Invest Clin 65(4): 476 - 494, 2024 https://doi.org/10.54817/IC.v65n4a09

Correspondence author. Mei Zhu, Department of Clinical Laboratory, the Affiliated Chaohu Hospital of Anhui Me-

dical University, No. 64 Chaohu North Road, Chaohu, Anhui, PR China. E-mail: zhumei@ahmu.edu.cn

High throughput sequencing technology

and its clinical application in circulating

tumor DNA detection in patients with

tumors.

Chonghe Xu

, Dangui Zhou

and Mei Zhu

School of Basic Medical Sciences, Capital Medical University, Beijing, People’s

Republic of China.

Department of Clinical Laboratory, the Affiliated Chaohu Hospital of Anhui Medical

University, Chaohu, Anhui, People’s Republic of China.

These authors contributed equally to this work.

Keywords: high throughput sequencing; tumor; circulating tumor DNA; tumor

diagnosis; tumor treatment; tumor prognosis.

Abstract. The high-throughput sequencing (HTS) is now a highly favoured

technology in the field of genome research. A distinctive feature of this sequenc-

ing method is its data-yielding capability, which is capable of generating more

than 100 times than the first-generation Sanger sequencing platform. HTS

technology has been widely adopted for its advantages, including high through-

put, sensitivity, automaticity, information density and cost-effectiveness. Not

only does it help in the treatment and diagnosis of multiple diseases, but it

also provides new insights into the research in molecular biology of tumors.

Moreover, circulating tumor DNA (ctDNA) tests based on HTS technology are

increasingly extensively implemented for clinical purposes. In this review, we

will focus on the significant achievements and performances of the HTS, and

first-hand data from extensive experience will be summarized and analyzed to

discuss the advantages and specifics associated with each sequencing system

and further summarize the characteristics of their clinical applications.

Circulating tumor DNA detection by high throughput sequencing technology 477

Vol. 65(4): 476 - 494, 2024

Tecnología de secuenciación de alto rendimiento y su

aplicación clínica en la detección de ADN tumoral circulante

en pacientes oncológicos.

Invest Clin 2024; 65 (4): 476 – 494

Palabras clave: secuenciación de alto rendimiento; tumores; ADN tumoral circulante,

diagnóstico de tumores; tratamiento de tumores; pronóstico de tumores.

Resumen. La secuenciación de alto rendimiento (HTS) es una tecnología

popular en el campo de la investigación genómica. Una característica distintiva

de este método de secuenciación es su capacidad de generación de datos, que

puede generar 100 veces más datos que la Plataforma de secuenciación Sanger

de primera generación. La tecnología superconductora de alta temperatura es

ampliamente utilizada debido a sus ventajas de alto rendimiento, alta sensibi-

lidad, automatización, densidad de información y rentabilidad. No solo ayuda

a tratar y diagnosticar múltiples enfermedades, sino que también proporciona

nuevas ideas para la investigación de biología molecular tumoral. Además, la

detección de ADN tumoral circulante (ctDNA) basada en la tecnología HTS se

utiliza cada vez más ampliamente con fines clínicos. En esta revisión, nos cen-

traremos en los principales logros y rendimiento de HTS, y resumiremos y ana-

lizaremos datos de primera mano de una amplia experiencia, discutiremos las

ventajas y detalles específicos de cada sistema de secuenciación y resumiremos

aún más las características de su aplicación clínica.

Received: 15-08-2024 Accepted: 17-10-2024

INTRODUCTION

The past decade has witnessed the intro-

duction and broad application of HTS tech-

nologies. Not only can it perform chromo-

some mapping, but it can also conduct deep

sequencing and whole genome sequencing

analysis on blood, body fluids and excreta

such as urine, feces, sputum, cerebrospinal

fluid, sperm, saliva, vaginal secretions, milk

and effusions

1-7

. This revolutionary tech-

nology facilitates diagnosis at the genetic

level and is especially suitable for complex

diseases that are highly heterogeneous and

involve both genes and mutations, such as

tumors

. ctDNA is an important biomarker, a

circulating cell-free DNA (cfDNA) generated

by the apoptosis, necrosis and secretion pro-

cess of tumor cells, and it contains relatively

complete genetic information about tumour

cells

. Additionally, it contains the same mu-

tations as the DNA in tumor cells, including

insertions, deletions, rearrangements, copy

number variations and methylations. There-

fore, using HTS technology to test for ctDNA

can provide crucial information on the diag-

nosis, treatment and prognosis of tumors

In this review, we introduced the main

features of the HTS, such as first-generation

Sanger sequencing, second-generation HTS

platforms (454 Life Sciences pyrosequenc-

ing, Illumina/Solexa technology and SOLiD

ligase-mediated sequencing) and third-gen-

eration high throughput - next generation

sequencing (HT-NGS) platforms (Ion Tor-

rent technology, Single-molecule real-time

(SMRT) sequencing and Nanopore sequenc-

ing technology). In addition, this article also

478 Xu et al.

Investigación Clínica 65(4): 2024

describes the application of ctDNA in the di-

agnosis, treatment and prognosis of tumors.

Overview of high-throughput sequencing

platform

First-generation Sanger sequencing

In the mid-1970s, DNA sequencing saw a

major technological innovation, the Sanger di-

deoxy synthesis method, proposed by Sanger.

The advent of this method provided scientists

with a new means of determining four differ-

ent nucleotide bases in single-stranded DNA

using radio-labelling

11,12

. Sanger sequencing

technology, as a landmark development in

the field of DNA sequencing, has dramatically

advanced the process of genomics research.

However, this technology has certain limita-

tions in practical application, i.e., the amount

of DNA that can be processed in each experi-

ment is limited. Although this limitation

made Sanger sequencing unable to meet the

demand for high throughput in some cases,

it laid the foundation for developing subse-

quent sequencing technologies. In pursuing

higher throughput and more efficient se-

quencing technologies, scientists have made

continuous efforts and eventually succeeded

in developing the second, third and even

higher throughput sequencing platforms

13-15

Their high throughput and accuracy enable

researchers to access genetic information

more quickly and accurately, providing pow-

erful support in areas such as disease diagno-

sis, personalized medicine and biotechnology.

Second-generation HTS platforms

The second-generation HTS uses a dif-

ferent analysis principle than the first-gener-

ation Sanger sequencing. The key technolo-

gies of the second-generation HTS platform

include bridge sequencing and synthetic se-

quencing. These technologies have enabled

the platform to have a wide range of appli-

cations in areas such as genomics research,

variant detection and gene expression anal-

ysis. However, despite the significant ad-

vances in sequencing length and accuracy of

the second-generation platforms, they still

have some limitations, such as shorter read

lengths and higher error rates.

454 Life Sciences pyrosequencing

A unique sequencing method has at-

tracted much attention in exploring the

early development of NGS technologies.

In 2000, Jonathan Rothberg successfully

developed the first commercially available

NGS platform, which was innovative in that

it used a unique mechanism to read the

signals of individual nucleotides added to

a DNA template. It utilizes the properties

of luciferase, which generates light signals

when new nucleotides are added to a DNA

strand, and these signals are subsequent-

ly captured and converted into readable

data

. This method combines pyrosequenc-

ing technology with single-molecule emul-

sion PCR; sequencing is done through a

synthetic process in which four nucleotide

bases are added one by one to a DNA tem-

plate. Each time a new nucleotide is added,

it triggers the production of a different co-

loured light, which is caused by the release

of pyrophosphate from the microwells

12,17

In general, pyrosequencing is centred on

the concept of “sequencing by synthesis”,

which is in sharp contrast to the traditional

Sanger sequencing method, which is done

by detecting the release of pyrophosphate

to determine whether a specific nucleotide

has been added to the DNA strand. Pyro-

phosphate is released when a nucleotide

is added to a growing DNA strand. These

pyrophosphate releases are detected, and

a signal is generated. By monitoring these

signals, we can determine the bases on the

DNA template strand at each position. The

essential advantage of this method is that

it does not require ddNTPs, which means

that no terminating strand synthesis is

required during sequencing, thus increas-

ing the accuracy and efficiency of sequenc-

ing

14,18

. During the nucleotide doping, more

than one nucleotide may be doped into the

same position. This situation leads to the

Circulating tumor DNA detection by high throughput sequencing technology 479

Vol. 65(4): 476 - 494, 2024

formation of a homopolymer because the

nucleotide used lacks molecules capable of

preventing further doping. This fully doped

homopolymer will form in one cycle

With the rapid development of NGS

technologies, various sequencing platforms

are emerging. Roche discontinued support

of the platform in 2016, which resulted in

the platform being phased out, and other

more efficient and accurate sequencing plat-

forms are gradually replacing it as technol-

ogy advances. This change reflects the rap-

idly evolving field of sequencing technology

and the importance of continually updated

platforms and technologies.

Illumina/Solexa technology

In 2006, Solexa introduced an innova-

tive sequencing technology that employs a

reversible terminator strategy to enhance

adapter-linked DNA fragments through

bridge amplification. This method allows the

bases on the template strand to be read em-

ploying a nucleotide-by-nucleotide process

that involves successive nucleotide doping,

a cleaning step, imaging, and a subsequent

cleavage step

14,15,20,21

. The extraction and

segmentation of DNA is the primary step

aimed at breaking down complex DNA mol-

ecules into smaller, manageable fragments.

This process usually involves using specific

enzymes to cut the DNA, resulting in frag-

ments of a certain length. Next, these DNA

fragments need to be ligated to specific

adapter sequences to facilitate subsequent

sequencing steps. Adapter sequences are

short pieces of DNA or RNA sequences that

bind to the ends of the DNA fragments and

provide an interface for connection to the

sequencing machine. Once the adapter se-

quences have been successfully ligated to

the DNA fragments, these conjugates are

transferred to a flow cell. The flow cell is a

unique device that precisely positions the

DNA fragments on the cell surface. The

DNA fragments are copied in large num-

bers through clonal amplification, forming

clonal “clusters”. These clusters consist of

many identical single-stranded DNA frag-

ments that are physically tightly packed to

facilitate high-throughput sequencing by se-

quencing machines

17,19,22

During each sequencing cycle, four

fluorescently labelled nucleotides compete

for the opportunity to bind to the template

strand. This is a competitive process in which

only nucleotides complementary to the cor-

responding nucleotide on the template

strand can be successfully doped. Once a

nucleotide has been doped, the laser detects

a signal due to fluorescent labelling, identi-

fying which nucleotide has been doped into

the template strand. After identification, the

next step is to remove the blocking group

and fluorescent marker from the doped nu-

cleotide. This step is in preparation for the

next sequencing cycle, making the template

strand available again for new nucleotides to

be doped. During this process, the nucleotide

sequence on the template strand is gradually

built up, with each sequencing cycle adding

a point of information for the final determi-

nation of the entire DNA sequence. The effi-

ciency of this sequencing strategy lies in its

ability to quickly and accurately determine

the DNA sequence by detecting fluorescent

signals and complementary nucleotide in-

corporation. As the sequencing cycle is re-

peated, sequence information of the entire

genome is gradually revealed

21-23

Illumina sequencing has become an

indispensable tool in modern genomics re-

search. This technology supports a wide range

of sequencing protocols, covering areas rang-

ing from comprehensive sequencing at the

genome level to more specific exon sequenc-

ing, targeted sequencing, and macrogenomics

for studying microbial communities. It is also

widely used for methods such as RNA sequenc-

ing, chromatin immunoprecipitation sequenc-

ing (CHIP-seq) and methylome analysis

However, despite the Illumina sequenc-

ing platform’s market-leading position due

to its high output capacity and broad appli-

cability, this short-read technology still has

limitations in certain areas. In particular, in

480 Xu et al.

Investigación Clínica 65(4): 2024

many applications in genomics, short read

lengths limit resolution and accuracy. This

means that when high precision is required

to resolve genome structure or identify low-

frequency variants, short-read technologies

may not provide sufficient information

Therefore, for these specific research needs,

it may be necessary to use a combination

of other sequencing technologies, such as

long reads or single molecule sequencing, to

obtain higher-resolution sequence data. De-

spite these limitations, Illumina sequencing

technology continues to be essential in ad-

vancing genomics and biomedical research.

SOLiD ligase-mediated sequencing

In 2007, Applied Biosystems introduced

a new sequencing platform, Supported Oli-

gonucleotide Ligation Detection (SOLiD).

This platform is similar to other sequencing

technologies in that it detects the fluores-

cence intensity of dye-labelled molecules to

determine the sequence of DNA fragments.

However, the SOLiD platform employs a

unique sequencing technique known as DNA

ligase-based sequencing

12,24

. A distinguishing

feature of this technique is that it generates

relatively short read lengths, typically 35 base

pairs

25,26

. Nonetheless, the SOLiD platform

was a significant breakthrough at the time,

providing new tools for genomics research.

The technology involves cutting the

template DNA into small fragments and li-

gating them to a known junction sequence.

This process ensures that the DNA fragments

can be efficiently captured and sequenced.

Next, these junction-connected DNA frag-

ments are transferred to a particular type of

beads, which are subsequently immobilized

on a glass surface. These fragments can be

clonally amplified on the beads using emul-

sion PCR, resulting in many identical DNA

fragments. During the sequencing stage, the

sequence of each DNA fragment is deter-

mined by a two-base colour coding method.

This method relies on different dye pairs

to identify and record each nucleotide in

the DNA sequence. The SOLiD sequencing

platform is particularly adept at detecting

single nucleotide polymorphisms (SNPs),

which can be detected with an astonishing

99.85% accuracy. This high level of accuracy

makes SOLiD a powerful tool for genomics

research, especially for SNPs detection. With

this kind of precise sequencing, research-

ers can better understand genetic variations

and their relationship with diseases, provid-

ing a scientific basis for personalized medi-

cine and disease diagnosis

12,27,28

Like other NGS systems, SOLiD’s com-

putational infrastructure is more costly and

less convenient to operate. Nonetheless,

SOLiD technology has been widely used in

several fields, including but not limited to

whole genome resequencing, transcrip-

tomics research, targeted resequencing, and

epigenomics analysis. Currently, these three

leading second-generation high-throughput

sequencing platforms are available on the

market, as shown in Fig. 1. The commercial-

ization of these platforms provides powerful

tools for researchers and promotes research

progress in related fields. Meanwhile, with

the continuous development of science and

technology, more sequencing platforms are

under development and are expected to join

this competitive market in the future. This

will help further promote the development

of genomics research and provide more pos-

sibilities for biomedical research.

Third-generation HTS platforms

Third-generation sequencing technol-

ogy is gradually changing our understanding

of genomics. Compared with the previous

two generations of platforms, the advantage

of third-generation sequencing is that it pro-

vides longer read lengths and higher accu-

racy. The third generation of HTS platforms

uses single-molecule sequencing technology,

capable of simultaneously sequencing mil-

lions to billions of DNA molecules. The core

of this technology is represented by the Ion

Torrent technology, Single Molecule Real-

Time Sequencing and Nanopore sequencing

technology, as shown in Fig. 2.

Circulating tumor DNA detection by high throughput sequencing technology 481

Vol. 65(4): 476 - 494, 2024

Ion Torrent technology

Ion Torrent technology is a nucleotide

synthesis-based sequencing (SBS) method,

which has many similarities in principle with

the 454 pyrophosphate sequencing platform,

but the technical means used in detecting

the correct insertion of nucleotides differs

between the two methods. In Ion Torrent

technology, molecules are first fragment-

ed, and then these fragmented molecules

are bound to the surface of specific beads.

Next, the target molecules on these beads

are clonally amplified by emulsion PCR, re-

sulting in a large number of identical target

molecules on each beads. This step is a core

component of the Ion Torrent technology,

ensuring effective capture of target mole-

cules and providing efficient sequencing. Af-

ter this process, each bead can be regarded

as an independent sequencing reaction unit,

which lays the foundation for the subsequent

sequencing steps

12,17,20,22

Fig. 1. Characteristics of the three leading second-generation HTS platforms.

Fig. 2. Characteristics of the three leading third-generation HTS platforms.

482 Xu et al.

Investigación Clínica 65(4): 2024

Treated beads are dispensed into tiny

holes on the chip during sequencing. These

microholes form a microarray, with each

hole corresponding to a specific sequencing

reaction. When beads are placed in these mi-

croholes, a synthetic-based sequencing reac-

tion is performed on each bead

22,29

. During

DNA replication, each newly added nucleo-

tide causes a slight change in the pH of the

solution. This change is captured by the

sensor and converted into a voltage signal,

enabling the monitoring of nucleotide ad-

dition. Specifically, when a nucleotide pairs

successfully with a complementary base on

the DNA strand, it releases a hydrogen ion,

causing the pH to drop. This pH change is

detected by the sensor and converted into

a voltage signal. No voltage spike occurs if

no nucleotides are added in this round. Two

hydrogen ions are released when two neigh-

bouring nucleotide sites are filled with the

same nucleotide simultaneously, causing the

voltage signal to double. This doubled volt-

age change provides a direct signal to dis-

tinguish between neighbouring nucleotides.

By continuously monitoring the addition of

nucleotides and the corresponding voltage

changes during each sequencing cycle, we

can accurately determine the sequence of

bases on a DNA strand

17,22

Ion Torrent technology is favoured in

the sequencing field for its highly efficient

detection system, which does not rely on ex-

pensive cameras, light sources or scanners,

resulting in a significant increase in detec-

tion speed compared to traditional 454 py-

rophosphate sequencing

. This advantage

has made the Ion Torrent method the pre-

ferred choice for various applications. How-

ever, the technology is not without its limi-

tations. Studies have shown that while there

is a correlation between the number of base

integrations and voltage changes, it is not

a perfect relationship. As a result, the prob-

lem of homopolymer template elongation

remains, which is caused by cumulative light

intensity changes

. Despite this challenge,

the Ion Torrent technology remains essen-

tial in modern gene sequencing due to its

speed and accuracy.

Single-molecule real-time (SMRT)

sequencing

In 2011, Single Molecule Real-Time

Sequencing (SMRT) technology was intro-

duced by Pacific Biosystems, marking a new

advancement in the field of gene sequencing

on long-read platforms. This technology is

unique because it can sequence up to 30-50

kb or longer DNA fragments, far beyond what

conventional sequencing technologies can

handle

17,31

. SMRT sequencing centres on the

tight binding of a specially formulated DNA

polymerase to the target DNA, a process that

occurs in SMRT cells

. The design of these

SMRT cells is unique in that they contain

tens of thousands of tiny chambers, each

equipped with a DNA polymerase and a zero-

mode waveguide (ZMW) well

. This innova-

tive design allows the sequencing process to

be performed at the level of individual mol-

ecules, resulting in efficient and accurate

sequencing of DNA sequences. ZMW plays a

crucial role, which is a tiny structure that

precisely directs light energy to a specific,

relatively small-sized region. This property

makes the ZMW a critical factor in enabling

single-molecule sequencing

. In prepara-

tion for sequencing, the SMRT technology

takes an innovative approach, unlike tradi-

tional methods of fixing DNA strands. Spe-

cifically, high-fidelity DNA polymerase and

single-stranded DNA templates are added

to the SMRT cell chamber to serve as tem-

plates for DNA replication at the bottom of

the ZMW

23,27

. This design allows the DNA

replication process to occur at the level of

individual molecules, resulting in efficient

and accurate sequencing of DNA sequences.

An essential step in this process involves the

addition of different phosphorylated fluores-

cein markers to these tiny chambers. These

markers are present to enable precise moni-

toring of DNA polymerase activity. When

DNA replication occurs, DNA polymerase

must recognize and integrate nucleotides

Circulating tumor DNA detection by high throughput sequencing technology 483

Vol. 65(4): 476 - 494, 2024

complementary to the template DNA. Phos-

phorylated fluorescein markers added to the

ZMW minicells play a crucial role in this pro-

cess. They can interact specifically with DNA

polymerase activity, thus enabling research-

ers to distinguish and detect different events

during DNA replication

In ZMW technology, integrating nucleo-

tides is a crucial step that triggers the re-

lease of fluorescein. Once the fluorescein is

released from the bottom of the ZMW, it is

no longer in the detection state and emits a

specific fluorescent signal. The uniqueness

of this fluorescent signal means that each

nucleotide produces a different fluorescence

pattern. In practice, the fluorescence signals

from the tiny chamber of the ZMW are cap-

tured by a high-definition video system and

converted into digital signals. These digital

signals are then analyzed to determine the

nucleotide sequence on the DNA template.

Since each nucleotide has unique fluores-

cence properties, the sequence of the DNA

can be determined by identifying and inter-

preting these fluorescence signals

15,17,23

. In

SMRT cells, up to one million ZMWs are inte-

grated on a single chip. These tiny chambers

are the core units in the sequencing process,

performing both nucleotide integration and

imaging. Each ZMW independently captures

and records real-time images of nucleotide

integration. This ability to process in par-

allel dramatically increases the speed and

throughput of sequencing

15,17

Nanopore sequencing technology

As an innovative single-molecule se-

quencing method emerging in recent years,

Nanopore sequencing technology has made

significant progress in genomics research.

Unlike traditional sequencing techniques

based on nucleotide integration, nanopore

sequencing technology offers an entirely new

strategy. This technology utilizes nanopores

as sensors to directly detect biological mac-

romolecules, such as DNA and RNA, at the

single-molecule level, thus enabling real-time

monitoring at the single-molecule level

15,32

Nanopore sequencing technology elimi-

nates the cumbersome PCR amplification

and chemical labelling steps during opera-

tion. This means that researchers do not

need to perform complex pre-processing of

samples when performing sequencing, great-

ly simplifying the experimental process.

More importantly, this technology allows di-

rect use of cell lysates for sequencing with-

out the need for additional sample prepara-

tion, which not only saves experimental time

but also reduces experimental costs. These

advantages make nanopore sequencing tech-

nology more efficient and more widely appli-

cable in single-molecule sequencing

Nanopore sequencing technology is an

innovative DNA sequencing method that uti-

lizes protein nanopores embedded in a poly-

mer membrane. In this process, when a DNA

molecule passes through a nanopore known

as a molecular motor protein, it causes a

disorder in the nanopore protein, which gen-

erates electrical signals. The conversion of

these electrical signals is the core principle

of nanopore sequencing technology, which

enables researchers to determine the DNA

sequence by analyzing these signals

14,27,33,34

Based on nanopore sequencing technology,

researchers can determine the sequence of

a DNA molecule by detecting changes in the

electrical currents it generates as it passes

through the nanopore. The key to this tech-

nique is that each nucleotide causes unique

current changes that can be detected and

recorded precisely. By analyzing these cur-

rent changes, we can tell the sequence of

nucleotides in a DNA molecule

The nanopore technology could offer a

wide range of applications in fields such as

personalized medicine, agriculture and sci-

entific research. In addition, the portability

of nanopore sequencing technology allows se-

quencers to be taken into the field for direct

sequencing, greatly expanding the range of

sequencing applications and increasing the

efficiency of field studies

14,32-37

. This technol-

ogy provides a direct and efficient means to

study the properties of DNA and is essential

484 Xu et al.

Investigación Clínica 65(4): 2024

for research in genomics, molecular biology

and other related fields.

Application of HTS technology in tumor

diagnosis and treatment

Tumors have claimed numerous lives

as malignant cells continuously mutate and

evolve as they divide. Most patients died

from fatal metastasis and cancer recurrence.

However, with mature HTS technology, re-

searchers have obtained significant genetic

information on tumor pathogenesis, drug

resistance and metastasis, which will help in

the early screening and identification of tu-

mors, selection of therapies and monitoring

of prognosis.

Diagnosis of tumors

Early diagnosis and prompt treatment

are critical to controlling the growth of tu-

mors. Genetic testing is characterized by

the ease of sampling, which allows research-

ers to minimize the harm to their subjects.

In order to alleviate the pain associated with

traditional invasive pathological biopsies,

researchers have turned to body-fluid-based

sequencing to assist in the diagnosis of can-

cer. Circulating tumor DNA (ctDNA) is de-

rived from single- or double-stranded DNA

and DNA-protein complexes shed by tumor

tissue. The presence of mutated DNA frag-

ments is observed at relatively high concen-

trations in the circulation of most patients

with metastatic cancer and low but detect-

able concentrations in a significant propor-

tion of patients with localized cancer. This

characteristic ensures that ctDNA exhibits

particular specificity and can serve as a bio-

marker for clinical purposes

Lung cancer is one of the most com-

mon cancers, and its early diagnosis plays a

crucial role in improving patients’ quality of

life. However, low-dose computed tomogra-

phy (LDCT), a widely used method for early

detection of lung cancer, cannot accurately

distinguish between malignant and benign

lung nodules. Many studies have shown that

HTS technology can detect specific muta-

tions strongly associated with lung cancer in

ctDNA extracted from body fluid samples

39-41

Sumitra et al. identified tumor-related alter-

ations in 94% of patients with limited-stage

small cell lung cancer (LS-SCLC) and 100%

of patients with extensive-stage small cell

lung cancer (ES-SCLC) by performing tar-

geted ctDNA sequencing and whole genome

sequencing on their body fluid samples. This

study demonstrates that targeted ctDNA se-

quencing can identify potential treatment

targets in more than half of the patients and

shows advantages over traditional invasive

biopsy

Additionally, Peng et al. have developed

a method to determine whether a lung tu-

mor can be surgically removed by perform-

ing ultra-deep sequencing on the targeted

mutations in ctDNA. Moreover, the accura-

cy of ctDNA testing is 63%, 83%, 94%, and

100% for stages I, II, III, and IV lung cancer,

respectively. The overall sensitivity comes to

80%, considering age and serum biomarker

tests, and the specificity rises to 99%

. This

body-fluid-based screening can reduce the

risk of unwanted damage and metastasis and

assess the tumor progression by analyzing

tumor mutation load.

Based on previously mentioned evi-

dence, it can be concluded that HTS tech-

nology is also widely used to diagnose

other types of tumors. Himisha et al. have

discovered that the genome characteriza-

tion of castration-resistant neuroendocrine

prostate cancer (CRPC-NE) can be identi-

fied by analyzing ctDNA in patients’ plasma

samples. This reduces the trauma associated

with traditional invasive pathological biop-

sies and provides timely guidance for clini-

cal adjustment of medication

. Cai et al.

developed a genetic diagnosis model based

on whole-genome analysis of 5-hydroxymeth-

ylcytosine (5hmC) obtained from cfDNA

samples of 2,554 individuals. The model

demonstrated superior diagnostic perfor-

mance to AFP and could distinguish between

early-stage hepatocellular carcinoma (HCC)

and non-HCC with an AUC of 0.884 in the

Circulating tumor DNA detection by high throughput sequencing technology 485

Vol. 65(4): 476 - 494, 2024

validation set

. In addition, Yurika et al.

found that the methylated SEPT9 gene in

serum ctDNA was highly significant in the

early diagnosis of HCC with both high sensi-

tivity and specificity

. However, it should be

noted that although HTS-based body fluid bi-

opsy technology provides a new direction for

tumor diagnosis, most of the test results still

need to be combined with other imaging or

serological indicators and cannot be used in-

dependently as a diagnostic gold standard.

Furthermore, it is necessary to vali-

date the test in a larger population. In ad-

dition, the lack of research on related gene

test chips has severely limited its application

scope. However, this problem can likely be

solved with the development of technology

and the related industrial chain.

Treatment for tumors

It is important to note that low-frequen-

cy mutations can occur during the division of

tumor cells. Therefore, tumor cells can carry

different genetic mutations even if they ap-

pear histologically similar, while different tu-

mor cells can carry the same mutations. By

comparing the gene sequences of primary

and metastatic neoplasms, physicians can

assess the effectiveness of their treatment

and develop personalized therapies. They

also gain insight into the potential mecha-

nisms behind tumor drug resistance, laying

a solid foundation for accurate diagnosis and

treatment of tumors.

Targeted therapy

Today, several signalling pathways and

associated mutations associated with tumor

formation have become effective drug tar-

gets. As a result, targeted therapies based

on tumor genes have become mainstream.

As the most common BRAF mutation, the

V600E mutation is strongly associated with

the particular invasiveness of rectal cancer

cells in metastatic colorectal cancer cases.

Resistance to chemotherapy has been ob-

served in cases with V600E mutations, and

resistance to EGFR inhibitors is gradually

increasing. Scott et al. demonstrated that

using NGS technology, the concomitant use

of EGFR and BRAF inhibitors in combina-

tion with irinotecan was effective in patients

with the V600E mutation. The discovery

informed the development of new targeted

therapeutic regimens and laid a solid foun-

dation for new targeted therapies

Dasatinib is a small-molecule tyrosine

kinase inhibitor that can be used as a first-

and second-line treatment for gastrointesti-

nal mesenchymal tumors (GIST). Zhou et al.

found that dasatinib can be employed as a

preferred treatment option for patients with

wild-type GIST or GIST with the D842V mu-

tation who are unable to take regorafenib, as

they identified genetic variations related to

the tumor signalling pathway by NGS tech-

nology

. Christian et al. investigated the

clinical efficacy of the combination of ibru-

tinib, rituximab and high-dose methotrexate

(HDMTX) in central nervous system lym-

phoma (CNSL). The results proved that the

combination is safe and effective. In addi-

tion, the analysis of ctDNA in cerebrospinal

fluid samples enables researchers to monitor

disease progression in patients with CNSL

and to adjust targeted therapy

promptly.

In recent years, site-specific therapies (in-

cluding HTS-guided targeted therapies)

emerged as a promising option for patients

with metastatic cancer of unknown primary

site (CUP). A phase 2 clinical trial conduct-

ed at 19 institutions in Japan by Hidetoshi

et al. demonstrated that CUP site-specific

therapy based on NGS technology had favor-

able survival outcomes.

Moreover, targeted therapy based on the

tumor-associated mutations showed excel-

lent therapeutic responses, even in patients

with CUP

. In conclusion, NGS technology

can provide a reference for clinical drug se-

lection and efficacy evaluation by detecting

target genes and other tumor-related genes,

which has great potential in pursuing indi-

vidualized treatment today. Nevertheless, it

is essential to acknowledge the current limi-

tations of the evidence base, primarily de-

486 Xu et al.

Investigación Clínica 65(4): 2024

rived from small, single-ethnic clinical trials.

When the mutation type is considered rare,

the subjective decision to administer chemo-

therapy over targeted therapies in the clinic

may also affect the study results.

Chemotherapy

Chemotherapy has consistently been

the focus of research. as one of the leading

traditional means of treating tumors. In ad-

dition, gene sequencing technologies, rep-

resented by HTS technology, have undoubt-

edly facilitated the chemotherapy of tumors.

Most patients diagnosed with triple-negative

breast cancer (TNBC) receive neoadjuvant

chemotherapy. Approximately one-third of

them can achieve complete pathological

remission with neoadjuvant chemotherapy,

but two-thirds will have residual disease and

a high risk of recurrence. Milan et al. con-

ducted ctDNA sequencing using HTS tech-

nology in 196 early-stage TNBC patients

with residual disease after neoadjuvant che-

motherapy and circulated tumor cell (CTC)

analysis in 123 patients. The results showed

that the presence of ctDNA and CTC in ear-

ly-stage TNBC patients after neoadjuvant

chemotherapy was associated with distant

tumor metastasis. The detection of ctDNA

and CTC in early-stage TNBC patients after

neoadjuvant chemotherapy was found to be

independently associated with disease recur-

rence and serve as an important indicator

for assessing patient status after future neo-

adjuvant chemotherapy

. Colorectal cancer

is one of the most common tumors world-

wide, with surgical resection, chemotherapy,

radiotherapy, and targeted therapy repre-

senting the principal treatment modalities.

Resistance to chemotherapy drugs in some

tumor patients has also posed a significant

challenge for clinicians. Li et al. from Pe-

king University combined the i-CR platform

with HTS technology to construct a new in

vitro tumor model, enabling personalized

drug testing and personalized treatment for

patients within 2-3 weeks and providing an

entirely new treatment option for colorectal

cancer

Osteosarcoma is one of the malignant

tumors for which early and effective preopera-

tive chemotherapy is crucial to the survival of

patients. However, the previous combination

of methotrexate (MTX), doxorubicin (DOX),

and cisplatin (DDP) has been found to have

significant differences in therapeutic efficacy

among patients, with a higher incidence of

adverse effects. Thus, there is an urgent need

to develop new drug combinations. Zhang et

al. from Central South University revealed

the heterogeneity of potential therapeutic

target genes. They elucidated the synergistic

mechanism of DOX and HDACs inhibitors for

treating osteosarcoma through RNA sequenc-

ing and second-generation sequencing (HTS)

of osteosarcoma samples. This provides a

foundation for developing entirely new che-

motherapeutic drug combinations and will

undoubtedly inspire the subsequent research-

ers

. Researchers can also develop effective

chemotherapy sensitizers by studying drug-

resistance genes and resistance mechanisms.

A team of researchers has already demon-

strated that SMOi inhibitors can release the

drug resistance of breast cancer tumor cells

and improve their chemosensitivity to doxo-

rubicin by studying the resistance mecha-

nism in breast cancer dependent on the Hh

signalling pathway

. Based on sequencing

relevant tissue or body fluid samples from pa-

tients using HTS technology, physicians can

comprehensively assess the expected thera-

peutic effects before chemotherapy to ap-

propriately risk-stratify patients in a clinical

setting. However, it is important to consider

whether the relevant experimental results ex-

clude potential interactions with other types

of treatment given after chemotherapy, and

another vital influence is the duration of fol-

low-up, as a short follow-up period may lead

to biased experimental data. In conclusion,

further clinical studies of NGS results are re-

quired before they can be relied upon as a risk

assessment tool.

Circulating tumor DNA detection by high throughput sequencing technology 487

Vol. 65(4): 476 - 494, 2024

Radiotherapy

Radiotherapy is an effective and cost-

efficient treatment. In recent years, with the

continuous improvement of radiotherapy

equipment and technology, the cure rate

of radiotherapy has increased, with the side

effects gradually decreasing. While genetic

therapies are gradually replacing chemo-

therapy and targeted therapy, radiotherapy

remains an important treatment option.

However, based on the patient’s sensitivity

to radiotherapy, HTS technology can still

guide the use of radiotherapy. In a recent

study, Raffaello et al. statistically employed

HTS technology to investigate the relation-

ship between ctDNA and tumor regression

grade (TRG) of surgical specimens in 25

consecutive patients with locally advanced

colorectal cancer (LARC) who had under-

gone long-term neoadjuvant chemo-radio-

therapy (Na-ChRT). The results showed that

the side effects of Na-ChRT were significant-

ly associated with positive liquid biopsies on

the day of surgery. This suggests that ctD-

NA assessment using HTS technology may

identify LARC patients with a poor response

to NA-ChRT to avoid potentially ineffective

treatment

Radiotherapy is one of the critical

tools in treating lymphoma, which can im-

prove the therapeutic effect and alleviate

the symptoms. However, some patients still

experience relapse or a poor prognosis due

to resistance to radiotherapy. Luo et al. em-

ployed CRISPR to construct an activated cell

line library, screened for radiotherapy-resis-

tant cells and performed HTS and bioinfor-

matics analyses to identify genes associated

with radiotherapy resistance. Sixteen of the

screened genes were identified as potential

genes associated with lymphoma radiother-

apy resistance. These genes are not only ex-

pected to be used as potential biomarkers or

new targets for therapy but have also dem-

onstrated the advantages of HTS technology

in potential target screening

. The assess-

ment of the overall condition and prognosis

of patients after radiotherapy is another cru-

cial part of the treatment, and it has been a

hot topic of research in recent years to in-

directly assess the prognosis of patients by

monitoring the relevant genes in their pe-

ripheral blood-free DNA (cfDNA) using NGS

technology. Jae et al. found the value of HPV

cfDNA in evaluating treatment response by

dynamically monitoring the cfDNA of cervi-

cal cancer patients using targeted HTS. They

found that HPV cfDNA is valuable for moni-

toring and predicting treatment response,

providing new insights for management and

evaluation after radiotherapy

Radiotherapy is an effective cancer

treatment, but it still inevitably has some

side effects. Most patients tolerate and re-

spond well to radiotherapy after surgery for

early-stage breast cancer. However, a propor-

tion of long-term survivors still develop ra-

diotherapy-related complications, with sub-

cutaneous fibrosis and capillary dilatation

being the most common cutaneous compli-

cations of radiotherapy for breast cancer. In

order to identify their susceptibility genes

and genotoxicity, Sarah et al. performed tar-

geted HTS on germline DNA samples from

48 breast cancer patients with extreme late-

stage cutaneous toxicity phenotypes and

identified a total of five single-nucleotide

variants in three genes (TP53, ERCC2, and

LIG1) with possible effects. This discovery

can provide a possible way for radiotherapy

to avoid patients susceptible to the side

effects and serious consequences of inap-

propriate radiotherapy58 and promote the

development of individualized medication.

HTS technology accelerates the discovery

of radiotherapy-resistant genes, effectively

avoiding ineffective radiotherapy and signifi-

cantly improving the effectiveness of treat-

ments. However, there are fewer studies on

applying HTS to radiotherapy complications,

and radiotherapy is mainly used as an adju-

vant treatment. Therefore, the influence of

chemotherapy or targeted therapy cannot be

excluded from the research, and its applica-

tion as a risk assessment tool still needs to

be further explored.

488 Xu et al.

Investigación Clínica 65(4): 2024

Tumor prognosis

Currently, following surgical interven-

tion and related treatments, tumor patients

need to be assessed for treatment and moni-

tored for tumor recurrence through regular

serum tumor marker tests and conventional

imaging tests. If tumor recurrence can be

anticipated at an earlier stage than the tra-

ditional examinations, early interventions

can be carried out, thus prolonging the sur-

vival time of patients. Remaining tiny tumor

foci after surgery or radiotherapy treatment

are important factors affecting patients’

prognosis, but it is more difficult to detect

their presence by traditional imaging and se-

rology screening. Fortunately, ctDNA detec-

tion based on HTS technology can provide

a new idea to solve this problem. Based on

this, Jeanne et al. performed ctDNA analysis

on stage III colon cancer patients who had

undergone chemotherapy and surgery and

found that ctDNA analysis in patients un-

dergoing surgery can serve as a marker of

prognosis.

In contrast, ctDNA analysis in chemo-

therapeutic patients could screen out pa-

tients who have completed the standard

therapy but are still at high risk of recur-

rence

. HTS technology, as an emerging

test method, has advantages over traditional

serological and imaging tests. Ma et al. as-

sessed ctDNA in breast cancer patients using

NGS technology while using the molecular

tumor burden index (mTBI) in the samples

to monitor tumor burden and found that it

may have a higher sensitivity in indicating

disease progression and distant metastasis

than CT imaging

Although most patients with advanced

tumors have lost the opportunity for radi-

cal surgical treatment, appropriate radio-

therapy or interventional therapy, as well as

an assessment of prognosis, are still neces-

sary. By sequencing the RAS gene in ctDNA

from 47 plasma samples from 37 patients

with RAS-mutated colorectal cancer (CRC)

with unresectable metastases, Elena et al.

found that the RAS-mutated allele score had

an independent prognostic value for CRC

survival and could be used as a non-invasive

decision-making tool in first-line treatment

for cancer

. Zhao et al. performed target-

ed capture sequencing on 1,021 genes fre-

quently mutated in unresectable primary

HCC cases. The results showed that ctDNA

abundance correlated more closely with tu-

mor size than AFP levels. It was also associ-

ated with the Barcelona Clinic Liver Cancer

(BCLC) staging system. Dynamic changes of

ctDNA showed consistent or higher sensitiv-

ity compared with imaging in assessing the

response to interventions and a high degree

of consistency with tumor mutational load

of tissue and blood samples

As a focus in the realm, immunother-

apies represented by immune-checkpoint

inhibitor therapies have revolutionized the

treatment of late-stage solid tumors. Im-

mune checkpoint inhibitors and the immu-

notherapy they represent have revolution-

ized the treatment of advanced solid tumors

as a hotspot in cancer therapy. Valsamo et al.

analyzed the clonal dynamics of ctDNA and

tumor exogenous TCR parameters during

immune checkpoint blockade in non-small

cell lung cancer (NSCLC) employing the

NGS technology. They assessed the value of

liquid biopsy monitoring as a surrogate in-

dicator of treatment response. The research

results indicate that ctDNA testing after

treatment can enable patients with immune

checkpoint blockages and primary drug re-

sistance to be quickly identified for alterna-

tive treatment

Because of the side effects and drug

resistance that immunotherapy can cause,

only a small proportion of patients can

benefit from it in the long term, and it is

particularly important to provide the neces-

sary monitoring throughout the process

By measuring ctDNA levels and dynamic

changes using HTS technology, the progno-

sis and outcome of immunotherapy can be

predicted both before and during treatment

to avoid the trauma and misinterpretation

that traditional monitoring can cause. How-

Circulating tumor DNA detection by high throughput sequencing technology 489

Vol. 65(4): 476 - 494, 2024

ever, it should be noted that monitoring tu-

mors after treatment is a long-term process,

and there is still room for improvement in

terms of price and variety compared to tra-

ditional means. Before HTS can be promoted

and applied as a decision-making tool, it is

still necessary to carry out comparative ex-

periments on a large scale with traditional

means of detection in order to further prove

its reliability.

CONCLUSION

As a high-throughput detection tech-

nology, HTS can still be applied to large-scale

genetic or genomic testing, thus providing

a powerful tool for researching the mecha-

nisms of tumor genesis and clinical diagno-

sis and treatment. As sequencing technology

continues to evolve, it is anticipated that the

goal of routine screening, prevention, diag-

nosis, treatment and prognosis of tumors

will be achieved through this technology.

ACKNOWLEDGMENTS

The authors thank all the Department

of Clinical Laboratory colleagues for their

comments on earlier versions of this manu-

script.

Funding

This study received funding from the

Major Project of Humanities and Social Sci-

ences Research in Anhui Universities (grant

no. SK2021ZD0032) and Key Project of Nat-

ural Science Research of Higher Education

Institutions in Anhui Province (Grant No.

2024AH050739).

Availability of data and materials

Not applicable.

Author’s ORCID numbers

• Chonghe Xu (CX):

0009-0001-1377-6946

• Dangui Zhou (DZ):

0000-0003-2805-3553

• Mei Zhu (MZ):

0000-0003-3130-3672

Authors’ contributions

CX and DZ wrote the original draft and

edited and critically revised the manuscript.

MZ substantially contributed to the concep-

tion and revision of the work.. All authors

read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors have declared that no com-

peting interest exists.

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