# 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.

![Liquid biopsy workflow from blood collection through cfDNA extraction to genomic analysis](images/liquid-biopsy-workflow.jpg)

## 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.

| FDA-Approved Test | Technology | Key Indications |
|---|---|---|
| Guardant360 CDx | NGS (74 genes) | EGFR in NSCLC, pan-tumor TMB-H |
| FoundationOne Liquid CDx | NGS (300+ genes) | Multiple companion Dx indications |
| cobas EGFR Mutation Test v2 | PCR-based | EGFR mutations in NSCLC |
| Epi proColon | Methylation PCR | CRC screening (SEPT9) |
| Shield (Guardant) | cfDNA methylation | CRC screening |

## 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.

![ctDNA kinetics showing serial monitoring during treatment with decline, nadir, and resistance emergence](images/ctdna-kinetics.jpg)

### 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.

![Comparison of tissue biopsy versus liquid biopsy showing complementary genomic information from tumor heterogeneity](images/tissue-vs-liquid-biopsy.jpg)

## 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

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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.