analytical, and postanalytical considerations of cfDNA in oncology testing, as well as the limitations and benefits of cfDNA as a biomarker outside of the plasma compartment.