Residency · Residency · Pathology

Liquid Biopsy and Circulating Tumor DNA

Introduction

Liquid biopsy refers to the analysis of tumor-derived biomarkers in body fluids, most commonly circulating tumor DNA (ctDNA) in peripheral blood. This minimally invasive approach provides real-time genomic information for therapy selection, monitoring treatment response, detecting resistance, and identifying minimal residual disease.

Biology of Circulating Tumor DNA

Sources and Characteristics

ctDNA is released into the bloodstream through tumor cell apoptosis, necrosis, and active secretion. Fragment size is typically approximately 167 bp, corresponding to mononucleosome-associated DNA. ctDNA constitutes a variable fraction of total cell-free DNA (cfDNA), ranging from less than 0.01% to over 50% depending on tumor burden, stage, and type. Its half-life is approximately 1-2 hours, which means it reflects real-time tumor dynamics. Higher ctDNA levels are found in advanced and metastatic disease, while early-stage tumors shed lower amounts.

Other Liquid Biopsy Analytes

Beyond ctDNA, other analytes include circulating tumor cells (CTCs), which are intact tumor cells shed into blood at low frequency (1-10 per 10 mL), with CellSearch being FDA-cleared for enumeration. Exosomes and extracellular vesicles contain DNA, RNA, and proteins from tumor cells. Cell-free RNA (cfRNA) includes tumor-derived mRNA and microRNA. Tumor-educated platelets are platelets that have taken up tumor-derived RNA. While blood is the primary specimen, ctDNA is also detectable in CSF, urine, pleural fluid, and ascites.

Pre-Analytical Considerations

Specimen Collection and Processing

Blood should be collected in cell-stabilizing tubes (Streck Cell-Free DNA BCT, PAXgene) to prevent white blood cell lysis, which dilutes the ctDNA signal. Standard EDTA tubes are acceptable if processed within 2-4 hours. A two-step centrifugation removes cells first, then debris. Plasma is preferred over serum because serum contains DNA released during clotting. Plasma should be stored at -80 degrees C if not extracted immediately.

cfDNA Extraction

Specialized kits optimized for short cfDNA fragments (such as QIAamp MinElute and MagMAX) are used. Typical yield is 5-30 ng per mL of plasma. An input of 10-30 mL of whole blood is recommended for sensitive ctDNA analysis. Quantification is performed by fluorometry (Qubit) or qPCR.

Analytical Methods

PCR-Based Approaches

Droplet digital PCR (ddPCR) provides absolute quantification of specific mutations with sensitivity down to approximately 0.01% VAF, but is limited to known targets. BEAMing (beads, emulsion, amplification, magnetics) is a digital PCR variant with very high sensitivity. These methods are best suited for monitoring known mutations such as EGFR T790M resistance.

NGS-Based Approaches

Targeted panels covering cancer-relevant genes use error correction with unique molecular identifiers (UMIs). Examples include Guardant360, FoundationOne Liquid CDx, and Tempus xF. They achieve sensitivity of 0.1-0.5% VAF with UMI-based error correction and provide broader genomic coverage than PCR-based methods, detecting SNVs, indels, CNAs, and fusions.

Ultra-Sensitive Approaches

CAPP-Seq combines hybrid capture with computational error suppression. Tumor-informed assays (such as Signatera) design custom panels based on the patient's tumor whole exome sequencing, monitoring specific clonal mutations with sensitivity of approximately 0.01% VAF. Methylation-based assays detect cancer-specific methylation patterns in cfDNA for early detection and tissue-of-origin determination.

FDA-Approved Liquid Biopsy Tests

Current Approvals

Guardant360 CDx was the first comprehensive liquid biopsy companion diagnostic, approved for EGFR mutations in NSCLC and pan-tumor TMB-H. FoundationOne Liquid CDx is a 300-plus gene panel with multiple companion diagnostic indications. cobas EGFR Mutation Test v2 is a PCR-based test for EGFR mutations in NSCLC. Epi proColon detects methylated SEPT9 in cfDNA for colorectal cancer screening, though adoption has been limited. Shield (Guardant Health) is a cfDNA-based colorectal cancer screening test.

Clinical Applications

Therapy Selection

Liquid biopsy identifies actionable mutations when tissue biopsy is insufficient, inaccessible, or delayed, covering targets such as EGFR, ALK, ROS1, BRAF, KRAS, PIK3CA, BRCA1/2, and NTRK. It captures tumor heterogeneity from multiple metastatic sites and is complementary to tissue biopsy. Guidelines recommend tissue biopsy as first-line when feasible, with liquid biopsy if tissue is unavailable or insufficient.

Resistance Monitoring

Liquid biopsy detects emergence of resistance mutations during targeted therapy, such as EGFR T790M arising during first or second-generation EGFR TKI therapy in NSCLC, and ESR1 mutations in ER-positive breast cancer during aromatase inhibitor therapy. Serial monitoring enables early detection of resistance before radiographic progression.

Minimal Residual Disease (MRD)

Post-surgical or post-treatment ctDNA detection predicts relapse before clinical or radiographic evidence. Tumor-informed assays based on the patient's own tumor mutations achieve the highest sensitivity. Demonstrated utility exists in colorectal, lung, breast, and bladder cancers. ctDNA-positive patients after curative-intent surgery may benefit from adjuvant therapy, with clinical trials ongoing.

Early Cancer Detection (Multi-Cancer Early Detection)

Galleri (GRAIL) is a cfDNA methylation-based test that can detect over 50 cancer types with tissue-of-origin prediction. It has population-level screening potential but requires validation in prospective clinical trials. Current limitations include lower sensitivity for early-stage (stage I) cancers and false-positive rate considerations. It holds FDA breakthrough device designation but is not yet standard of care.

Limitations and Challenges

False negatives occur with low tumor burden, non-shedding tumors (such as some CNS and mucinous tumors), and early-stage disease. Clonal hematopoiesis of indeterminate potential (CHIP) involves age-related somatic mutations in hematopoietic stem cells (DNMT3A, TET2, ASXL1, TP53, JAK2) that are detected as cfDNA variants and can be misattributed to tumor. Matched buffy coat/WBC sequencing is recommended to filter CHIP variants. Tumor heterogeneity means liquid biopsy may detect mutations not present in the primary biopsy and vice versa. Not all detected variants are actionable, and variant interpretation frameworks apply.

Clinical Pearls

ctDNA analysis captures spatial tumor heterogeneity by sampling DNA shed from multiple tumor sites, providing a more comprehensive genomic picture than a single-site tissue biopsy. Clonal hematopoiesis is a major confounding factor in liquid biopsy, and concurrent white blood cell sequencing is recommended to distinguish true tumor-derived variants from CHIP. Tumor-informed MRD assays achieve the highest sensitivity for detecting residual disease after curative-intent treatment and are increasingly used to guide adjuvant therapy decisions. A negative liquid biopsy result does not exclude the presence of actionable mutations, and tissue biopsy should be pursued when ctDNA testing is negative and clinical suspicion is high.

References

1. Wan JCM, et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017;17(4):223-238.
2. Merker JD, et al. Circulating tumor DNA analysis in patients with cancer: American Society of Clinical Oncology and College of American Pathologists joint review. J Clin Oncol. 2018;36(16):1631-1641.
3. Razavi P, et al. High-intensity sequencing reveals the sources of plasma circulating cell-free DNA variants. Nat Med. 2019;25(12):1928-1937.
4. Klein EA, et al. Clinical validation of a targeted methylation-based multi-cancer early detection test using an independent validation set. Ann Oncol. 2021;32(9):1167-1177.

Read this lecture as Markdown