Dongguang Wei, MD. Pathology Resident
Jeffrey Paul Gregg, MD, Senior Director of Clinical Pathology, Director of Molecular Diagnostics


What is liquid biopsy: A test done on circulating tumor cells or shed fragments of tumor DNA from a sample of blood to determine the genomics or molecular portrait of a tumor1. It is also known as a liquid biopsy (LBx), or circulating tumor (ct) DNA assay (ctDNA).

Why liquid biopsy:
Limitations of single tissue biopsy:

  1. Represents only a spatial and temporal snapshot of the tumor
  2. Suboptimal to represent tumor heterogeneity, especially when the tumor is not easily accessible or multiple tissue biopsies are not feasible
  3. Direct biopsy is difficult, expertise not available (pulmonology or interventional radiology) or unsafe for the patient
  4. Small biopsies may not be sufficient for molecular analysis; this can occur in up to 20% of biopsy samples
  5. The tissue may have significant necrosis or minimal tumor content that is not amendable to molecular profiling
  6. Decal tissue (e.g bone metastasis) causes significant DNA degradation such that it cannot be utilized for tissue-based molecular profiling.
  7. The overall turnaround time (TAT) for molecular analysis of a tumor, from scheduling the biopsy, to histological analysis, and finally molecular results, can take a significant amount of time that may impact therapeutic decision and clinical care

Advantages of liquid biopsy2:

  1. Minimal invasive procedure, can be applied to many body fluids (blood, urine, ascites, pleural effusion, etc.)
  2. Solving the issue of “insufficient tissue for molecular analysis”
  3. Can be used for monitoring of efficacy and progression
  4. Repeated at different time points to ensure “real-time” follow-up of the molecular changes during the disease course, i.e., driver mutation
  5. To monitor the responses to treatment, i.e., early detection of drug resistance and progression
  6. Can be used to identify therapeutic targets without invasive biopsy

Clinical applications for liquid biopsies:

  1. Establish the molecular fingerprint of the tumor at diagnosis
  2. When tissue is insufficient or cannot be sampled
  3. When the patient is too ill for biopsy
  4. At the time of progression to identify resistance mutations
  5. Confirmation of targeted therapy and mechanisms of drug resistance
  6. Real-time monitoring of disease (MRD)
  7. Early detection of metastasis
  8. Provide Prognostic information
  9. Can complement tumor molecular profiling before, at the same time, or after the tumor biopsy


Although circulating (ctDNA) can be isolated from many non-plasma body fluids, i.e., urine, cerebrospinal fluids, ascites, pleural effusion, and saliva, however, due to the intrinsic limitations of abovementioned specimen, blood represents one of the best sources for ctDNA sampling.

Compared to the normal cells, cancer cells shed higher concentrations of circulating nucleic acids (CNA) 3, 4. The underlying mechanisms could be inefficient clearance of apoptotic or necrotic cells within the tumor mass, leading to the accumulation of tumor debris and excess ctDNA.

Circulating cfDNA is composed mainly of highly fragmented apoptotic DNAs strands (between 200 and 160 base pairs) from both tumor cells and germline cells5. Being part of the cfDNA, the ctDNA can be distinguished from the non-tumor cfDNA (germline) by identifying the presence of somatic driver mutations, which, by definition, can be hall markers of tumor cells. Interestingly, the ctDNA demonstrates considerable variations in tumor fraction (ctDNA/cfDNA), between 0.01% and more than 90%5. Other technical challenges are: different tumor types shed variable amount of ctDNA, and, even in patients with the same tumor type, the concentration of ctDNA may vary dramaticially6.

Currently, we still have technical limitations of measuring circulating ctDNA2:

  1. Lack of standardized and widely approved methods for analysis
  2. Contamination with cell-free DNA (cfDNA) from healthy cells
  3. Low levels of ctDNA (False Negative)
  4. Accurate quantification of the mutant allele in the sample

Multiple pre-analytical variables, i.e., blood sampling and handling, ctDNA extraction procedures, and storage temperature may dramatically affect the quantity and quality of ctDNA fragment yields7-10.

Plasma is preferred as the matrix of choice for ctDNA extraction by the majority of clinical trials 7-11. For the collection of liquid biopsy samples, plasma should be used over serum as it has greater sensitivity. Following blood draw, plasma is obtained by centrifugation of the sample. To prevent cellular degradation, ethylenediaminetetraacetic acid (EDTA) tubes should only be used if the sample can be processed within 1 to 2 hours from collection. If longer, stabilization tubes (e.g., Cell-Free DNA BCT® [Streck, NE, US], The Cell-Free DNA Collection Tube [Roche, Basel, SUI] and PAXgene® Blood cfDNA tube, which use unique preservatives to prevent the release of genomic DNA, can stabilize blood at room temperature for >7 days, after which mutations can still be detected. Stabilization tubes allow for greater flexibility in processing time and reduce the risk of degradation and contamination. Fresh plasma should be stored at −20°C or −80°C (on dry ice for shipping), with long-term stability of DNA in plasma best demonstrated at −80°C.

Another issue following ctDNA extraction is the validation of quantification. The application of reliable and efficient protocol for cfDNA quantification is fundamental for the clinical evaluation of cfDNA as a liquid biopsy in order to obtain consistent data, comparable between laboratories. However, the standardization of the quantification method is not established yet. The frequently used protocols include spectrophotometric methods, fluorescent dyes, or quantitative PCR-based methods15.

Two different analytical approaches are applied for plasma ctDNA study: targeted approach or single gene (limited number of genes) and comprehensive genomic profiling approach (CGP and >60 genes). The targeted approach is applied to analyze known hotspot genetic mutations of specific genes aimed on therapy decisions; i.e., KRAS, EGFR, and BRAF genes in lung, colon, and melanoma tumors. The reported methods for this purpose include: real-time PCR; digital PCR (dPCR); droplet digital PCR (ddPCR); beads, emulsions, amplification, and magnetics (BEAMing); and targeted next-generation sequencing (NGS). The CGP approach is applied to investigate ctDNA over a broad number of genes. The reported method for this purpose is generally hybrid-capture NGS platforms. This approach can be more expensive and take longer to complete to the targeted approaches. In all of these approaches with liquid biopsy, the ability to identify amplifications is reliant on tumor fraction (higher the tumor fraction the better the sensitivity) and homozygous deletions and LOH are difficult to detect. There is high sensitivity and specificity for short variants.

NGS is emerging as a revolutionary approach for molecular testing, this cost-effective approach can analyze tens to hundreds of genes from multiple patients at the same time. One recent study developed cancer personalized profiling by deep sequencing (CAPP-Seq)16. CAPP-Seq method is capable to detect ctDNA in all stage II–IV non–small-cell lung carcinoma patients and in 50% of patients with stage I. The specificity of the test was 96% for mutant allele fractions down to approximately 0.02%16. In a recent study by UC Davis, they observed a significant association between tumor metabolic burden and the ability of an NGS-based liquid biopsy to detect gene mutations in patients with advanced solid tumors, suggesting that sufficient plasma ctDNA shed from metabolically active tumors is required for the successful detection of gene mutations in plasma ctDNA.17

The adoption of liquid biopsy is accelerating and this is not more evident in non-small cell lung cancer (NCSCL). CAP/IASLC/AMP and ASCO recommend, in NSCLC, when tissue is limited and/or insuf?cient for molecular testing, a ctDNA assay can be used to identify EGFR mutations. A recent IASLC statement paper recommends that liquid biopsy can be considered at the time of initial diagnosis in all patients with advanced NSCLC who need tumor molecular profiling, particularly when tumor tissue is scarce, unavailable, or for patients in whom invasive procedures may be risky or contraindicated. It is also recommended that liquid biopsy be conducted at the time of initial diagnosis if the turnaround time for tissue biopsy is anticipated to be longer than 2 weeks. Following the CAP/IASLC/AMP recommendations and the recent IASLC statement paper on liquid biopsy, there is now more familiarity and clinical interest in liquid biopsy for NSCLC. Two commercial NGS-based liquid biopsies, FoundationOne™ Liquid (Foundation Medicine, MA, US) and PGDx elio™ (Personal Genome Diagnostics, MD, US), were granted US FDA breakthrough device designation in April 2018 and July 2018, respectively. A 3rd commercial NGS-based liquid biopsy, Guardant360® assay (Guardant Health, CA, US), was granted US FDA expedited access pathway designation in February 2018. All three assays include the genes recommend by NCCN and CAP/IASLC/AMP including MSI-H.



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  17. Cathy Zhou*, Zilong Yuan*, Weijie Ma, Lihong Qi, Angelique Mahavongtrakul5, Ying Li,, Hong Li, Jay Gong, MS2, Jin Li, MS, Michael Molmen, Travis A. Clark, Matthew Cooke, Garrett M Frampton, Vincent A. Miller, Elizabeth H. Moore, David K. Shelton, Jeffrey P. Gregg, Philip J. Stephens, Tianhong Li. Detection of genomic alterations in plasma circulating tumor DNA in patients with 18fluorodeoxyglucose-avid solid tumors. J Hematol Oncol. 2018 Nov 6;11(1):129. doi: 10.1186/s13045-018-0671-8. PMID:30400986.