1076062
review-article2022

CIX0010.1177/11769351221076062Cancer InformaticsHirahata et al

Liquid Biopsy: A Distinctive Approach to the
Diagnosis and Prognosis of Cancer
Tetsuyuki Hirahata , Reeshan ul Quraish , Afraz ul Quraish,
Shahan ul Quraish, Munazzah Naz and Mohammad Abdul Razzaq

Cancer Informatics
Volume 21: 1–7© The Author(s) 2022
Article reuse guidelines:
sagepub.com/journals-permissions
DOI: 10.1177/11769351221076062
https://doi.org/10.1177/11769351221076062

Hirahata Gene Therapy Laboratory, HIC Clinic, Tokyo, Japan.

ABSTRACT: Cancer is a leading cause of morbidity and mortality worldwide. Over the past decades, the concept of precision cancer medicine
has emerged as a novel approach in the field of oncology that aims to tailor the most effective treatment options to each individual cancer patient
based on the genetic profile of the tumor of each individual patient. Recently, tissue biopsy has become an essential part of cancer care andis widely used to characterize the tumor. However, tissue biopsy techniques face different challenges due to their invasiveness, cost, time, andadversity in potential sampling due to tissue heterogeneity. To overcome these issues, a non-invasive approach has developed, which is known
as liquid biopsy. It is a simple, fast, and worthwhile technique based on the analysis of circulating tumor DNA (which is a fraction of cfDNA),circulating tumor cells (CTCs), and other tumor-derived material in blood plasma. This review provides an overview of the concept of liquidbiopsy and briefly discusses the role of ctDNA and CTC analysis as tools for early diagnosis and prognosis of cancer. In this review, we also
speculate on the advantages of liquid biopsy as opposed to tissue biopsy and postulate that liquid biopsy may be a comprehensive approachto overcome the current limitations associated with costly, invasive, and time-consuming tissue biopsy.
Keywords: Cancer, liquid biopsy, tissue biopsy, circulating tumor DNA (ctDNA), circulating tumor cells (CTCs)
RECEIVED: November 9, 2021. ACCEPTED: January 7, 2022.
Type: Review
Funding: The author(s) received no financial support for the research, authorship, and/or
publication of this article.

Introduction

Cancer is a term for a distinctive disease characterized byuncontrolled abnormal cell proliferation, which can invade
nearby tissues and metastasize to distant organs through theblood and lymph systems.1,2 Metastatic spread of cancer to distant sites remains the main cause of death for cancer patients in~90% of cases.3 For cancer treatment, conventional cancer
therapies practiced in the clinic include systemic chemotherapy(including hormonal therapy and molecular targeted drugs),
surgery and radiotherapy,4 and the choice of therapy depends
on the type and stage of the tumor, as well as the general state
and age of the patient.4,5 These conventional therapies are generally effective against early-stage localized tumors, whereas
their effectiveness is not the same against a later stage of cancer
and there are chances of frequent relapse.4,6Drug resistance appears to be a serious problem in treatment of cancer; currently, 90% of chemotherapy failures occur
during the invasion and metastasis of cancers related to drug
resistance. In chemotherapy, by following the administration ofa certain drug, a large number of patient tumor cells become
resistant to the drug.7
Although many types of cancer are initially susceptible tochemotherapy, over time they can develop resistance to traditional therapies through different mechanisms, such as drug
inactivation, epigenetics, drug target alteration, drug efflux,
DNA damage repair, cell death inhibition, and epithelialmesenchymal transition (EMT). These mechanisms promote
direct or indirect drug resistance in human cancer cells, acting
independently or in combination and through various signaltransduction pathways.8

Declaration of Conflicting Interests: The author(s) declared no potential
conflicts of interest with respect to the authorship, and/or publication of this article.
CORRESPONDING AUTHOR: Tetsuyuki Hirahata, Hirahata Gene Therapy Laboratory,
HIC Clinic #1105. Itocia Office Tower 11F, 2-7-1. Yurakucho, Chiyoda-ku, Tokyo 100-0006,
Japan. Email: hirahata@hicc.jp

One study showed that mutations in self-controlling negative feedback in signal transmission can produce resistancefor drugs targeting such signaling pathways. Furthermore,tumor cells may also lack antigenic mutations, or some key
molecules of the immune system are perhaps absent, such as
HLA, and these cells can become unsusceptible to immunotherapy drugs.9
In addition, various agents used in conventional therapies
are harmful both to normal cells and cancer cells, which canlead to prominent side effects. These conventional cytotoxic
treatments also lack specificity for tumor cells, making themineffective in providing lifelong protection against cancer.4,6
Therefore, there was an urgent need for new therapeuticstrategies that allow more effective monitoring of cancer status,
thereby improving the efficacy of treatment by specificallyeliminating cancer cells and sparing normal cells from toxicity
to develop long-lasting protection.10
As a result of recent advancements in medical and pharmacogenetic research, the field of oncology has introduced aprofound innovative approach that overcomes the old onesize-fits-all strategy; this new paradigm is known as precision
medicine, which is designed to customize therapies for patientsin relation to the personalized molecular profile of thetumor.11,12 So far, the path of survival of cancer patients is tissue dependent, known as tissue biopsy, which has several
advantages over conventional cancer treatments.

Tissue Biopsy

Tissue biopsy involves the examination of tumor tissue by
obtaining tissue through a fine needle or open surgery,13 these

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Cancer Informatics ﻿2tissue samples are genetically analyzed to define the type oftumor and then the best personalized treatment regimen isdesigned.

Drawbacks of tissue biopsy
Despite the informative nature of tissue biopsy, it is painful,invasive, costly, time consuming, and, most importantly, sometimes inappropriate to capture tumor heterogeneity.14
Apart from other issues related to tissue biopsy, intratumoral heterogeneity is always a potential issue in the precision of this procedure, which induces significant challenges inselecting an effective treatment strategy based only on tissue
biopsy.15
Tissue biopsies typically obtain a sample of only a part of
the tumor and therefore encompass only a fraction of tumor
heterogeneity, as serial tumor sampling is not clinically practical with invasive tissue biopsy; therefore, complete informationon the levels of genetic and epigenetic variability of a patient’s
cancer is not achieved.16,17
Invasive tissue biopsy has also been studied to be impossible
after surgical resection and if there is no detectable metastatic
mass.18
Moreover, unsatisfactory detection of early-stage tumor orresidual lesions, also causing limitations of this therapy.19
Therefore, a worthwhile and noninvasive approach has
been developed that could proficiently capture the genomic
content of tumors in fluids and detect tumor heterogeneity inthe course of cancer treatments; this approach is known as
liquid biopsy that provides noninvasive multifunctional biomarkers for early diagnosis, accurate prognosis, selection oftherapeutic targets, monitoring of metastases, as well as monitoring response and resistance to treatment.20

Liquid Biopsy

The term liquid biopsy is basically introduced for the analysis
of CTCs21 but is now also associated with ctDNA and otherbiomarkers such as miRNAs.22
In addition to blood, several body fluids such as blood,plasma, serum, saliva, urine, and gastric juice could be used toperform liquid biopsy for non-invasive clinical evaluation.23-25
The clinical use of liquid biopsy has increased significantly
since 2004, when Guardant Health released the first commercially available liquid biopsy test.26 Liquid biopsy (restricted to
ctDNA analysis) was officially adopted as a diagnostic tool inthe main reference center laboratories during the course of
2016.17
Currently, there are several commercially available assaysthat are FDA approved; some are considered proficient intreatment capability by insurance companies. For example, in
2016, the FDA approved cobas® EGFR Mutation Test v2 todetermine the eligibility of patients with non-small cell lungcancer to receive certain EGFR tyrosine kinase inhibitors.27-29

Liquid biopsy: A comprehensive approach toovercome the current limitations associated withtissue biopsy
Liquid biopsy represents a contemporary diagnostic and prognostic tool in precision oncology that has received considerable
recognition over the past years to overcome the current limitations associated with tissue biopsies.30
For example, cancer is often found in organs or tissues of the
body that are difficult to access, such as in lung cancer, tissueresident biomarker measurements are clinically often not possible due to significant clinical risk, such as bleeding, injury, or
infection as a consequence of an invasive tissue biopsy; therefore, liquid biopsy has the advantages of being a noninvasive
and sustainable technique to solve this problem.31
In the perspective of tumor heterogeneity, serial liquidbiopsy can be a better representative of the entire population oftumor cells than a tissue biopsy sample, can monitor tumor
heterogeneity longitudinally, and allows successive monitoringof the tumor genetic profile.15,18,32-34
Furthermore, unlike tissue biopsies, liquid biopsy enables
the detection of tumor at an early stage; early detection oftumor not only prevents cancer mortality, but also reduces
morbidity and costs.35

Approaches to liquid biopsy analysis
Liquid biopsy comprises the noninvasive analysis of ctDNA
and CTCs. In addition to ctDNA and CTC biomarkers, otherblood components recently discovered as sources of bloodbased biomarkers, such as exosomes or platelets,36 but in thisreview we will focus on circulating tumor DNA (ctDNA) andcirculating tumor cell (CTC).
Cell-free DNA (cfDNA)/circulating tumor DNA (ctDNA). Circulating cell-free DNA (cfDNA) and circulating tumor DNA
are significant non-invasive blood biomarkers used to assess
tumor diagnosis and prognosis.37
Discovery of cfDNA and ctDNA. Mandel and Metais discovered the presence of cfDNA in the blood of healthy individuals
in 1948.38 Decades later, many researchers have extended Mandel and Metais’ work to the identification of tumor-derived
cfDNA (also known as circulating tumor DNA-ctDNA) inthe blood of cancer patients.38,39
According to previous reports, ctDNA possesses many cancer-associated molecular characteristics, such as single nucleotide mutations, methylation changes, cancer-derived viralsequences, rearrangements, amplifications, MSI and LOH,40-43
therefore, it was considered derived from tumor tissue.
Presence of cfDNA and ctDNA in the body. It is mainly foundin the bloodstream; now it can be derived from cerebrospinal
fluid, saliva, and can be excreted through urine with extremely
low content. Recently, ctDNA has been shown to be present in
the CSF of patients with brain tumors, and a high percentage

Hirahata et al

3

Figure 1. Cell-free DNA (cfDNA) concentration in healthy and cancer patients and its relation to circulating tumor DNA (ctDNA). ctDNA originates mainly
from necrosis, apoptosis, and/or circulating tumor cells (CTCs). Fragments of this tumor-derived DNA circulate in the blood with cfDNA(derived from alltypes of cells, cancerous and noncancerous). A portion of this cfDNA can also come from live cells when they spontaneously release DNA.

of tumor DNA has also been observed in the saliva of patientswith oral cavity cancer.44-46
Release of cfDNA and ctDNA in the blood stream. Twomechanisms for the release of DNA into the blood circulation
have been postulated. The first is the passive release of DNA
through cell death either by apoptosis or necrosis.47
Another mechanism of ctDNA release is active secretion by
viable tumor cells,47,48 secretion occurs by the release of extracellular vesicles, such as exosomes and prostasomes, which contain DNA pieces of around 150 to 250 bp.49
Elevated level of cfDNA and ctDNA in cancer patient. In general, cancer patients have an elevated level of circulating freeDNA (Figure 1) than healthy individuals.50-52
In 1977, scientists made the dynamic observation of abnormally high levels of cfDNA in plasma and serum from cancer
patients compared to healthy individuals, and this fraction of
cfDNA originated from cancer patients was assumed to represent mainly circulating tumor DNA (ctDNA).51,53
Concentration and size of cfDNA and ctDNA fragments incancer patient. Initial studies showed that the concentrationof cfDNA in cancer patients increased significantly compared
to a healthy individual that ranges from 0 to 1000 ng/mL ofblood, with an average of 180 ng/mL. In contrast, cfDNA in
healthy individuals ranges from 0 to 100 ng/mL of blood, withan average of 30 ng/mL.49,51
Another amazing feature of ctDNA is the wide variability
in the fragment size. This variability can be attributed to thevariety of mechanisms that underlie dead cancer cells. In
healthy individuals, cfDNA is released predominantly throughapoptosis with small and uniform fragments of around 185 to
200 bp.54,55
In cancer patients, due to incomplete and random digestionof genomic DNA by the nuclease enzyme, necrosis creates aspectrum of DNA fragments of various sizes, interestingly;
shorter fragments of ctDNA have been reported in sometumor types (eg, hepatocellular carcinomas) as well as large
fragments of cfDNA of thousands of base pairs.50,54-58
These studies show that the concentration and length of the
ctDNA fragment can be used for the prediction, early diagnosis, and prognosis of cancer.59
Clinical application of cfDNA and ctDNA. Conventionally,
protein biomarkers are used in cancer diagnosis and in theevaluation of therapeutic responses, such as carcinoembryonicantigen (CEA), prostate specific antigen (PSA), cancer antigen (CA) 19-9 and CA-125, but the specificity and reliabilityof these protein biomarkers are not satisfactory.60,61 Recently,
ctDNA has been the most commonly used type of blood-based
biomarker candidate in clinical practice. Specifically, ctDNA
analysis has been used primarily to monitor response to therapy, detect minimal residual disease throughout treatment, andevaluate the development of resistance to therapy.62-67
Of many potential clinical benefits of ctDNA, early detection in cancer diagnosis can be an important approach to
reduce cancer mortality,68 and several studies have also reported
the detection of genetic alterations in patients with early stage
cancer that support the role of ctDNA in early detection ofcancer.69-73
Many studies have also shown that ctDNA can be a powerful prognostic factor. For example, in the case of triple-negative
breast cancer patients, the detection of ctDNA during or after
neoadjuvant chemotherapy, and prior to surgery, has beenshown to have a strong prognostic value.74
Another study in pancreatic cancer showed that higher levels of total percentage of ctDNA were an independent prognostic factor for overall survival.75
In addition, several studies have compared cfDNA levels incancer patients with healthy individuals and those with benign
conditions, showing promising diagnostic and prognostic
applications of cfDNA. For example, plasma cfDNA levels
have been reported to be elevated in metastatic RCC relative to4localized disease,76 and in the case of bladder cancer, urine
cfDNA levels were found to be significantly elevated compared
to controls.77
Plasma levels of cfDNA have also been reported to be elevated compared to benign prostatic hyperplasia (BPH).78
Similar to some studies in melanoma, breast, ovarian, and coloncancers,79-82 elevated levels of cfDNA can serve as a noninvasive predictor and prognostic biomarker of cancer.
Furthermore, ctDNA can potentially help detect cancer
recurrence much earlier than current conventional procedures.83
For example, a study of breast cancer patients shows that
ctDNA monitoring is a precise method for the early detectionof asymptomatic metastatic recurrence in patients diagnosed
with primary breast cancer.84
According to another study, patient-specific ctDNA analysis can be a more sensitive and specific approach to detecting
recurrence in breast cancer compared to standard clinical andradiological surveillance, and provides evidence that ctDNA
can be detected in most patients with breast cancer several
months before clinical relapse.85
Postoperative detection of ctDNA has also been shown toremain strongly predictive of recurrence among patients withboth lower risk (pathological complete response—pCR) andhigher risk (pathologic node-positive—pN+) disease.86
Another potential role for analyzing ctDNA levels is toassess treatment response or resistance; which involves quantitative measurement of the level of ctDNA over time in response
to cancer treatments.87
Monitoring of treatment response is essential to avoid
continuing ineffective therapies, to prevent unnecessary side
effects, and to determine the benefit of new therapies.
Treatment response is generally assessed using serial imaging,but radiographic measurements often do not detect changes intumor burden. Dawson et al62 reported that circulating tumor
DNA levels showed a greater correlation with changes intumor burden, and provided the earliest assessment of treatment response in 10 of 19 women (53%) measuring tumor
burden with high sensitivity and specificity.
Circulating tumor cells (CTCs). CTCs discovered by Ashworth
in 1869 during an autopsy of a patient with metastatic cancer.88
These intact tumor cells are released into the bloodstream fromprimary and secondary tumor sites,89-91 and their migration to
the circulatory system results in the development of distantmetastases.90-92
Morphology of CTCs. CTCs are a highly effective cell population, characterized by high heterogeneity at the genetic, transcriptomic, proteomic, and metabolomic levels.93 They do nothave well-defined morphological aspects and may vary according to cancer type and stage,92 and are present as single cell orclusters of cells.94 They may cluster with parental tumor cells
or with fibroblasts, leukocytes, endothelial cells, or platelets andform aggregates with a higher propensity to develop distantmetastases.95,96

Cancer Informatics ﻿
Numbers of CTCs present in the blood stream. They are rarely
found in clinically healthy people or in people with non-malignant tumors. A study showed that patients with metastases
have 1 to 10 CTCs per mL of blood,89,91 with even lower numbers in early-stage diseases.97
Basically, the low number of CTCs isolated from a patient,
especially at the beginning of the disease, is the main challenge
in using CTCs for diagnostic purposes.98
However, with the development of technologies capable ofdealing with minimal cell numbers, such as microfluidics andNGS,99 the CTC analysis is now in a position to address highly
relevant questions.
Isolation and detection of CTCs. CTCs are observed in different types of cancer, such as melanoma, breast, ovarian,
prostate, lung, colorectal, pancreatic, head, neck, and bladdercancer.100,101
They are present in various biological fluids of cancer
patients, such as peripheral blood (Figure 2), urine, pleural
effusion, ascites, and cerebrospinal fluid.47
Techniques for the detection and isolation of these biomarkers were first described in 1960 and gradually improved
over the next 40 years.102,103
Detection of CTC has been improved through the development of specialized technologies based on various principles,
including antibody capture,104-106 size exclusion,107 red andwhite blood cell depletion,108 or dielectrophoresis.109
The CellSearch system is reported as a validated method for
CTC detection that has been approved by the US Food andDrug Administration. The CellSearch system, designed for the
CTC enumeration in 7.5 mL of blood, was first introduced in
2004 where the analytical accuracy, reproducibility, and linearity of the system were demonstrated.110
Clinical application of CTCs. Analysis of CTCs provides
information on cellular components, including DNA, RNA,and protein,7 and can be used as an independent prognostic
factor for tumors such as prostate cancer, breast cancer, andcolon cancer.111-114
CTC analysis also has the potential to be used as a biomarker for the screening and early detection of cancer.115 Early
detection and characterization of CTCs is an important tool
for monitoring and preventing the development of overt metastatic disease.116,117
In patients with metastatic breast cancer, the detection of
CTC has been reported to have a great prognostic value.118
Another role of circulating tumor cell (CTC) analysis has beenreported to monitor response to therapy in patients withadvanced non-small cell lung cancer (NSCLC). Changes in
CTCs number during treatment have been proposed as a predictive biomarker of response to both chemotherapy and targeted therapies119.
CTCs have also been reported to replace tissue biopsies topredict tumor recurrence120 and in cases of inaccessible neoplastic sites or unsuccessful sampling; CTCs would be a perceptible approach over tissue biopsy.121

Hirahata et al

5

Figure 2. This is a schematic diagram showing the collection resource for genetic material from various gravitational layers of blood. The genetic material
for liquid biopsy can be collected from various body fluids, including blood compartments, ctDNA from plasma, and CTC from buffy coat. Different typesof change can be detected as tumor markers from CTC-derived RNA, such as oncogene expression and mutation. ctDNA can be used for the detection of
mutations, the promotor methylation of tumor suppressor genes, and the length of DNA fragments.

Conclusion

In summary, liquid biopsy is a significant innovation in thefield of precision medicine and has made a competent potential
in the treatment of cancer; this revolutionary non-invasive
technique consists of the detection and isolation of circulatingtumor DNA, circulating tumor cells, and exosomes, as a sourceof genomic and proteomic information in cancer patients.121
Analysis of these biomarkers offers a valuable range of information that allows non-invasive diagnosis, prognosis, andtreatment of cancer.
Both ctDNA and CTCs are interesting complementarybiomarkers of liquid biopsy that can be used in parallel in various aspects of cancer management. Analysis of each biomarker
has its own advantages and disadvantages; specifically, ctDNA
analysis is an attractive approach because it is simple to isolate
and analyze ctDNA, but it is limited to the analysis of DNArelated abnormalities; in contrast, CTC analysis provides profiling of the entire cell; however, it is difficult to isolate apopulation of rare cells10,17 and is not easily adopted in mostlaboratories. Thus, its clinical applicability will require further
research and verification.35
Clearly, liquid biopsy opens up a new possibility of noninvasive, safe, and easily repeatable practice in cancer treatment.
Despite the advantages of liquid biopsy, more studies are
required to address the challenges and limitations of this technique, which restricted its implementation in clinical practice.
However, the multifaceted advantages of liquid biopsy demonstrate its application as a promising prognosis and diagnostic
tool in precision oncology, and it may be a comprehensive
approach to overcome the current limitations associated withinvasive tissue biopsy and can identify multiple tumor genes
present in the bloodstream at multiple time points.

Authors Contributions

R.Q. coceptualized, researched and wrote this manuscript. All
authors have read and agreed to the published version of the
final manuscript.

Ethics Approval and Consent to Participate

This manuscript does not involve the use of animal or humandata or tissue, so ethical approval is not applicable in this section.

ORCID iDs

Tetsuyuki Hirahata-754X
Reeshan ul Quraish-7583
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