Liquid biopsies: genotyping circulating tumor DNA

Abstract

Genotyping tumor tissue in search of somatic genetic alterations for actionable information has become routine practice in clinical oncology. Although these sequence alterations are highly informative, sampling tumor tissue has significant inherent limitations; tumor tissue is a single snapshot in time, is subject to selection bias resulting from tumor heterogeneity, and can be difficult to obtain. Cell-free fragments of DNA are shed into the bloodstream by cells undergoing apoptosis or necrosis, and the load of circulating cell-free DNA (cfDNA) correlates with tumor staging and prognosis. Moreover, recent advances in the sensitivity and accuracy of DNA analysis have allowed for genotyping of cfDNA for somatic genomic alterations found in tumors. The ability to detect and quantify tumor mutations has proven effective in tracking tumor dynamics in real time as well as serving as a liquid biopsy that can be used for a variety of clinical and investigational applications not previously possible.

Fig 1.

Methodologies for detecting circulating tumor DNA (ctDNA). Sanger sequencing (dideoxy-terminator sequencing), amplification refractory mutation system (ARMS),^, pyrosequencing, pyrophosphorolysis-activated polymerization (PAP), tagged-amplicon deep sequencing (TAM-Seq), digital polymerase chain reaction (PCR), and beads, emulsion, amplification, and magnetics (BEAMing).

Fig 2.

Genetic alterations detectable in circulating cell-free tumor DNA. Tumor cells release small fragments of cell-free DNA into circulation by multiple mechanisms. Cancer-associated genetic alterations such as point mutations, copy number variations, chromosomal rearrangements, and methylation patterns can be detected in circulating cell-free DNA.

Fig 3.

Detection of tumor-specific DNA mutations in the blood of patients to monitor response and relapse with targeted therapies. This schematic depicts a representation of a patient with metastatic colorectal cancer. At the time of presentation, DNA from the primary tumor is used to identify the baseline mutation profile; in this case, the tumor is found to be APC mutant and KRAS wild type (WT). At baseline, evaluation of the patient's plasma DNA only identifies KRAS WT fragments. This patient is treated with an anti–epidermal growth factor receptor (EGFR) monoclonal antibody, experiences a clinical response, and has a corresponding decrease in APC mutation level, further indicating a decrease in tumor burden. Continuous monitoring of plasma DNA shows the emergence of KRAS and NRAS mutations and/or MET amplification, indicative of the emergence of multiple different resistance clones. Clinical resistance becomes manifest at a later time point. Test tubes represent samples of plasma from which circulating free DNA is extracted and used to monitor the presence of cancer-specific aberrations.