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STROKOVNI ČLANEK
Liquid biopsy – a diagnostic and therapeutic tool in the treatment of non-small cell lung cancer
Copyright (c) 2023 Slovenian Medical Journal. This work is licensed under a
Creative Commons Attribution-NonCommercial 4.0 International License.
Liquid biopsy – a diagnostic and therapeutic tool in the 
treatment of non-small cell lung cancer
Tekočinska biopsija – diagnostični in terapevtski pripomoček pri zdravljenju 
nedrobnoceličnega raka pljuč
Maša Majcen,1,2 Aleš Rozman,2,3 Izidor Kern2,3
Abstract
Lung cancer is one of the most prevalent cancers and the leading cause of cancer mortality worldwide. The past decade 
has brought important progress in drug treatments by discovering the driver mutations. The evolution of targeted on-
cological treatments directed to the biological properties of lung cancer in the individual patient has led to a significant 
increase in survival. During treatment, new mutations accumulate in the tumour, which prevents the long-term success 
of the therapy. Liquid biopsy is a method that has established itself in recent years as a less invasive diagnostic procedure 
that allows monitoring the response to treatment and identifying the mechanisms of resistance. The circulating tumor 
DNA is the most prevalent biomarker in lung cancer, but research on other biomarkers is also active. In this review article, 
we present the use of liquid biopsy in the clinical treatment of patients with non-small cell lung cancer. Its use is increas-
ingly recognized in early detection of lung cancer, identifying resistant mutations, potential assessment of disease burden, 
and longitudinal monitoring.
Izvleček
Rak pljuč je ena najbolj pogostih in smrtonosnih rakavih bolezni na svetu. V zadnjih 10 letih je z odkritjem vodilnih muta-
cij prišlo do velikega napredka pri zdravljenju. Zdravljenje postaja vse bolj prilagojeno bolniku oz. biološkim lastnostim 
raka, proti katerim so usmerjena tarčna onkološka zdravila, ki so bolnikom z napredovalo rakavo boleznijo pomembno 
podaljšala preživetje. Med tarčnim zdravljenjem se v tumorju razvijajo nove genetske spremembe, ki vodijo v odpornost 
tarč v rakavem tkivu, s čimer dolgoročno omejujejo uspešnost zdravljenja. Tekočinska biopsija (angl. liquid biopsy) je 
metoda, ki se je v zadnjih letih uveljavila kot manj invazivni diagnostični postopek, ki omogoča spremljanje odziva na 
zdravljenje in prepoznavanje mehanizmov odpornosti. Pri raku pljuč se najbolj uveljavlja vloga cirkulirajoče tumorske 
DNK (ctDNK), aktivne pa so raziskave tudi drugih bioloških označevalcev. V preglednem članku predstavljamo uporabo 
1 General hospital Celje, Celje, Slovenia
2 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
3 University Clinic of respiratory and allergic diseases Golnik, Golnik, Slovenia
Correspondence / Korespondenca: Maša Majcen, e: masa.majcen@gmail.com
Key words: lung cancer; liquid biopsy; circulating tumor DNA; PCR; NGS
Ključne besede: rak pljuč; tekočinska biopsija; cirkulirajoča tumorska DNK; PCR; NGS
Received / Prispelo: 3. 8. 2021 | Accepted / Sprejeto: 2. 3. 2022
Cite as / Citirajte kot: Majcen M, Rozman A, Kern I. Liquid biopsy – a diagnostic and therapeutic tool in the treatment of non-small cell 
lung cancer. Zdrav Vestn. 2023;92(1–2):59–69. DOI: https://doi.org/10.6016/ZdravVestn.3294
eng slo element
en article-lang
10.6016/ZdravVestn.3294 doi
3.8.2021 date-received
2.3.2022 date-accepted
Respiratory system Dihala discipline
Professional article Strokovni članek article-type
Liquid biopsy – a diagnostic and therapeutic 
tool in the treatment of non-small cell lung 
cancer
Tekočinska biopsija – diagnostični in terapevtski 
pripomoček pri zdravljenju nedrobnoceličnega 
raka pljuč
article-title
Liquid biopsy Tekočinska biopsija alt-title
lung cancer, liquid biopsy, circulating tumor 
DNA, PCR, NGS
rak pljuč, tekočinska biopsija, cirkulirajoča tu-
morska DNK, PCR, NGS kwd-group
The authors declare that there are no conflicts 
of interest present.
Avtorji so izjavili, da ne obstajajo nobeni 
konkurenčni interesi. conflict
year volume first month last month first page last page
2023 92 1 2 59 69
name surname aff email
Maša Majcen 1,2 masa.majcen@gmail.com
name surname aff
Aleš Rozman 2,3
Izidor Kern 2,3
eng slo aff-id
General hospital Celje, Celje, 
Slovenia
Splošna bolnišnica Celje, Celje, 
Slovenija 1
Faculty of Medicine, University 
of Ljubljana, Ljubljana, Slovenia
Medicinska fakulteta Ljubljana, 
Univerza v Ljubljani, Ljubljana, 
Slovenija
2
University Clinic of respiratory 
and allergic diseases Golnik, 
Golnik, Slovenia
Univerzitetna klinika za pljučne 
bolezni in alergijo Golnik, Golnik, 
Slovenija
3
Slovenian Medical Journallovenian Medical Journal
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1 Introduction
In recent years, the development of medicine has 
been directed towards patient-focused, personalized 
treatment. It is an individual approach to disease pre-
vention and treatment, based on genetic and molecular 
diversity and the influence of the environment on the 
individual’s health (1). The greatest progress can be seen 
in the field of oncology, which has been one of the most 
active branches of medicine in terms of research in the 
last decade. Cancer is a genetic disease that contains a 
patient-specific and unique mutation profile. With the 
development of increasingly sensitive, specific and af-
fordable technologies for the isolation and analysis of a 
tumour’s genetic material, the role of personalized treat-
ment is increasingly included in oncology treatment 
guidelines worldwide. With the discovery of mutations 
and thus new possibilities for effective targeted drugs, 
even in the treatment of patients with lung cancer, the 
successes of the personalized approach are already visi-
ble with prolongation of survival (2).
2 Lung cancer
Lung cancer is one of the most common and deadly 
cancers worldwide (3). In Slovenia, lung cancer ranks 
3rd among cancer types in men and 4th in women, with 
the proportion of female cancer patients still increasing 
(4). It is important to suspect and diagnose lung cancer 
as soon as possible (5). Despite medical progress, in most 
cases, the disease is detected at an advanced stage with 
subsequent poor survival and limited curative treatment 
options. In the last 10 years, with the discovery of driv-
er mutations, significant progress has been made with 
treatment, which is becoming more and more tailored 
to the biological properties of the patient’s cancer (6,7).
Lung cancer is a heterogeneous disease with sever-
al histological subtypes, according to the World Health 
Organization classification (8). Due to differences in 
treatment methods, the division into small cell and non-
small cell lung cancer, which includes adenocarcinoma 
and squamous cell carcinoma, is particularly important. 
Due to the emergence of many targeted treatments, an 
active and targeted search for driver mutations is recom-
mended in lung adenocarcinoma. In patients with small 
tekočinske biopsije pri klinični obravnavi bolnikov z nedrobnoceličnim rakom pljuč, ki se kaže v možnosti zgodnjega 
odkrivanja raka pljuč, identifikaciji vodilnih in odpornih mutacij, potencialni oceni bremena bolezni ter dolgoročnega 
spremljanja bolnika.
cell or squamous cell carcinoma, specific targeted ther-
apy is not available, so routine identification of driver 
mutations is not necessary (9).
2.1 Lung adenocarcinoma
The most studied and common mutation in non-
small cell lung cancer is a mutation in the epidermal 
growth factor receptor (EGFR) gene; other types of mu-
tations, such as ALK (anaplastic lymphoma kinase) re-
arrangements, V600E point mutation in the BRAF gene 
(v-raf murine sarcoma viral oncogene homolog B1), 
rearrangements of ROS1 (c-ros oncogene 1), RET (re-
arranged during transfection), NTRK (neurotrophic ty-
rosine receptor kinase), and a skipping mutation in exon 
14 of the MET gene are rarer (6,7,10). When patients are 
diagnosed with lung adenocarcinoma in Slovenia, EG-
FR, KRAS (Kirsten rat sarcoma viral oncogene homo-
log), and BRAF mutations and ALK, ROS 1, and NTRK 
rearrangements are determined in the tumour sample 
(9). As a result of targeted therapy, the survival of pa-
tients with advanced cancer and their quality of life have 
significantly improved (2). During targeted therapy, new 
genetic changes which prevent the long-term success 
of this treatment develop in the tumour. Extensive re-
search is being conducted around the world to identify 
the mechanisms of treatment resistance as precisely and 
quickly as possible with minimal invasiveness. The need 
for repeat tumour biopsies represents a limitation; a new 
method, the liquid biopsy, is becoming available for this 
purpose (11).
2.2 Squamous cell lung carcinoma
At the oncologist’s request, the aforementioned mu-
tations, though rarely present, are also tested in selected 
younger and non-smoking patients with squamous cell 
carcinoma and in other rare histological types of non-
small cell lung cancer. As a rule, in patients with squa-
mous cell carcinoma (and adenocarcinoma with nega-
tive markers), due to the possibility of immunotherapy, 
only immunohistochemical staining for PD-L1 (pro-
grammed death-ligand 1) is performed (9).
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Liquid biopsy – a diagnostic and therapeutic tool in the treatment of non-small cell lung cancer
Legend: CT – computed tomography.
Tkivna biopsija Tekočinska biopsija
Sample type tumour tissue biopsy (bronchoscopy with 
biopsy, CT-guided needle biopsy, surgical 
biopsy)
blood sample (less frequently pleural 
effusion, saliva, urine, ascites, cerebrospinal 
fluid)
Procedure invasiveness Invasive Minimally invasive, non-invasive
Procedure difficulty Difficult Less difficult
Complications Rare, potentially life-threatening Very rare, less dangerous
Tumour heterogeneity detection No Yes
Patient burden Yes No
Longitudinal follow-up of tumour 
characteristics
No Yes
Monitoring treatment response No Yes
Sensitivity High High, but it differs depending on the 
authors and the type of method used
Table 1: Comparison of tissue and liquid biopsy in lung cancer (13-26).
3 Liquid biopsy
3.1 A comparison of liquid and tissue biopsy
A liquid biopsy is a procedure for the detection of 
cancer and molecular tumour characteristics in body 
fluids (12). Unlike traditional tissue biopsy using bron-
choscopy or other minimally invasive procedures, a 
liquid biopsy is much less burdensome for patients. 
Around the world, different types of body fluids are 
used for analysis (e.g. pleural effusion, urine, sputum); 
most frequently, samples of peripheral blood or plas-
ma are used (13-16). The role of liquid biopsy in the 
treatment of cancer is mainly in the identification of 
driver mutations, which allow for targeted treatment, 
monitoring the treatment response and success, as well 
as early detection of disease recurrence (17).
Tissue biopsy is still the gold standard for cancer 
diagnosis, defining the tumour type and its character-
istics. By tumour tissue sampling and processing, driv-
er mutations have been discovered in recent decades, 
allowing for the development of targeted therapy. By 
taking tissue samples before and after the appearance 
of resistance to targeted therapy, researchers identi-
fied the mechanisms of new, secondary mutations. 
Despite the advantages offered by tissue biopsy, it is 
still a time-consuming invasive method that requires 
an experienced team of specialists to perform the 
procedure and process the samples. There are several 
contraindications for invasive tissue biopsy, limiting 
its use in patients with advanced cancer. Complica-
tions are also possible (18). Time required for the bi-
opsy and intra- and intertumour heterogeneity are the 
most significant limitations. Accurate identification of 
mutations with a tissue biopsy is frequently not possi-
ble at all, as due to tumour heterogeneity, the biopsy 
might not capture the entire set of mutations (19). Ad-
ditionally, tissue biopsy does not allow for monitoring 
changes in the tumour’s genetic profile over time and 
the possible discovery of new mutations that pose an 
increased risk of treatment resistance (20).
On the other hand, as a newer method, liquid bi-
opsy is more accessible, reproducible, easier and faster 
to perform, either minimally or even non-invasively, 
and less burdensome for the patient. Compared to tis-
sue biopsy, the risk of complications is negligible (18). 
Repeat sample collection is possible, giving immediate 
insight into the tumour’s changes and allowing longitu-
dinal follow-up (21,22). Despite the many advantages 
that place liquid biopsy alongside the established tissue 
biopsy (Table 1), its use in everyday practice is gaining 
ground slowly, mainly due to dilemmas regarding the 
reliability of the test to prove the presence of a muta-
tion and current high costs when using highly sensitive 
techniques. Liquid biopsy has already found a place in 
routine clinical practice in non-small cell lung cancer. 
In international recommendations (23), liquid biopsy 
is already the method of choice for the detection of 
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treatment-resistant T790M mutation in patients with 
EGFR-mutated lung cancer treated with EGFR tyro-
sine kinase inhibitors. The latest recommendations of 
the IASLC (International Association for the Study of 
Lung Cancer) include an expanded use of liquid biopsy 
in patients with lung cancer (24). The test’s sensitivi-
ty and specificity undoubtedly depend on the size of 
the tumour mass, the presence of metastases and the 
type of method used to prove or determine mutations 
(25,26).
4 Types of liquid biopsy
Liquid biopsy is based on the knowledge that can-
cer cells present in the bloodstream are identical to 
primary tumour cells, which was first demonstrated by 
Australian scientist Thomas R. Ashworth in 1869 (27). 
It is estimated that thousands of cells are released into 
the circulation daily with an average half-life of 1–2.5 
hours. As early as 1948, researchers Mandel and Metais 
described circulating free DNA fragments (cfDNA) in 
plasma, but the association between cfDNA concentra-
tion and disease states was not proven until many years 
later (28). cfDNA is the total freely circulating DNA 
originating from all cells in the organism, healthy and 
diseased. With the improvement of techniques for the 
isolation of genetic material, it is now possible to detect 
and monitor various types of mutations that serve as 
specific biological markers for the presence of cancer 
in the bloodstream.
4.1 Molecular technologies
In liquid biopsy, several types of molecular tech-
niques are used, which are roughly divided into two 
groups. The first, the target group, represents a fast, more 
affordable, highly sensitive and specific technique that 
can identify even a small number of point mutations 
in one gene. This group includes methods based on the 
use of polymerase chain reaction (PCR). As narrowly 
focused methods, prior knowledge about the type of 
tumour and mutation is required. The second group 
includes high-performance methods that identify 
mutations in several genes (next-generation sequenc-
ing, NGS). Since they are aimed at identifying a large 
part of the genome and can identify both specific and 
non-specific mutations, prior knowledge about the 
type of tumour and mutation is not required. Their 
biggest limitations are high cost, long procedure time, 
increased technology demands, and analysis (23,29). 
According to data from the literature, the sensitivity of 
cfDNA detection methods using NGS is between 75% 
and 90% with high tissue biopsy concordance (30).
4.2 Types of biological markers
Liquid biopsy involves different methods of circu-
lating marker detection. From a clinical point of view, 
among these markers, cfDNA, of which tumour DNA 
is a component, has been by far the most widely re-
searched and used for genotyping solid cancer types, 
including lung cancer (mainly non-small cell lung 
cancer), where it is already included in routine man-
agement (24). There are several other biomarkers, but 
their clinical use in lung cancer is rare and currently 
limited to research.
4.2.1 Circulating tumour DNA
Circulating tumour DNA (ctDNA) is a subtype of 
cfDNA that is released from tumour cells and con-
tains a transcript of extracellular tumour DNA (dou-
ble-stranded or mitochondrial). It represents only a 
part of the total cfDNA, which also originates from 
non-tumour cells. It is found in body fluids (most of-
ten in blood), into which it is released through various 
mechanisms, such as necrosis, apoptosis, active secre-
tion, or cancer cell senescence (31). The concentration 
of ctDNA in body fluids is not constant. Its amount de-
pends on several factors, mainly on the size of the tu-
mour. The half-life of ctDNA is short (from a few min-
utes to two hours), allowing monitoring the tumour’s 
current state. Its concentration is unfortunately low, 
therefore highly sensitive and advanced isolation and 
detection molecular techniques are needed to identify 
ctDNA (32).
An additional obstacle with peripheral blood anal-
ysis is cfDNA of non-cancerous origin, released from 
leukocytes. It dilutes the ctDNA present, further re-
ducing the detection method’s sensitivity. In practice, 
adapted test tubes for sample collection and centri-
fuging the sample as soon as possible after collection 
are used to solve this problem (33). In addition to false 
negative results, record false positive results of liquid 
biopsy with ctDNA are also possible. In healthy sub-
jects, cancer-unrelated somatic mutations can arise 
from haematopoietic cells through the process of clon-
al haematopoiesis, which complicates the interpre-
tation of liquid biopsy results. These mutations most 
frequently arise from genes associated with haema-
tological malignancies, but less frequently, mutations 
associated with lung cancer can also arise through this 
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Liquid biopsy – a diagnostic and therapeutic tool in the treatment of non-small cell lung cancer
process, e.g. KRAS mutations. In patients undergoing 
liquid biopsy for suspected lung cancer, a KRAS muta-
tion can thus be detected despite the absence of lung 
cancer (34,35). Some specific tumour mutations, e.g. 
EGRF and BRAF, do not have a haematopoietic origin, 
so detection of a false mutation due to clonal haemato-
poiesis is not possible.
Despite excellent prospects, before the expected 
wider clinical use of ctDNA in oncology, the result’s 
credibility will require a precise standardization of the 
entire procedure from sample collection to final analy-
sis (12). In the literature, studies that use targeted and 
even more affordable PCR methods are introducing 
ctDNA into clinical practice. Due to increasing sensi-
tivity and gradually more affordable NGS methods, the 
use of these methods in clinical practice is expected to 
expand in the future.
4.2.2 Circulating tumour cells
Circulating tumour cells (CTCs) are individual tu-
mour cells that leave their site of origin (primary tu-
mour or metastasis). They travel throughout the body 
to distant locations via the bloodstream (36). Blood 
is therefore a suitable sample for their identification, 
cancer diagnosis and cancer monitoring. CTCs already 
play an important role in the diagnosis of metastatic 
breast, prostate, and colon cancer, and they already 
have a possible role in lung cancer management (37-
39). Similar to ctDNA, their blood concentration is 
unfortunately low (0.1-10 CTC/mL whole blood), and 
the half-life is short (1-2.4 hours). Detection requires 
highly sensitive techniques that distinguish CTCs from 
other circulating cells based on their physical, mor-
phological, and biological characteristics (40).
4.2.3 Other biological markers
There are more types of biomarkers, but compared 
to ctDNA, they are currently used in lung cancer only 
for research purposes. Due to the extensive research 
on their possible use and predicted important future 
role in the treatment of lung cancer, they are only men-
tioned in this article. Since they are not currently used 
in clinical practice, a detailed presentation is beyond 
the scope of this article.
Various biological markers with possible roles in 
cancer development are used for research purposes. 
Exomes are small, 30–100 nm membrane-bound ves-
icles that are produced by endocytosis or release from 
cells. They contain proteins and nucleic acids that can 
be transported between cells. Due to their lipid mem-
brane, they are protected from degradation, can pass 
through the membrane, and persist in the bloodstream 
for longer periods. They play many different roles in 
the development of cancer: in carcinogenesis, the for-
mation of metastases and the transfer of oncogenic 
factors between cells. The use of TEP (tumour educat-
ed platelets), tumour-associated autoantibodies (TA-
Ab) and microRNA is also mentioned (36,41).
5 Use of liquid biopsy in patients with lung 
cancer in clinical practice
Liquid biopsy has established itself in clinical prac-
tice in patients with lung cancer. With the discovery 
of critical molecular and cellular mechanisms of lung 
cancer development and the emergence of new target-
ed therapy methods, new techniques have been devel-
oped and are being used, particularly in the treatment 
of non-small cell lung cancer. Liquid biopsy has es-
tablished itself as a method to identify specific EGFR 
mutations in patients who develop targeted treatment 
resistance. Due to its clinical availability and cost-ef-
fectiveness, the current most widely used method is 
the detection of ctDNA with PCR, hence the reason 
for this article’s focus upon it. However, it is import-
ant to emphasize that in recent years, the number of 
scientific publications investigating its use in patients 
with cancer other than lung cancer is rapidly increas-
ing (Figure 1). We expect that in the coming years, the 
field of liquid biopsy will advance professionally and 
technologically, particularly with the expansion of the 
NGS method.
5.1 Early lung cancer detection
Due to its non-invasiveness and potential low cost, 
liquid biopsy has shown its potential for use in screen-
ing and early detection of lung cancer. Currently, the 
only recognized and recommended method for lung 
cancer screening is the use of low-dose computed to-
mography (CT) with confirmatory tissue biopsy. Due 
to the low sensitivity and specificity for early cancer 
detection, ideas on improving screening with a com-
bination of CT imaging and a blood test (liquid biop-
sy) have appeared in the literature (42). According to 
data from the TRACERx study, due to early genomic 
and chemical changes, ctDNA should be present in the 
bloodstream 6-12 months before the first radiological 
signs of lung cancer, and the concentration of cfDNA is 
higher in cancer patients compared to healthy controls 
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Figure 1: Graphic representation of the temporal distribution of search results in the PubMed digital database up to 2021. 
The search keywords used were »lung cancer« and »liquid biopsy« with synonyms.
Se
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 h
its
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er
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Year
1945    1949      1953    1957     1961     1965     1969     1973     1977     1981     1985     1989     1993     1997     2001     2005     2009     2013     2017     2021
0
5
10
15
20
Časovna razporeditev rezultatov iskanja z izbranimi ključnimi besedami
v zbirki PubMed
(številke podane na 100.000)
and patients with benign lung changes (43,44). Ma-
ny studies in recent years have been concerned with 
obtaining the most favourable biological markers for 
screening use, e.g. ctDNA, CTC, microRNA, exomes, 
proteins, etc. (45-47). One of the first studies, conduct-
ed in 2014, to isolate CTCs from patients with chronic 
obstructive pulmonary disease, who represent a group 
at higher risk of developing lung cancer, was the first to 
show promising results for detecting patients at high 
risk of developing lung cancer with a normal CT scan 
result (48). Some similar follow-up studies have failed 
to demonstrate similar statistical reliability for predict-
ing lung cancer risk (49,50). Currently, the combina-
tion of a low-dose CT and liquid biopsy with CTC and/
or ctDNA cells is an important research topic for the 
early detection of lung cancer (studies by Spira’s group, 
Lecia et al) (51). A recent larger study by Chen et al, 
which included more than 120,000 healthy, asymp-
tomatic individuals, highlighted the potential for early 
detection of multiple specific ctDNA methylations in 
477 regions, which research suggests may be specific 
to certain solid tumours, including lung cancer. They 
reported correct identification of 88% of solid tumours 
(including lung cancer) demonstrated over a 4-year 
follow-up and 95% specificity for identifying the type 
of cancer (52). Despite high expectations, a problem 
arises when using ctDNA as a biological marker due 
to the low frequency of mutated alleles in early, cur-
able forms of lung cancer, as their detection exceeds 
the sensitivity of the currently available methods (53).
Today, liquid biopsy as a screening method does not 
yet seem viable from an organizational and cost point 
of view. False-negative tests are an additional problem; 
currently, it would be reasonable to use the test only in 
patients with risk factors for the development of lung 
cancer. Considering the extensive research into liquid 
biopsy, it is expected that the method will establish it-
self as a new approach to disease management in the 
future.
5.2 Identifying mutations and treatment 
resistance
Several different types of mutations have been iden-
tified that participate in lung cancer development. 
Identifying mutations in cancer cells is key to select-
ing an appropriate and effective targeted therapy. Ac-
cording to the recommendations of the European and 
65
STROKOVNI ČLANEK
Liquid biopsy – a diagnostic and therapeutic tool in the treatment of non-small cell lung cancer
American professional guidelines, treatment should be 
directed according to the detected resistance mecha-
nism; before first-line treatment, a tissue biopsy is still 
in place today (54,55). It is particularly important to 
identify the mutations for which targeted treatment is 
available. The biggest limitation in the use of targeted 
therapy are secondary mutations resulting in resistance 
to primary treatment, which require a repeat biopsy to 
change treatment. According to data from Nosaki et al, 
a repeat lung tumour biopsy is not feasible in as many 
as 20% of patients with treatment resistance, and many 
studies also report insufficient or unrepresentative tu-
mour tissue samples (56,57).
Based on the favourable study results and the afore-
mentioned problems with repeat tissue biopsies in the 
event of resistance, the European Medicines Agency 
(EMA) was in 2015 the first to issue an approval for 
the use of plasma ctDNA in lung cancer; a year later, 
the US Food and Drug Administration (FDA) did the 
same (58,59). Its use is currently limited for EGFR sta-
tus determination in patients with newly diagnosed 
non-small cell lung cancer where tissue biopsy is not 
possible or in the event of resistance during treatment 
with primary or second-generation tyrosine kinase 
inhibitors (TKIs) for the detection of the point mu-
tation in exon 20 T790M. This is the most common 
treatment-resistant mutation (proven in approximate-
ly 50% of patients) and is an indication for treatment 
with third-generation TKIs. Two standardized, high-
ly sensitive and specific mutation tests (Cobas EGFR 
mutations Test v2, Roche; Therascreen, Qiagen) based 
on the use of qPCR have been approved for detection 
(20,60-62).
However, according to the AURA study, caution 
is required when interpreting the results of T790M 
mutation detection (63). In the study, they found in-
consistencies between the presence of the mutation in 
tissue and liquid samples as a consequence of tumour 
heterogeneity. In case of a positive result (i.e. proven 
resistance mutation), the type of treatment should be 
changed. Additionally, due to low method sensitivity, it 
is necessary to take into account the possibility of false 
negative results, so another sample should be taken in 
the event of a negative result (64-66).
In the case of EGFR-independent mutations, can-
cer cells acquire an EGFR-independent alternative 
pathway for cancer cell survival and proliferation by 
acquiring new mutations, e.g. KRAS, HER2, MET, 
PI3KCA, etc. Transformation from non-small cell 
to small cell lung cancer is rarely seen. With the ex-
pansion of advanced techniques in everyday clinical 
practice, ctDNA analysis with NGS with which we 
can identify a wider range of cancer cell mutations is 
becoming more and more popular. Since the publica-
tion of the latest IASLC recommendations, a number 
of additional studies have emerged to support the use 
of broad-spectrum NGS analysis (24). It can be used 
to demonstrate the presence of other important muta-
tions for which we have available targeted treatments, 
e.g. ALK (67) and ROS 1 (68) rearrangements, BRAF 
mutations (69), etc. In 2020, the ESMO expert group 
already recommended NGS for the detection of ge-
netic alterations (EGFR, ALK, etc.) in advanced lung 
cancer in regular clinical practice (70). At the end of 
2020, the FDA was the first to issue approval for two 
commercial NGS tests (Guardant360, FoundationOne 
Liquid CDx) (24).
So far, the use of NGS worldwide is still limit-
ed compared to PCR methods. However, its role will 
undoubtedly increase significantly in the future with 
lower costs and better availability. Despite the increas-
ing role of liquid biopsy compared to tissue biopsy, it 
should be taken into account that ctDNA analysis does 
not detect the emergence of some other resistance 
mechanisms, e.g. histological transformations of can-
cer tissue (71).
5.3 Assessment of disease burden and long-
term monitoring of treatment response
With a liquid biopsy, adequate treatment response, 
disease progression, and assessment of patient survival 
could be determined early. Studies examining liquid 
biopsy as a method for quantitative and qualitative 
monitoring of tumour burden and molecular profile 
are emerging in the literature. The concentration of 
ctDNA in plasma changes, and depends on the type 
of cancer, its location, size, and tumour blood supply, 
ergo monitoring trends and not the absolute concen-
tration is more appropriate (72). Due to this variability, 
different concentrations of ctDNA can be found in pa-
tients with the same tumour type and the same disease 
stage. Despite the variability, in one large Chinese mul-
ticentre prospective cohort study from 2020, signifi-
cantly shorter non-small cell lung cancer survival was 
reported in patients with higher pre-treatment ctDNA 
concentrations and mutations (73,74). This was associ-
ated with a higher baseline disease burden. However, 
on the other hand, data can be found in the literature 
that baseline ctDNA concentrations do not predict re-
sponse to treatment (75).
With the introduction of a new drug, sequential 
66
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blood analyses could be used to monitor the response 
to treatment and rate of decline in ctDNA concentra-
tions to assess treatment effectiveness. In their 2016 
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survival in patients with undetectable EGFR mutation 
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6 Conclusion
Liquid biopsy is a method that is slowly taking an 
important place in diagnosis and monitoring the re-
sponse to lung cancer treatment. Although its role in 
the treatment of lung cancer is currently still limited, 
it is gradually establishing itself in global guidelines 
(55,56). Currently, ctDNA is already used in clinical 
practice in patients with non-small cell lung cancer for 
the primary detection of driver mutations (mainly EG-
FR) and resistance mechanisms during targeted thera-
py. The method also shows great promise in the early 
detection of cancer and in monitoring the response to 
treatment (24,55). Due to the increasing sensitivity and 
more affordable methods, the use of the NGS molec-
ular method is expected to expand into clinical prac-
tice, and other types of biological markers also show 
great potential in research (24). Further technological 
advancements and research are needed, to improve the 
reliability and applicability of the method in regular 
clinical practice, and above all, the standardization of 
the entire procedure is essential - from sample collec-
tion to reporting of results and their evaluation.
Conflict of interest
None declared.
67
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