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 Table of Contents  
Year : 2022  |  Volume : 2  |  Issue : 2  |  Page : 49-53

Liquid biopsy-detecting molecular targets in cancer management

Department of Histopathology, NHS Trust, Dorset, UK

Date of Submission12-Mar-2022
Date of Acceptance05-Apr-2022
Date of Web Publication17-May-2022

Correspondence Address:
Dr. Nithya Ravindran
17 The Woodpeckers, Weymouth, Dorset DT3 5RS
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jalh.jalh_10_22

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Liquid biopsies are newer diagnostic tests conducted on blood samples to detect biomarkers shed by tumors, such as cancer cells or portion of tumor DNA. Liquid biopsies are not to replace surgical biopsies but are complementary in the diagnosis and treatment of cancer. The circulating tumor DNA and intact circulating tumor cells are the two main components that are targeted during a liquid biopsy. Information from the liquid biopsy is used to determine the best-individualized cancer therapy to track how a patient is responding to treatment, or to discover whether there is recurrence of cancer. This review summarizes the main techniques and applications of liquid biopsy in cancer.

Keywords: Biomarkers, circulating tumor cells, liquid biopsy, tumor DNA

How to cite this article:
Ravindran N. Liquid biopsy-detecting molecular targets in cancer management. J Adv Lung Health 2022;2:49-53

How to cite this URL:
Ravindran N. Liquid biopsy-detecting molecular targets in cancer management. J Adv Lung Health [serial online] 2022 [cited 2022 Jul 6];2:49-53. Available from: http://www.jalh.com/text.asp?2022/2/2/49/345370

  Introduction Top

Liquid biopsies are newer diagnostic tests to detect certain cancers using blood samples by identifying biomarkers shed by tumors, such as cancer cells or portion of tumor DNA. Liquid biopsy is a revolutionary technique consisting of the detection and isolation of circulating tumor cells (CTC), circulating tumor DNA (ctDNA), and exosomes (EXOs). A liquid biopsy will help in deciding patient-tailored therapies that has significantly improved all measurable outcomes. Tissue samples collected from surgical biopsies are considered the gold standard in cancer diagnosis and treatment because they offer a true representative sample of the tumor. However, there are limitations for tissue biopsy [Table 1]. It is an invasive test, and many tumors are not accessible to tissue samplings such as deeper lesions or highly vascular lesions. Sometimes, the condition of the patient may not permit an invasive procedure when the patient is too sick. Moreover, tissue samples retrieved from the tumor may be inadequate for processing or the biopsy may not pick up the true representative samples. Liquid biopsies overcome many of the obstacles that come from surgical biopsies. Since these biopsies are obtained with a simple blood sample, they are noninvasive, allowing for data even when a surgical biopsy is not possible. In addition, liquid biopsies are often less expensive than surgical biopsies.
Table 1: Comparison between standard tissue biopsy and liquid biopsy

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Another advantage of liquid biopsies is their potential to detect cancer recurrence or relapse before a cancer becomes visible or a patient exhibits clinical symptoms.[1] Following treatment, levels of ctDNA that break off from the tumor and enter the bloodstream drop after the tumor is surgically removed or as the tumor's size decreases. If the tumor begins to grow back, ctDNA levels increase, and this increase can be detected with a liquid biopsy. DNA fragments and cancer cells collected from liquid biopsies originate from throughout the body, rather than from a single tumor site as with a tissue biopsy. As a result, liquid biopsies may pinpoint genetic mutations that would have been missed with a localized surgical biopsy.

Liquid biopsy is a relatively new procedure. As such, present understanding restricts its use to certain forms of cancer, including breast cancer, prostate cancer, colon cancer, and non-small cell lung cancer (NSCLC). Another newer application of liquid biopsy is in early cancer detection and evaluation of cancer at early stages.

  Advantages Over Surgical Biopsy Top

Liquid biopsy is non invasive and can be used where surgical biopsy is not possible. Liquid biopsy is often less expensive than surgical lung biopsy. Another advantage is the potential to detect cancer recurrence or relapse before a cancer becomes visible or the patient exhibit clinical symptoms.

  Liquid Biopsy Techniques Top

Apoptotic or necrotic cancer cells release circulating cell-free DNA fragments, designated as ctDNA, as well as EXOs, namely membrane-encapsulated subcellular structures containing proteins and nucleic acids released by the tumor cells.[2],[3] Primary tumor and metastatic sites are also able to exfoliate vital cells that enter the bloodstream, which are called CTCs. Liquid biopsy currently offers a high specificity, allowing the collection of robust and reproducible data in a simple and noninvasive way using a blood sample.[4]

The current landscape of liquid biopsy includes three primary techniques:

  1. ctDNA or cell-free DNA tests: Also known as ctDNA tests or cfDNA tests, these tests analyze DNA from a tumor found in the bloodstream
  2. CTC tests: Also referred to as CTC tests, these liquid biopsy tests look for whole tumor cells found in the bloodstream. These cells work as metastasis agents that can lead to the growth of additional tumors in sites other than that of the primary tumor
  3. EXO tests: Other liquid biopsy techniques are being investigated, including those examining molecules of noncoding RNA and extracellular vesicles known as EXOs.

CTC, ctDNA, and EXOs are analyzed for information such as DNA abnormalities, RNA expression, protein expression, amplifications, deletions, translocations, chromosome abnormalities, and point mutations [Figure 1].
Figure 1: Cancer targets in the blood sample and the informations they provide for cancer management

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Liquid biopsy tests approved by the Food and Drug Administration could identify specific changes in single genes only. The newly approved tests use a technique called next-generation sequencing (NGS), which can evaluate many different genes at the same time.

To detect somatic point mutations, several polymerase chain reaction (PCR)-based technologies such as beads, emulsion, amplification, and magnetic (BEAMing) and droplet digital PCR (ddPCR) are used. In BEAMing technologies, magnetic beads in water-in-oil emulsions are used to perform a single-molecule amplification by PCR, followed by a flow cytometry to quantify the genetic variants. In ddPCR, the sample is divided into thousands of droplets representing a partition of single molecules, each of them undergoing a PCR analysis by selected primers against known regions of ctDNA. The molecular alterations are then measured by fluorescent probes, which bind to the amplified region. Digital PCR technologies are limited by scalability for larger studies and may miss substantial information. To overcome this issue and obtain a larger and more comprehensive analysis of genomic regions, several NGS-based technologies are developed.

  Clinical Applications of Liquid Biopsies Top

The genetic information provided by tumor biomolecules offers insight into the tumor. This information can be used in the following:

  • Early detection of cancer in susceptible individuals
  • Prognosis of the cancer
  • Determining individualized treatments
  • Measuring a cancer's response to treatments
  • Looking for cancer relapse or recurrence after treatment.

  1. Early intervention: Liquid biopsy can be used to diagnose cancer earlier through screening. This is especially important in susceptible individuals such as those who are having a strong family history of cancer or who are exposed to risk factors to develop cancer
  2. Localized disease: This will help to determine the risk of recurrence after treatment
  3. Metastatic disease: Used to determine treatment selection based on the presence of biomarkers. This is the basis of precision medicine or individualized treatment
  4. Refractory disease: Liquid biopsy can also give information regarding mechanisms of treatment resistance, disease progression, and identification of newer treatments.

An ideal scenario where liquid biopsy helps the clinician to make an appropriate clinical scenario is NSCLC. The identification of oncogenic activation of particular tyrosine kinases in some advanced NSCLC tumors, especially mutations in the epidermal growth factor receptor (EGFR), rearrangements of the anaplastic lymphoma kinase (ALK) gene or c-ROS oncogene 1 gene, has led to the development of specific molecular treatments for patients. Identification of these patient subsets has led to an ongoing effort to identify biomarkers and treatments that can be used for other patients with advanced NSCLC. It is unfortunate to see that number of patients with advanced adenocarcinoma (NSCLC) subjected to tumor genetic testing remains low, and it is therefore important to increase awareness regarding potentially targetable genetic alterations.

In NSCLC, as well as with other malignancies, selecting a specific targeted drug to the identified driver mutation has significantly improved therapeutic efficacy, often in conjunction with decreased toxicity. Screening for driver mutations thus has become an increasingly standard part of the diagnostic workup for NSCLC, and the resultant information is useful in choosing between standard chemotherapy (in the absence of a targetable driver mutation) and newer targeted therapies. According to a French study in which all lung cancers were subjected to molecular profiling, approximately 50% of tumors exhibited a genetic alteration, which led to the use of a targeted agent as first-line therapy in these cases.[5] The presence of a genetic alteration was associated with improved first-line progression-free survival (10 vs. 7.1 months) and overall survival (16.5 vs. 11.8 months). The Lung Cancer Mutation Consortium in the United States analyzed samples using multiplex genotyping from 733 patients with adenocarcinoma at 14 centers, identifying a targetable driver mutation in 466 cases (64%).[6] The median survival for 260 patients with an oncogenic driver who received a targeted agent was 3.5 years, the median survival for 318 patients with a driver but without targeted therapy was 2.4 years, and the median survival for 360 patients without a driver mutation was 2.1 years. These studies underscore the potential clinical benefit and prognostic utility provided by large-scale utilization of molecular profiling in lung cancer.

The most useful biomarkers for predicting the efficacy of targeted therapy in advanced NSCLC are somatic genome alterations known as “driver mutations.” These mutations occur in cancer cells within genes encoding for proteins critical to cell growth and survival. Many other recurrent molecular alterations have been identified in NSCLC that are much less essential to maintain the oncogenic phenotype, and are often referred to as “passenger mutations.”

Guidelines from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association of Molecular Pathologists recommend analysis of either the primary tumor or of a metastasis for EGFR and ALK for all patients whose tumor contains any component of adenocarcinoma histology, or who have light or never-smoking history.[7],[8]

The principle behind liquid biopsies is that cell-free ctDNA and/or CTCs are often present in the blood of patients with lung cancer.[9] Platforms available for clinical use focus almost exclusively on isolating and detecting ctDNA rather than CTCs. PCR-based platforms include allele-specific PCR, which preferentially amplifies a mutant DNA molecule over wildtype DNA, and emulsion PCR assays, which perform PCR reactions in thousands of droplets of a sample to quantify mutant and wildtype DNA.[10] While such assays may have a theoretical turnaround time of as little as 1 day, they generally cover a limited number of common mutations, such as EGFR, KRAS, BRAF, and ALK. NGS-based plasma genotyping platforms are much broader in scope but take 1–3 weeks for results. In general, all of these methods are highly specific, although some platforms may detect allelic alterations that are present at such a low frequency that they may be clinically insignificant or represent low-level sequencing background noise or clonal mutations in hematopoietic stem cells.[9],[10],[11]

  The Future of Liquid Biopsy Top

The potential of the liquid biopsy in the field of cancer research is being clearly recognized and the role of liquid biopsy in the course of cancer diagnosis and management is being evaluated in several clinical trials. However, for the actual implementation of the liquid biopsy in routine clinical practice, it is necessary to develop standardized methodologies such as blood collection, storage, processing, and DNA extraction. Progress in the clinical implementation can be achieved only if long-term studies with adequate sample sizes are performed and results obtained from such studies are correlated with disease-free survival or overall survival. The timing of ctDNA analysis in relation to therapy may be important. Multiple samples at different time points should be obtained from the same patients for monitoring ctDNA after and during drug administration which may provide valuable information on the kinetics of ctDNA release.

The limitation of ctDNA-based liquid biopsies is that the types of studies have a higher chance of false negativity than traditional biopsies because of the variable amounts of DNA that tumors may shed into circulation. The sensitivity of liquid biopsies ranges between 60% and 80%,[12],[13] as some cancers do not shed DNA into the bloodstream. Mutation detection in blood has been associated with more advanced disease characteristics, including worsened performance status and prognosis and the presence of more metastatic sites.[14] Irrespective of technical limitations, liquid biopsy is a fascinating area of cancer research, clinical standards of which should be validated by well-designed and sufficiently powered multicenter studies.

The detection of CTCs in the peripheral blood of patients with solid epithelial tumors (e.g., breast, prostate, lung, and colon cancer) has great potential in cancer management.[15] Currently, more than 400 clinical trials use CTCs as biomarkers and PubMed lists more than 14,000 publications on CTCs. Many publications deal with the patients in advanced stages, but there is also an increasing number of publications on patients at earlier disease stages without clinical and radiologic signs of overt metastases, especially in breast cancer,[16],[17] but also in other tumor entities such as bladder cancer.[18]

The detection of tumor-educated platelets (TEP) in the peripheral blood of patients is expected to be used in clinical tumor diagnosis. This is a new development based on the knowledge that under the stimulation of tumor cells and the tumor microenvironment, platelet precursor mRNA is spliced into mature RNA and converted into functional protein to respond to external stimuli, forming TEPs. This emerging liquid biopsy method can replace and supplement the current tumor detection methods.

  Conclusion Top

Liquid biopsies are gaining popularity as they provide the opportunity to genotype in a less invasive and less expensive manner, and may offer a chance to monitor the molecular features of cancer through the course of treatment or predict relapse after adjuvant treatment. Liquid biopsy and NGS tests provide enhanced genetic information about tumors and help determine whether targeted cancer drugs can be used to treat the tumors. It was specifically approved as a companion diagnostic for patients with a type of NSCLC and other types of cancers such as bladder cancer, breast cancer, colonic cancer, and prostate cancer. Unfortunately, the number of patients with advanced cancer subjected to tumor genetic testing remains low, and it is therefore important to increase awareness regarding potentially targetable genetic alterations.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Jamal-Hanjani M, Wilson GA, McGranahan N, Birkbak NJ, Watkins TB, Veeriah S, et al. Tracking the evolution of non-small-cell lung cancer. N Engl J Med 2017;376:2109-21.  Back to cited text no. 1
Perakis S, Speicher MR. Emerging concepts in liquid biopsies. BMC Med 2017;15:75.  Back to cited text no. 2
Siravegna G, Marsoni S, Siena S, Bardelli A. Integrating liquid biopsies into the management of cancer. Nat Rev Clin Oncol 2017;14:531-48.  Back to cited text no. 3
Crowley E, Di Nicolantonio F, Loupakis F, Bardelli A. Liquid biopsy: Monitoring cancer-genetics in the blood. Nat Rev Clin Oncol 2013;10:472-84.  Back to cited text no. 4
Barlesi F, Mazieres J, Merlio JP, Debieuvre D, Mosser J, Lena H, et al. Routine molecular profiling of patients with advanced non-small-cell lung cancer: Results of a 1-year nationwide programme of the French Cooperative Thoracic Intergroup (IFCT). Lancet 2016;387:1415-26.  Back to cited text no. 5
Kris MG, Johnson BE, Berry LD, Kwiatkowski DJ, Iafrate AJ, Wistuba II, et al. Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. JAMA 2014;311:1998-2006.  Back to cited text no. 6
Lindeman NI, Cagle PT, Aisner DL, Arcila ME, Beasley MB, Bernicker EH, et al. Updated Molecular Testing Guideline for the Selection of Lung Cancer Patients for Treatment with Targeted Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Thorac Oncol 2018;13:323-58.  Back to cited text no. 7
Leighl NB, Rekhtman N, Biermann WA, Huang J, Mino-Kenudson M, Ramalingam SS, et al. Molecular testing for selection of patients with lung cancer for epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors: American Society of Clinical Oncology endorsement of the College of American Pathologists/International Association for the study of lung cancer/association for molecular pathology guideline. J Clin Oncol 2014;32:3673-9.  Back to cited text no. 8
Haber DA, Velculescu VE. Blood-based analyses of cancer: Circulating tumor cells and circulating tumor DNA. Cancer Discov 2014;4:650-61.  Back to cited text no. 9
Oxnard GR, Paweletz CP, Sholl LM. Genomic analysis of plasma cell-free DNA in patients with cancer. JAMA Oncol 2017;3:740-1.  Back to cited text no. 10
Hu Y, Ulrich BC, Supplee J, Kuang Y, Lizotte PH, Feeney NB, et al. False-positive plasma genotyping due to clonal hematopoiesis. Clin Cancer Res 2018;24:4437-43.  Back to cited text no. 11
Oxnard GR, Thress KS, Alden RS, Lawrance R, Paweletz CP, Cantarini M, et al. Association between plasma genotyping and outcomes of treatment with osimertinib (AZD9291) in advanced non-small-cell lung cancer. J Clin Oncol 2016;34:3375-82.  Back to cited text no. 12
Sacher AG, Paweletz C, Dahlberg SE, Alden RS, O'Connell A, Feeney N, et al. Prospective validation of rapid plasma genotyping for the detection of EGFR and KRAS mutations in advanced lung cancer. JAMA Oncol 2016;2:1014-22.  Back to cited text no. 13
Wu YL, Sequist LV, Hu CP, Feng J, Lu S, Huang Y, et al. EGFR mutation detection in circulating cell-free DNA of lung adenocarcinoma patients: Analysis of LUX-Lung 3 and 6. Br J Cancer 2017;116:175-85.  Back to cited text no. 14
Alix-Panabières C, Pantel K. Circulating tumor cells: Liquid biopsy of cancer. Clin Chem 2013;59:110-8.  Back to cited text no. 15
Zhang L, Riethdorf S, Wu G, Wang T, Yang K, Peng G, et al. Meta-analysis of the prognostic value of circulating tumor cells in breast cancer. Clin Cancer Res 2012;18:5701-10.  Back to cited text no. 16
Lucci A, Hall CS, Lodhi AK, Bhattacharyya A, Anderson AE, Xiao L, et al. Circulating tumour cells in non-metastatic breast cancer: A prospective study. Lancet Oncol 2012;13:688-95.  Back to cited text no. 17
Rink M, Chun FK, Dahlem R, Soave A, Minner S, Hansen J, et al. Prognostic role and HER2 expression of circulating tumor cells in peripheral blood of patients prior to radical cystectomy: A prospective study. Eur Urol 2012;61:810-7.  Back to cited text no. 18


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