Introduction

Cancer is the second leading cause of death worldwide. Successful treatment hinges on early detection, but conventional screening and diagnostic approaches, often relying on tissue biopsies, come with limitations that hinder their effectiveness.

One promising solution is liquid biopsy, a groundbreaking technique that analyzes bodily fluids, such as blood, urine, lymph, and cerebrospinal fluid, for signs of cancer. When these fluids circulate near a tumor, they can acquire shed tumor cells and their components, such as DNA and RNA.

Liquid biopsies are innovative, non- or minimally-invasive medical procedures that detect and analyze these tumor-derived cells and components for cancer screening, treatment guidance, and follow-up care, potentially revolutionizing early detection and personalized treatment strategies. This article delves into the importance of early cancer detection, the shortcomings of existing screening methods, and the potential of liquid biopsy as a transformative advancement in the fight against cancer.

Commonly used biomarkers in cancer screening liquid biopsies include:

• Circulating tumor cells (CTCs) – Cancer cells that break free from the primary tumor, enter the bloodstream or lymphatic system, and, if not neutralized, can promote metastasis to distant organs.^[1], ^[2], ^[3] Tests like Cell Search® use CTCs. (See FAQ below.)
• Cell-free DNA (cfDNA) – Free-floating DNA fragments in the plasma that may reflect mutation and methylation pattern abnormalities indicative of abnormalities including cancer. Commercially available tests such as _ GRAIL Galleri, Epi proColon2.0 CE, FoundationOne Liquid CDx, and Guardant360 CDxFoundationOne Liquid CDx_ use cfDNA, leveraging a methylation database to identify patterns common to many cancers.^[4]. (See FAQ below.)
  It is important to note that cfDNA from non-cancerous cells can be used for a wide variety of health screening tests, including prenatal diagnostics; however, in the context of cancer, cfDNA from blood can be used to detect cancer at a lower threshold of cancer cells and an earlier cancer stage than circulating tumor DNA.
• Circulating tumor DNA (ctDNA) – A specific subset of cell-free DNA (cfDNA) used for identifying mutations and methylation patterns associated with cancer. It often retains the mutations of the original tumor.^[5] Tests like Agilent Resolution ctDX FIRST and Cobas EGFR Mutation Test v2 assess ctDNA. (See FAQ below.)
• Cell-free RNA (cfRNA) – Tumor-derived long, non-coding RNA and microRNA that modulate epigenetic alterations and regulate gene expression.^[6]
  

The importance of early detection in cancer treatment

"Even a small tumor may already harbor around one billion cells and have altered its phenotype and microenvironment, compromising the immune response and increasing the likelihood of metastasis." Click To Tweet

Early intervention and discovery in cancer care can reduce mortality rates and improve quality of life. As tumors progress, they can alter their microenvironment considerably, diminishing immune responses and heightening the risk of metastasis. Intervening early can increase the chances of survival for people with tumors that would otherwise continue their natural progression.

The significance of early cancer detection becomes even more critical when considering the varying survival rates across cancer stages. According to data from the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute, the average 5-year survival rate across cancer stages varies. For stage I-II cancer (local stage), the survival rate is notably higher at 71 percent, compared to 51 percent for stage II-III (regional stage) and 14 percent for stage IV (distant stage).^[7]

In this context, it is noteworthy that medical imaging techniques can only detect tumors equal to or larger than 1 centimeter (cm) in diameter.^[8] Remarkably, even at this small size, a tumor may already harbor around one billion cells and have altered its phenotype and microenvironment, compromising the immune response and increasing the likelihood of metastasis.^[2], ^[9]

Once detected using these conventional means, accessible tumors are biopsied or excised for staging (size and invasiveness) and grading (typical or atypical cell appearance). The tissue sample can identify relevant mutations and gene expression that guide selecting the most promising treatment.^[10]

The challenge of misdiagnosis

While offering promising advances in early detection, a common argument against frequent liquid biopsies is the risk of misdiagnosis due to an inaccurate false positive or false negative result.

False positives can drive people to make critical healthcare decisions based on erroneous information, potentially subjecting them to the emotional toll of further tests and unneeded treatments. Conversely, liquid biopsy false negatives, although not ideal, are of less concern because liquid biopsy screening is more effective at detecting disease than not screening, making it a valuable tool in early disease detection and monitoring. For example, Galleri® (by GRAIL), a well-known, commercially available multi-cancer early detection test, claims a false positivity rate (specificity) of 0.5 percent, suggesting that one in 200 people without cancer who take the test will be incorrectly identified as having cancer.

However, relying solely on specificity may present a skewed view of the test's accuracy. The use of predictive value may offer clearer insights. For example, the trial gauging the test's accuracy assessed blood samples with a cancer prevalence approximately 50 times higher than that observed in the general population, yielding a positive predictive value of 99 percent and a negative predictive value of 68 percent. In a more realistic setting, the test's positive predictive value would be 44.2 percent, and its negative predictive value would be 99.5 percent. The positive predictive value for early cancer stages alone would be even lower.^[11]

Therefore, striking the right balance between identifying true positives (sensitivity) and identifying true negatives (specificity) remains a critical challenge to overcome in developing and using liquid biopsy technologies. (For more information, see "Accuracy in screening" section below)

Liquid biopsy biomarkers

Liquid biopsy employs various biomarkers to detect and analyze cancer-related molecular alterations, including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and cell-free RNA (cfRNA).

Circulating tumor cells

Circulating tumor cells are cancer cells that have detached from a tumor and entered the bloodstream or lymphatic system. If not eliminated by immune cells, shear stress (frictional force in blood vessels), or other protective mechanisms, CTCs can spread to other body parts, driving metastasis.^[1], ^[2], ^[3]

Surviving CTCs can be detected and isolated using antibodies that interact with molecules expressed on tumor cell surfaces, but ideally not on healthy cells. Yet, relying on specific surface markers can pose challenges. Some commonly used markers are not exclusively associated with cancer cells and vary among individuals with different or even the same types of cancer. Additionally, cancer cells have the potential to alter their marker expression over time.^[1], ^[2]

Notably, tumor cell shedding can occur even in the early stages of cancer.^[12], ^[2] However, the rarity of CTCs makes their detection and isolation difficult. One CTC can hide in a billion other cells; even in advanced cancer, one milliliter of blood may contain fewer than 10 CTCs.^[7], ^[1] Nevertheless, CTCs possess unique qualities that other liquid biopsy biomarkers do not. Rather than focusing on a single cellular component, CTC isolation facilitates the analysis of DNA, RNA, and other cellular molecules, providing a more detailed picture of the tumor's traits and characteristics.^[1]

Cell-free DNA

Cell-free DNA (cfDNA) is a broad category of extracellular strands of DNA found in various body fluids and may reflect mutation and methylation pattern abnormalities indicative of cancer. By scrutinizing cfDNA for specific mutations associated with cancers, malignancies are detectable at earlier stages, improving the chances of successful intervention. However, utilizing cell-free DNA for cancer screening poses many challenges, including the need for improved sensitivity and specificity to ensure accurate detection of subtle genetic and epigenetic alterations.^[13]

Circulating tumor DNA

Circulating tumor DNA (ctDNA) is a fragment of tumor DNA present in body fluids that retains the mutations of the parent tumor. The mechanisms that drive cellular DNA release likely involve programmed cell death (apoptosis), unprogrammed, "accidental" cell death (necrosis), and the elimination of cells by the immune system.^{[14] [6]}

Circulating tumor DNA is vulnerable to enzymatic degradation, leaving only fragments protected through histone and non-histone protein binding or by encapsulation in extracellular vesicles undamaged,^[6] limiting the amount of DNA accessible to analyze mutations and epigenetic modifications.

Even so, as little as one milliliter of blood plasma can contain tens of billions of fragments of cfDNA, corresponding to 0.01 to 0.015 milligrams per milliliter, with only a fraction representing tumor DNA in a person with cancer.^[15], ^[7] In advanced cancers, the concentration of ctDNA can be ten times lower than that of cfDNA; in early cancer stages, it can be 1,000 times lower, posing significant technical challenges for detection.^[15] Up to 25 percent of metastatic cancers have undetectable ctDNA levels measured via current technology.^[7]

• Learn how epigenetics influences cancer risk in this overview article.

Cell-free RNA

Cell-free RNA (cfRNA) includes long, non-coding RNA and microRNA released from apoptotic and non-apoptotic cells.^[6] These RNA molecules, which are transcribed from DNA, serve as epigenetic modulators and regulators of cellular gene expression. Like ctDNA, cfRNA is a fragile molecule that relies on vesicles or binding proteins to shield it from degradation.^[6] In contrast to ctDNA analysis, tumor-derived cfRNA liquid biopsies offer insights into tumor biology by scrutinizing not only tumor-related changes in cfRNA originating from tumor cells but also from immune cells and the tumor microenvironment.^[16]

Beyond detecting cancer-relevant mutations, cfRNA can reveal alterations in gene expression, such as the upregulation of the cell membrane protein programmed death-ligand 1 (PD-L1). Elevated PD-L1 expression on cancer cells is a mechanism to dampen immune cell activation and is a target for immunotherapy.^[17]

Cancer screening

Cancer screening is a crucial component of preventive healthcare, involving systematic assessment of early signs of cancer before symptoms manifest. Effective cancer screening programs are pivotal in reducing cancer-related mortality and improving long-term patient outcomes.

Sensitivity, specificity, prevalence, positive and negative predictive value

Sensitivity, specificity, prevalence, and predictive value are statistical measures that characterize the accuracy of tests for screening and diagnosis.^[18], ^[19], ^[20] Resolving issues regarding these statistical measures is essential for validation and wide-scale implementation of liquid biopsies.

• Sensitivity, the proportion of true positives among positive test results. For example, a 90 percent sensitivity in a cancer test detects 90 out of 100 cancer patients but misses 10 (false negatives).
• Specificity, the proportion of true negatives among negative test results. For example, a 90 percent specificity identifies 90 out of 100 non-disease individuals as healthy but wrongly identifies ten as having the disease (false positives).
• Prevalence, the percentage of a population with a specific characteristic, such as a disease. Without consideration of prevalence, sensitivity and specificity only partially convey test performance.
• Positive and negative predictive values incorporate sensitivity, specificity, and prevalence to determine the probability that individuals with positive or negative test results have the disease or not. This calculator determines the positive predictive value of a given test.
  

Accuracy and reliability of traditional cancer screening tests and their influence on clinical outcomes

Lung cancer

Low-dose computed tomography (LDCT) scans are currently the only recommended screen for lung cancer. The prevalence of lung cancer in people with a heavy smoking history falls between 0.8 percent and 1.7 percent, resulting in LDCT's positive predictive value of less than 20 percent. LDCT scans have an estimated sensitivity of 85 percent, specificity of 94 percent, and negative predictive value of 99.87 to 99.72 percent in people with a heavy smoking history.^[21] Even with the hypothetical achievement of a 99 percent sensitivity and specificity in LDCT scans, the positive predictive value would still span from 44.39 percent (at 0.8 percent prevalence) to 63.13 percent (at 1.7 percent prevalence). Put simply, while LDCT scans excel at detecting lung cancer cases, they tend to produce positive results for many people who do not have the disease.

Nevertheless, a meta-analysis of randomized controlled trials, spanning up to 10 years of follow-up, revealed a 17 percent reduction in the risk of lung cancer-related mortality among high-risk populations undergoing LDCT screening compared to standard care.^[22]

Breast cancer

Mammography is considered the gold standard for breast cancer screening. Data from a study involving 55,000 Danish women demonstrated that mammography sensitivity varies, ranging from 47 percent for high-density breasts to 78 percent for low-density breasts, while specificity remains relatively consistent, ranging from 97 to 99 percent across breast density categories.^[23] Another extensive study encompassing more than 500,000 women concluded that mammography can lower the 10-year risk of breast cancer-related mortality by 34 percent or more.^[24]

Accuracy in cancer screening

A meta-analysis of several studies using CTCs, cfDNA, ctDNA, nucleosomes, microRNA, and multiple biomarkers evaluated the sensitivity and specificity of liquid biopsy in early cancer detection. The overall sensitivity and specificity were 76 percent and 92 percent, respectively, with better results for cfDNA than ctDNA and CTCs. The pooled sensitivity for early detection of lung cancer was 58 percent, and the pooled specificity was 86 percent.^[25]

Another meta-analysis involving patients with tissue biopsy-confirmed breast cancer and healthy controls evaluated the accuracy of cfDNA-based cancer screening. Liquid biopsy correctly identified 70 percent of cancer patients and 87 percent of healthy controls. The accuracy improved when the reviewers included only studies with modern assessments of genetic and epigenetic irregularities in cfDNA. The sensitivity reached 88 percent, while the specificity reached 98 percent.^[26]

The influence of liquid biopsy on reducing the risk of cancer-related mortality and other clinical outcomes remains uncertain. Currently, liquid biopsy cannot substitute conventional screening tests but may serve as a valuable complement.

Treatment guidance

"Liquid biopsies offer the advantage of near real-time monitoring of tumor evolution." Click To Tweet

Targeted therapy in cancer treatment involves using drugs specifically engineered to focus on cancer cells while sparing normal cells from harm. Knowing whether a cancer has targetable genetic mutations may enhance treatment outcomes in cancer patients.^[27] Currently, mutational analysis relies on tissue biopsies, which may not be recommended or feasible in some cases and may provide an incomplete assessment when tumors lack homogeneity.^[7]

Liquid biopsies offer a less stressful alternative for critically ill patients, allowing the collection of circulating biomarkers from various body sites through a single blood draw.^[28] This technology has demonstrated promise in guiding treatment decisions, including screening for mutations like KRAS in advanced colorectal cancer,^[29] EGFR in advanced non-small-cell lung cancer,^[30],^[31], and PIK3CA in advanced breast cancer^[32].

Monitoring the response to cancer treatment is crucial because it informs subsequent therapeutic decisions. Research indicates that liquid biopsy can detect changes in tumor burden earlier than conventional methods, such as imaging or protein biomarkers.^[33] A proof-of-concept analysis involving ctDNA in 30 breast cancer patients concluded that ctDNA outperformed the serum biomarker CA 15-3 in identifying changes in tumor burden.^[34]

Other studies have yielded mixed results. For example, in melanoma patients, a transient increase in ctDNA levels shortly after treatment initiation correlated with a positive treatment response,^[35] whereas responsive colorectal cancer patients exhibited decreasing ctDNA levels.^[36] Consequently, the measurement timing and accounting for potential variability in liquid biopsy biomarker levels in response to successful treatment across various cancer types and therapies are critical for interpreting test results.

Cancers often acquire mutations over time, leading to drug resistance.^{[37] [38]} Tissue biopsies, due to their invasiveness, are limited in terms of frequency and may cause delays in transitioning to potentially more effective treatments as resistance develops.^{[37] [38]} However, liquid biopsies offer the advantage of near real-time monitoring of tumor evolution.

For example, ctDNA analysis in advanced non-small cell lung cancer patients on EGFR mutation-targeted treatment revealed resistance mutations in 41.8 percent of cases.^[39] In a cohort study of 42 patients with gastrointestinal cancer, cfDNA analysis surpassed tissue biopsies in identifying clinically relevant resistance, with liquid biopsies detecting resistance not found in tissue samples in 78 percent of cases. ^[38]

Despite these promising findings, validating the preliminary results and assessing their influence on clinical outcomes requires further research.

Prognosis and recurrence

Neoadjuvant therapy encompasses chemotherapy, radiotherapy, or hormone therapy administered before surgery to reduce tumor size.^[40] Adjuvant therapy employs the same treatments to eliminate residual cancer cells and reduce the risk of cancer recurrence.^[40] However, these therapies often have severe side effects. Liquid biopsies may identify patients most suitable for (neo-)adjuvant therapy and enhance survival prediction.^[33]

For example, among 230 stage II colon cancer patients with ctDNA detected in their blood plasma, 79 percent experienced cancer recurrence 27 months after tumor resection without adjuvant therapy, whereas only 9.8 percent of patients without detectable ctDNA levels had recurrence. Those treated with adjuvant chemotherapy and having ctDNA in their plasma after treatment were 11 times more likely to experience cancer recurrence than those without ctDNA.^[41]

A meta-analysis of 21 studies involving non-metastatic breast cancer patients revealed that those with five or more CTCs per 7.5 milliliters of blood before neoadjuvant treatment were 6.25 times more likely to die during follow-up than patients with no detected CTCs. Patients with one CTC were 0.09 times more likely to die.^[42]

The diverse medical applications of liquid biopsy beyond oncology

While liquid biopsy research predominantly focuses on oncology, the potential applications of this technology extend to other medical fields. For example, screening for microbial cell-free DNA holds promise for diagnosing infectious diseases that may evade detection using conventional methods.^[43] The analysis of cfRNA and exosomes (small vesicles containing cellular components released by living cells) could provide a minimally-invasive alternative for identifying hypertensive disorders of pregnancy and gestational diabetes mellitus.^[44], ^[45]

Additionally, liquid biopsy can enhance the safety of prenatal screening for conditions like fetal growth restriction and congenital obstructive nephropathy, a urinary tract blockage that causes chronic kidney disease in children.^[44] In cardiovascular disease, exosome analysis may be a diagnostic tool for coronary artery disease, heart failure, cardiomyopathy, cardiac arrhythmia, valvular heart disease, and stroke.^[44] Moreover, changes in cfDNA levels and epigenetics hold promise for improving the diagnosis, prognosis, and treatment of neurological disorders, including traumatic brain injury, Alzheimer's disease, and multiple sclerosis.^[46]

Conclusion

Liquid biopsy is emerging as a significant innovation across various medical disciplines, primarily focusing on oncology but with potential applications in other areas. This approach, which involves the analysis of bodily fluids, offers a less invasive means of detecting and studying tumor-related biomarkers than traditional biopsies. Its applications span from early cancer detection to treatment guidance and monitoring of cancer recurrence. While recent studies underscore its potential, addressing existing accuracy challenges remains crucial.

As technology advances and our understanding of tumor biology deepens, liquid biopsy is poised to assume a central role in personalized medicine by delivering timely insights into tumor characteristics and progression. Ongoing research and clinical assessments are vital to enhance accuracy and expand its utility for improving patient outcomes in cancer management.

FAQ

Q: Which liquid biopsy tests currently have FDA approval or "breakthrough device designation"?

A: Several liquid biopsy tests have received FDA approval or breakthrough device designation, an identifier granted to specific innovative diagnostic tests or medical devices that may benefit diagnosing, monitoring, or treating severe medical conditions, expediting their availability to patients. Below is a list of some of these tests and their uses.

• Cell Search®

  • CTCs of epithelial origin (CD45-, EpCAM+, and cytokeratins 8, 18+, and/or 19+)
  • Whole blood
  • Monitoring of patients with metastatic breast, colorectal, or prostate cancer
  • Prognosis and prediction of progression-free survival and overall survival in patients with metastatic breast, colorectal, or prostate cancer

• Galleri Multi-cancer Early Detection Test (GRAIL, Inc.)

  • Not FDA approved, but available under a Clinical Laboratory Improvement Amendment (CLIA) waiver
    
  • cfDNA
  • Multi-cancer early detection test for 50+ types of cancers
  • Screening for abnormalities in methylation patterns that are specific to cancers

• Epi proColon2.0 CE (Epigenomics AG)

  • cfDNA
  • Plasma
  • Screening test for colorectal cancer
  • Identification of methylated Septin9 DNA
  • Positive results need to be verified by colonoscopy or sigmoidoscopy

• FoundationOne Liquid CDx (Foundation Medicine)

  • cfDNA
  • Plasma
  • Therapy guidance in patients with metastatic castration-resistant prostate cancer
  • Identification of BRCA1, BRCA2, ATM mutations
  • Companion diagnostic test with RUBRACA (rucaparib), LYNPARZA® (olaparib)
  • Therapy guidance in patients with non-small cell lung cancer
  • Identification of EGFR, MET mutations
  • Companion diagnostic test with IRESSA (gefitinib), TAGRISSO (osimertinib), TARCEVA (erlotinib), EXKIVITY (mobocertinib), TABRECTA® (capmatinib)
    
  • Therapy guidance in patients with metastatic colorectal cancer
  • Identification of BRAF V600E alterations
  • Companion diagnostic test with BRAFTOVI (encorafenib)

• Guardant360 CDx (Guardant Health, Inc.)

  • cfDNA
  • Whole blood
  • Complete genomic testing for all solid cancers
  • Therapy guidance in patients with advanced NSCLC
  • Identification of EGFR mutations, ERBB2 mutations, KRAS G12C mutations
    
  • Companion diagnostic test with TAGRISSO® (osimertinib), RYBREVANT® (amivantamab-vmjw), ENHERTU® (fam-trastuzumab deruxtecan-nxki) or LUMAKRAS™ (sotorasib)
  • Therapy guidance in patients with advanced breast cancer
  • Identification of ESR1 mutations
  • Companion diagnostic test with ORSERDU™ (elacestrant)

• Agilent Resolution ctDX FIRST (Resolution Bioscience)

  • ctDNA
  • Plasma
  • Therapy guidance in patients with advanced non-small cell lung cancer (NSCLC)
  • Identification of KRAS G12C mutation
  • Companion diagnostic test with KRAZATI (adagrasib)

• Cobas EGFR Mutation Test v2 (Hoffman-La Roche Ltd.)

  • ctDNA
  • Plasma
  • Therapy guidance in patients with metastatic non–small cell lung cancer (NSCLC)
  • Identification of epidermal growth factor receptor (EGFR) gene mutations
  • Companion diagnostic test with Tarceva (erlotinib) for metastatic NSCLC
    
  • Companion diagnostic test with IRESSA (gefitinib) for NSCLC

Other resources

• An Overview of Liquid Biopsy for Screening and Early Detection of Cancer
• Liquid biopsy enters the clinic — implementation issues and future challenges
• Liquid biopsy: a step closer to transform diagnosis, prognosis and future of cancer treatments
• Circulating cell-free nucleic acids: characteristics and applications

Relevant clips and episodes

• Q&A Session #14 (08/01/20) - Modifiable risk factors for reducing cancer risk.
• Q&A Session #03 (03/11/18)
• Q&A Session #34 (04/02/22) - Q: Would you stop taking a multivitamin if you were diagnosed with cancer?

The information presented within these Q&A segments or the broader article are not intended to serve as a substitute for the consultation, diagnosis, and/or medical treatment by a qualified healthcare provider.