Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 11, pp. 1484-1503 © Pleiades Publishing, Ltd., 2025.

Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 11, pp. 1579-1600.

1484

REVIEW

Circulating Tumor DNA And Its Potential Applications

for Assessing Effectiveness of Neoadjuvant Drug

Therapy in the Breast Cancer Patients

Tatiana M. Zavarykina

1,2,a

*, Irina V. Pronina

1,2

, Polina S. Mazina

1,2

Svetlana V. Khokhlova

, and Gennady T. Sukhikh

N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Science,

119334 Moscow, Russia

B. I. Kulakov National Medical Research Center of Obstetrics, Gynecology and Perinatology,

Ministry of Health of the Russian Federation, 117997 Moscow, Russia

e-mail: tpalievskaya@yandex.ru

Received July 18, 2025

Revised October 8, 2025

Accepted October 15, 2025

Abstract— The review discusses main characteristics and analytical features of the circulating tumor DNA

(ctDNA), which accounts for a minor fraction of the cell-free DNA (cfDNA) in cancer patients. Currently, ctDNA

is considered to be a promising biomarker for assessing treatment efficacy, prognosis, and disease monitoring

in oncology, including breast cancer (BC). A significant proportion of BC patients receive neoadjuvant drug

therapy, effectiveness of which largely determines necessity and extent of subsequent treatment. Determina-

tion of ctDNA could be the most sensitive method for evaluating response to neoadjuvant therapy, as it enables

real-time monitoring of molecular changes during the treatment, prediction of therapeutic response, and as-

sessment of recurrence risk. This approach could become an additional tool for personalization of BC therapy.

DOI: 10.1134/S0006297925602187

Keywords: circulating tumor DNA, breast cancer, neoadjuvant chemotherapy, pathologic response of tumor,

relapse

* To whom correspondence should be addressed.

INTRODUCTION

Breast cancer (BC) is the most common and so-

cially significant malignancy among women, requir-

ing substantial economic resources for the patient

treatment and rehabilitation. Development of cancer,

among other factors, is associated with accumulation

of somatic mutations in the tumor, particularly mu-

tations in the driver genes, which play a major role

in malignant transformation and serve as targets for

targeted therapies. Presence of tumor-associated dis-

turbances in the DNA isolated from blood plasma has

been reported in various types of cancer, including

breast cancer [1], lung cancer [2, 3], colorectal cancer

[4, 5], prostate cancer [6], gastric cancer [7], ovarian

cancer [8], and other malignancies [9-13]. Detection

of the circulating tumor DNA (ctDNA), which carries

genetic alterations unique to the tumor, is a basis for

a new form of liquid biopsy that is being integrated

into the clinical practice. Since most molecular aber-

rations identified in the plasma ctDNA reflect genet-

ic changes present in the primary tumor, the ctDNA

analysis serves as a convenient predictive and prog-

nostic biomarker, as well as a tool for monitoring dis-

ease progression [14-16].

DEVELOPMENT OF THE

CIRCULATING TUMOR DNA CONCEPT

The existence of extracellular nucleic acids was

first demonstrated in the mid-20th century, when

Mandel and Métais (1948) [17] detected and isolated

extracellular nucleic acids from human blood plasma.

In 1966, Tan etal. [18] performed quantitative analysis

of the cell-free DNA (cfDNA) levels in blood samples

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Fig. 1. Forms of cell-free DNA (cfDNA) in the bloodstream.

from the patients with systemic lupus erythematosus.

They were the first to confirm the hypothesis that

the disease-specific cfDNA can circulate in the blood-

stream. Elevated cfDNA levels in the samples of pa-

tient serum provided prospects of its molecular pro-

filing for prediction and management of autoimmune

disorders. Prognostic potential of cfDNA for assessing

cancer treatment efficacy was first demonstrated in

1977. In the study by Leon etal. [19], total cfDNA con-

centration in the serum of the patients with various

cancers was measured and found to be significantly

higher than in the healthy individuals. Furthermore,

the cfDNA concentration correlated with the tumor

response to neoadjuvant therapy, showing decrease

in the patients who responded positively to radiation

treatment [19]. These findings suggested that quanti-

tative cfDNA analysis could be employed to evaluate

efficacy of anticancer therapies, including comparison

of different treatment regimens. In 1999, Silva et  al.

[20] detected ctDNA in the plasma of patients with

BC, and its presence showed a statistically significant

correlation with the clinicopathological characteristics

of the patients.

Today, ctDNA is being actively investigated as a

potential biomarker for cancer diagnosis and as a

minimally invasive tool for assessing treatment effi-

cacy, prognosis, and disease monitoring in oncology.

CHARACTERISTICS OF ctDNA

There are various types of free-circulating nucleic

acids distinguished depending on the origin, such as

genomic DNA, mRNA, viral DNA and RNA, microRNA,

etc. Cell-free DNA (cfDNA) represents fragmented DNA

molecules circulating in biological fluids. It can exist

as a single- or double-stranded DNA with fragment

lengths ranging from 120 to 21,000  bp (on average

160-180  bp). In most cases, cfDNA circulates in the

bloodstream as part of nucleosomes – macromolecular

complexes of histones and DNA [21] – or within ves-

icles [22] (Fig.  1). In these forms, cfDNA is protected

from nuclease degradation and from eliciting immune

responses [22]. In addition, cfDNA can be bound to the

surface of blood cells, such as erythrocytes, through

specific membrane proteins.

In the healthy individuals, cfDNA originates from

several physiological processes, including apoptosis,

phagocytosis, DNA release from neutrophils, and enu-

cleation of erythroid precursors [23]. Plasma cfDNA

is actively secreted by leukocytes (55%), erythroid

precursors (30%), and lymphocytes (12%) [24]. Con-

centration of free-circulating cfDNA in the healthy

individuals typically ranges from 1 to 10  ng/mL [25,

26]. Due to the rapid nuclease degradation, half-life of

free-circulating cfDNA varies between 15  min and 2  h,

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which makes it suitable for use in modern medical

monitoring approaches [27].

In cancer patients, a fraction of cfDNA consists

of ctDNA, which accounts for approximately 0.01-1.0%

of the total cfDNA. Concentration of ctDNA depends

on the tumor stage and localization, ranging from

5 to 1500 ng/mL [25]. ctDNA could be released into

the bloodstream from the primary tumor cells, dis-

tant metastatic nodes, or circulating in the peripheral

blood tumor cells [28, 29]. Detection of tumor-specif-

ic mutations in cfDNA allows ctDNA to be identified

within the total pool of circulating nucleic acids. Both

qualitative and quantitative mutation composition of

ctDNA vary not only among the cancer types but

also between the patients with the same diagnosis.

For example, in the case of metastasis, ctDNA could

comprise more than 10% of cfDNA, whereas at ear-

ly stages of cancer or during minimal residual dis-

ease (MRD) monitoring, its fraction could be below

0.1% [30]. Although cfDNA is rapidly degraded in

bloodstream, a comparative study employing digital

PCR (dPCR) approach, targeted and whole-genome

sequencing (WGS), comparing different methods of

blood sample collection and storage for isolation

of cfDNA and analysis of ctDNA, demonstrated that

cfDNA remains stable for up to 24h in the EDTA-treat-

ed blood plasma samples at room temperature, up to

48 h at 4°C, and for extended periods when plasma

is appropriately processed and frozen [30]. Howev-

er, the reported acceptable processing intervals for

blood samples in EDTA tubes vary across studies,

ranging from 1-2  h after blood collection [31,  32] to

up to 24  h [30,  33,  34]. Therefore, in routine labora-

tory practice, blood samples collected in the EDTA

tubes for the ctDNA analysis are recommended to

be processed as soon as possible. The highest cfDNA

stability is achieved using specialized blood collection

tubes containing stabilizing reagents, whose perfor-

mance is comparable across different manufactur-

ers [30].

ctDNA has been detected in the patients with

early-stage BC [35-37] and could serve as a prog-

nostic biomarker [38-43]. It enables real-time mon-

itoring of the treatment response, including detec-

tion of minimal residual disease [38-44], and could

be applied to assess therapy efficacy in the patients

with BC [45-53].

DETECTION OF ctDNA

AS A VARIANT OF LIQUID BIOPSY

Analysis of ctDNA in the cancer patients – pri-

marily focused on identifying driver mutations that

determine tumor sensitivity to targeted therapy –

represents a form of liquid biopsy. Although ctDNA

is most commonly analyzed in the peripheral blood,

other biological fluids such as urine, saliva, pleural

or cerebrospinal fluid can also be used, depending

on the tumor type and localization [54]. Liquid bi-

opsy with ctDNA detection offers several advantages

over conventional tissue biopsy. Classical biopsy is an

invasive procedure that is not always feasible due to

the tumor localization. Moreover, it often fails to cap-

ture the complete molecular and genetic profile of the

malignancy because of tumor heterogeneity – that is,

coexistence of multiple tumor cell clones. Presence

of the so-called cancer stem cells contributes to the

ongoing evolution of genetic and biological tumor

properties during therapy and disease progression,

which is nearly impossible to monitor using conven-

tional tissue sampling. Performing repeated tissue

biopsies to assess tumor evolution and response to

therapy, is often complicated due to dispersion of tu-

mor foci, size or localization, and overall condition

of the patient. In particular, sampling from the meta-

static nodes may be technically challenging or risky.

Incontrast, liquid biopsy sampling for ctDNA analysis

is a simple, routine, and minimally invasive proce-

dure. Therefore, biological material can be collected

as frequently as needed to monitor disease dynamics

and to obtain real-time information about the tumor

[55, 56]. Thus, ctDNA analysis serves as a minimally

invasive alternative for the molecular characteriza-

tion of solid tumors.

METHODS FOR DETECTING

CIRCULATING TUMOR DNA

In most solid tumors, including BC, analysis

of ctDNA in blood samples is the most informative

and technically accessible approach. For quantitative

ctDNA analysis, blood plasma is preferred, as adher-

ence to the rigorously standardized pre-analytical

procedures involving double centrifugation minimiz-

es contamination with the genomic DNA from blood

cells, thereby increasing specificity and sensitivity of

the method [57].

Since ctDNA constitutes only a very small fraction

of the total cfDNA (Fig. 1), highly sensitive analytical

techniques are required for its detection. The most

common ctDNA detection approach relies on iden-

tifying tumor-specific mutations within the cfDNA

[58]. Quantitative ratio of normal cfDNA to ctDNA in

blood plasma of the cancer patients is expressed as

the mutant allele fraction (MAF). MAF of 0.1% means

that for every 999 molecules of cfDNA derived from

normal tissue, there is 1 molecule of ctDNA. It is

well established that the MAF values decline as tu-

mor size decreases [59]. For accurate quantitative and

qualitative analysis of ctDNA, various modifications

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of dPCR and next-generation sequencing (NGS) are

employed [60, 61].

The dPCR method allows detection of extreme-

ly small amounts of target DNA (in this case, ctDNA)

even in the presence of a large amount of other

DNA molecules [62], with sensitivity reaching MAF

values as low as 0.001% [63]. Major limitation of the

dPCR-based approaches is the requirement for prior

knowledge of potential mutations and their genomic

locations, which is necessary for primer and probe

design. This method efficiently detects the known

mutations, such as major driver mutations charac-

teristic of the primary tumor or variants associated

with therapeutic response in the specific tumor types

[64]. It correlates well with histopathological findings,

complements them, and provides additional informa-

tion for more precise tumor classification, recurrence

risk assessment, and therapy optimization.

Cancers lacking known mutations require a com-

bined methodological approach for ctDNA analysis,

involving prior identification of molecular–genetic

alterations in the primary tumor  [65]. This is partic-

ularly relevant for the triple-negative BC, in which

no driver mutations have been identified. Whole-ge-

nome sequencing (WGS)  [66] or whole-exome se-

quencing (WES)  [67] can be used to identify somatic

mutations in the tumor. Based on these findings, a

panel of most representative somatic mutations is

generated, including corresponding primer and probe

systems for their detection by dPCR [39].

NGS-based approaches enable detection of a

broad range of mutations – both previously described

and novel ones [68]. ctDNA analysis using NGS can be

performed in two formats: targeted sequencing of the

selected genes or gene panels, and whole-genome ap-

proaches [69]. Whole-genome sequencing allows iden-

tification of genetic alterations without prior analysis

of the primary tumor [58]. In particular, approaches

based on the copy number aberration analysis using

WGS data with different coverage depths have been

proposed [70]. Tumor-specific mutations were iden-

tified based on the ratio of detected variants at dif-

ferent depths of WGS coverage, without the need for

primary tumor analysis [70]. Some systems, such as

the Guardant Reveal multi-cancer assay  [71], evalu-

ate not only genetic but also epigenetic alterations,

namely, CpG methylation. Detection of ctDNA was

performed not by comparing tumor and leukocyte

DNA, but by comparing cfDNA sequencing results

with the reference databases  [72]. Despite numerous

advantages and substantial technological progress,

the NGS-based ctDNA analysis faces several challeng-

es. Detection of rare mutations present at very low

frequencies could be hampered by sequencing errors

[73]. Therefore, specialized bioinformatic approaches

and additional error-correction systems are required

for analysis of the sequencing data aimed at studying

ctDNA [1, 74-76].

An actively developing and increasingly clinically

implemented approach involves creation of the per-

sonalized marker panels for ctDNA analysis. In this

approach, both tumor biopsy material and blood sam-

ples are used for sequencing to identify somatic muta-

tions specific to the individual patient. Whole-genome

[66] or whole-exome [67] sequencing is typically ap-

plied, while targeted sequencing is used less frequent-

ly [77]. The phenomenon of clonal hematopoiesis [16,

78] should be considered when analyzing the paired

blood sample [79]. The resulting mutation panel, re-

flecting the most abundant mutations in the tumor, is

next used for ctDNA detection. An example of such

approach is the commercially available Signatera™

assay (Natera, USA), developed for monitoring MRD,

that is, molecular residual disease in BC and sever-

al other malignancies [80,  81]. Personalized systems

can also evaluate nucleotide substitutions, particular-

ly among the previously identified single-nucleotide

variants (SNVs) [82], and are capable of detecting in-

sertions and deletions [83]. The number of mutations

assessed across different systems ranges from 1-2 up

to 16 (in the Signatera™ assay). This approach enables

tracking of changes in the clonal composition of the

tumor, predicting therapeutic response. Furthermore,

detection of ctDNA in the patient plasma may indicate

the presence of micrometastases [83]. The CloneSight

system, developed in Spain, enables monitoring of BC

recurrence by detecting ctDNA at 6, 12, 18 months

and beyond after completion of therapy [84].

Combined approaches to ctDNA analysis are also

being actively developed [65]. These include the use

of ELISA or conventional molecular biology techniques

integrated with biosensor-based ctDNA detection plat-

forms, such as electrochemical and fluorescent sys-

tems. In these methods, particular attention is paid

to simplifying and accelerating sample preparation

by reducing quality requirements for the biological

material needed for analysis. Furthermore, electro-

chemical detection offers the potential for “point-of-

care” ctDNA testing, since this approach does not re-

quire expensive equipment and specialized laboratory

conditions. In the study by Wang et  al. [85], several

types of sensor elements are described, in which spe-

cific ctDNA-binding receptors – based on antibodies,

aptamers, enzymatic systems, etc. – are immobilized

on the sensor surface. Upon the ctDNA–receptor

binding, a signal is transmitted to the electrode, fol-

lowed by potentiometric or voltammetric analysis.

Photometric sensors could be designed based on the

same principle. Approaches for cfDNA detection in-

volving probe-mediated reactions in solution are also

under development. Such reactions are most often

based on redox reactions, with a resulting product

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Fig. 2. Methods for detecting circulating tumor DNA (ctDNA).

that could be detected electrochemically, photometri-

cally, or fluorimetrically [86]. The probes could include

single-stranded DNA molecules complementary to the

target ctDNA [87], RNA molecules [86], antibodies [88],

the latter allows identification of ctDNA methylation.

These studies focus on analyzing physical and chem-

ical properties, as well as on quantitative assessment

of the ctDNA content (Fig. 2) [89].

Due to the technical challenges in the develop-

ment of such systems, they have so far been imple-

mented only for the most frequently occurring driver

mutations in the genes such as PIK3CA [90, 91], KRAS

[92], EGFR [93], and MGMT [94].

At present, rapid progress in analytical technol-

ogies suggests that the diversity of approaches for

ctDNA analysis will significantly increase in the near

future, expanding the arsenal of available methods

for ctDNA detection.

POTENTIAL OF ctDNA ANALYSIS

FOR EVALUATING EFFICACY OF NEOADJUVANT

DRUG THERAPY IN BREAST CANCER PATIENTS

Clinicopathological prerequisites for ctDNA

analysis in neoadjuvant treatment of breast can-

cer patients. For the patients with BC, neoadjuvant

drug therapy constitutes an integral part of the man-

agement of both early-stage and locally advanced

disease. In these cases, achieving pathologic complete

response(pCR) is a crucial prognostic factor and is as-

sociated with improved overall survival(OS) and dis-

ease-free survival (DFS) [95-102]. This relationship is

most pronounced in the triple-negative breast cancer

(TNBC) and HER2-positive BC. Typically, neoadjuvant

therapy includes 6-8 cycles of chemotherapy, with

addition of the targeted therapy when indicated (an-

ti-HER2 therapy for HER2-positive tumors). Some on-

cologists believe that such treatment intensity may be

excessive for the early-stage patients with high che-

mosensitivity. Monitoring ctDNA levels during thera-

py may facilitate treatment personalization, enabling

optimization of the therapy duration for each patient.

The response to neoadjuvant chemotherapy

(NACT) provides prognostic information that comple-

ments standard clinicopathological parameters of the

primary tumor, such as molecular biological subtype,

disease stage, and malignancy degree [98, 99]. Cur-

rently, efficacy of NACT is assessed using clinical pa-

rameters and pathomorphological examination of the

resected tissue after surgery. Routine practice relies

on conventional clinical and instrumental methods

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(physical examination, palpation, ultrasound of breast

and regional zones, mammography, MRI of mammary

gland), which often fail to reflect the true therapeutic

effect. The rate of complete response detection may be

up to twofold lower when based on clinical and im-

aging assessment than when determined by pathomo-

rphological examination of postoperative tissue [103].

A key parameter for assessing NACT efficacy is

pCR, characterized by the absence of viable tumor

cells in both breast tissue and lymph nodes upon

pathomorphological examination. The standard meth-

od for evaluating the degree of treatment-induced

pathomorphosis is routine pathological/histological

examination, including determination of the ypT and

ypN categories (post-NACT pathomorphological stag-

ing of tumor to T and N categories) or assessment of

residual cancer burden (RCB). This method involves

manual review of serial histological sections by a pa-

thologist, which typically does not cover the entire

volume of resected tissue, thereby contributing to po-

tential errors due to heterogeneity of biological sam-

ple; furthermore, analytical precision varies widely

across laboratories. Nevertheless, information about

the degree of treatment-induced pathomorphosis is

crucial for determining post-neoadjuvant therapeutic

strategies in the TNBC and HER2-positive BC. A com-

bined analysis has shown that the patients achieving

pCR have a significant survival advantage compared

to those with residual disease after NACT (residual

tumor cells after treatment) [101, 102]. While pCR

is a strong predictor of low recurrence risk, studies

indicate that predicting early metastatic recurrence

in the patients with residual tumors remains less re-

liable [102, 104, 105]. For example, survival analysis

in the I-SPY2 Trial Consortium demonstrated that the

3-year DFS reached 95% among the patients achieving

pCR [102], whereas metastases occurred in 22% of pa-

tients without pCR.

In the United States, several prognostic gene ex-

pression panels associated with BC progression have

been developed, including MammaPrint (Agendia Inc.),

Oncotype DX (Genomic Health), and Prosigna (Vera-

cyte Inc.), which are designed to predict recurrence

and/or metastasis. Based on analysis of these mark-

ers, a decision can be made regarding the necessity of

adjuvant (postoperative) chemotherapy. These panels

have proven their utility for the luminal ER-positive/

HER2-negative tumors, which is the most common

subtype of BC [104]. However, these assays do not re-

flect dynamic state of the tumor, its response to NACT

(prediction of pCR), and therefore cannot be applied to

guide decisions regarding the timing of chemotherapy

completion.

Detection of ctDNA in blood may represent a

more sensitive approach for evaluating the effect of

NACT, showing a strong correlation with pathologi-

cal methods used to assess the degree of pathologi-

cal response. Development and implementation of a

reliable marker reflecting tumor pCR could become

an additional tool for therapy individualization and

could also help determine optimal timing for the

treatment completion. Moreover, a highly relevant

emerging direction in the early-stage BC management

is investigation of whether surgery could be safely

omitted in the patients who achieve pCR. In such

studies, pCR is typically confirmed by the tissue bi-

opsy; however, this approach cannot fully capture the

entire tumor burden. Analysis of ctDNA concentration

and characteristics in these cases may provide a more

informative, sensitive, and specific assessment, as it

reflects molecular tumor dynamics in real time. For

instance, in BC, ctDNA quantification enables a more

accurate evaluation of tumor response to therapy

and facilitates detection of minimal residual disease

(MRD) and tumor resistance to therapy, i.e., molecular

relapse [40, 41, 44, 105, 106]. ctDNA monitoring could

be used for the early detection of the disease recur-

rence [65] and, in the treatment of metastatic disease,

for selecting the most effective treatment regimens

[46, 47, 81]. In this review, we focus on the potential

of ctDNA analysis for assessing NACT efficacy in BC

patients.

CtDNA analysis for detection of minimal re-

sidual disease during primary treatment. Analy-

sis of ctDNA provides opportunities for detection of

MRD, that is, presence of ctDNA in the absence of

other clinical signs of recurrence in the patients who

have completed therapy. Assessing treatment response

based on MRD detection could become an important

component of personalized management strategies,

helping to guide further therapeutic decisions in sub-

sequent observation of BC patients. Feasibility of us-

ing ctDNA to determine MRD following NACT and

surgery has been demonstrated in several studies [1,

52, 105, 106]. Of particular importance is the dynamic

change in ctDNA levels relative to baseline. As early

as 2015, it was shown that ctDNA detection in the

serial samples obtained from the patients after NACT

and subsequent surgery was associated with early

recurrence [39]. These findings were later confirmed

by additional studies involving various subgroups

of BC patients.

CtDNA assessment during NACT. Monitoring

ctDNA during NACT enables prediction of treatment

response, showing correlation with pCR, as well as

with the risk of recurrence and metastasis. Moreover,

the ctDNA positivity at mid-NACT (12 weeks after

treatment initiation) was found to be strongly associ-

ated with the decreased OS and DFS (p  =  0.0002 and

p  =  0.0034, respectively) [81].

Using a personalized whole-genome approach, the

ctDNA analysis allows evaluation of the relationship

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between the ctDNA presence, NACT response, recur-

rence risk, metastasis development, and 3-year surviv-

al. In this study, individualized mutation panels were

designed for each patient based on the whole-exome

sequencing of tumor tissue. The early treatment pe-

riod of NACT (3 weeks after therapy initiation) was

identified as a significant time point for pCR predic-

tion (odds ratio (OR)  =  4.33; p  =  0.012). After comple-

tion of NACT, all patients who achieved pCR were

ctDNA-negative. Among those who did not achieve

pCR, the ctDNA-positive patients exhibited a higher

risk of metastasis (OR  =  10.4). Patients who failed

to reach ctDNA negativity showed poor treatment

response and increased metastatic risk (OR  =  22.4;

95% CI; p  <  0.001), whereas the ctDNA-negative state

was associated with improved survival even in the

absence of pCR [44]. These findings are consistent

with the results of previous works [105,  107] and

the study by Chen et  al. [81], which demonstrated

that the ctDNA absence at mid-NACT (12 weeks after

treatment initiation) was associated with pCR achieve-

ment (p  =  0.02).

Subsequent studies revealed relationship be-

tween the ctDNA-negative status and pCR achieve-

ment in the TNBC patients [108], as well as a posi-

tive correlation between the ctDNA levels and RCB

[1, 109]. A correlation was also observed between the

ctDNA positivity and reduced DFS regarding distant

metastases, particularly at mid-NACT (12 weeks af-

ter treatment initiation) and after NACT but before

surgery (p  <  0.0001) [108].

Personalized ctDNA monitoring during NACT may

provide a real-time assessment tool for treatment

response and predict pCR as a surrogate survival

marker [44, 104].

CtDNA assessment after NACT. CtDNA detection

after completion of NACT but before surgery could

be considered as a prognostic marker. The presence

of ctDNA has been shown to be associated with poor-

er DFS [76,  110]. One of the earliest studies demon-

strated an increased risk of early recurrence in the

ctDNA-positive patients both after surgery and during

subsequent observation of the patient, with hazard

ratios (HR) of 25.0 and 12.0, respectively. 96% of the

patients without disease recurrence had no detectable

ctDNA (p  <  0.0038) [39].

This correlation was most prominent in the

study by Cailleux et  al. (HR  =  53; p  <  0.01) [106].

In the ctDNA-positive patients after NACT who did

not achieve pCR, a higher risk of distant metastasis

was observed (HR  =  5.89 for hormone receptor-posi-

tive BC and HR = 3.79 for TNBC) [108]. These findings

were subsequently confirmed by additional studies

involving various BC patient subgroups [41, 76, 109].

Using common mutations in the signaling path-

ways associated with BC progression is suggested

to be a promising approach for ctDNA quantification

and treatment response evaluation. It was found that

in the BC patients carrying PIK3CA and/or TP53 mu-

tations in the tumor tissue, absence of these muta-

tions in the cfDNA analysis after NACT corresponded

to achievement of pCR in 93.33% of cases [111]. For the

HER2-positive BC subtype, the analysis of TP53 mu-

tations in cfDNA has been shown to be a marker of

resistance to anti-HER2 therapy with monoclonal an-

tibodies [112], as well as the presence of PIK3CA and/

or TP53 mutations in cfDNA has been identified as a

predictor of poor response to anti-HER2 treatment [64].

CtDNA assessment after NACT and surgery. For

the ctDNA-positive patients after NACT and surgical

treatment, an earlier disease recurrence (reduced RFS)

was observed [113, 114]. CtDNA monitoring in the BC

patients with residual tumors after NACT demonstrat-

ed that persistence of ctDNA positivity after NACT

and surgery was associated with the decreased RFS.

This approach could enable identification of the pa-

tient subgroups requiring more intensive therapy, im-

proving their survival outcomes [115]. Association of

ctDNA with the disease recurrence and progression

was also observed during the post-NACT observation

[72, 114].

Baseline ctDNA levels. It should be noted that

ctDNA serves both as a predictive and as prognostic

marker when analyzed during or after NACT, prior

to surgery, and throughout subsequent treatment or

observation. However, the pretreatment ctDNA lev-

els are not significant in the TNBC or heterogeneous

BC cohorts, as no association has been observed be-

tween the presence of ctDNA before NACT and RFS

[44, 105, 106]. At the same time, correlation between

the ctDNA levels and clinicopathological character-

istics of BC – such as tumor size, lymph node me-

tastasis, and tumor grade – has been reported [108].

However, in the HER2-positive subgroup, the ctDNA

positivity (based on detection of PIK3CA and/or TP53

point mutations) before initiation of anti-HER2 thera-

py was found to be associated with the lower rate of

pCR achievement [64].

Potential of ctDNA analysis in TNBC patients.

The triple-negative subtype represents the most ag-

gressive form of BC, lacking known targets for hor-

mone or targeted therapies, with chemotherapy con-

tinuing to be the standard therapeutic approach. In

recent years, growing interest has been directed to-

ward evaluating response to NACT in the TNBC pa-

tients through changes in the blood levels of ctDNA.

For the TNBC patients, detection of the ctDNA-posi-

tive status has been associated with high incidence

of metastatic disease (nearly 72%) [38]. The potential

of ctDNA analysis for this most unfavorable BC sub-

type has been demonstrated in the long-term study

with up to 12 years of observation, where the

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Table 1. Key studies on ctDNA analysis for assessing the efficacy of NACT

Patients in the study Main results Reference

Early-stage BC ctDNA positivity after NACT was associated with an increased

risk of early recurrence, both after surgery and during

subsequent observation, with hazard ratios (HR) of 25.0 and 12.0,

respectively (p  <  0.0001);

ctDNA was undetectable in 96% of patients who remained

recurrence-free, both at the postoperative time point (p < 0.0038)

and during subsequent observation (p  <  0.0001)

[39]

TNBC absence of ctDNA in the middle of NACT (12 weeks after

the initiation of therapy) was associated with achieving pCR (p  =  0.02);

ctDNA positivity at mid-NACT (12 weeks after the initiation of therapy)

was strictly associated with the decreased OS and DFS (p  =  0.0002

and p  =  0.0034, respectively)

[81]

Stage I and II BC presence of ctDNA after completion of NACT and prior to surgery

was associated with shorter DFS

[111]

Early-stage BC presence of ctDNA after completion of NACT and prior to surgery

was associated with shorter DFS (HR  =  53; p  <  0.01)

[106]

High-risk stage II

and III BC

before treatment, 73% of the patients were ctDNA-positive,

and this proportion decreased during therapy;

importantly, the early stage of NACT (3 weeks after treatment

initiation) was identified as a critical time point for predicting pCR

(odds ratio (OR) =  4.33; p  =  0.012);

a ctDNA-negative status at this early stage correlated

with achieving pCR;

after completion of NACT, all patients who achieved pCR

were ctDNA-negative (n  =  17; 100%);

among those who did not achieve pCR (n  =  43), the ctDNA-positive

patients (14%) had an increased risk of metastasis (OR  =  10.4);

the patients who failed to reach ctDNA negativity demonstrated

poor response to treatment and higher risk of metastasis (OR  =  22.4;

95% CI, 2.5-201; p  <  0.001), whereas ctDNA negativity was associated

with improved survival even in the patients who did not achieve pCR

[44]

Localized BC patients exhibited significantly higher levels of cfDNA

from mammary gland compared to the healthy donors

(p  =  5.3  ×  10

−12

; AUCROC  =  91.25% (83.79–98.71%));

BC patients could be identified with 80% sensitivity

while maintaining 97% specificity;

presence of mammary gland specific cfDNA at the end

of chemotherapy reflected the presence of residual disease

[107]

Hormone receptor

(HR)-positive/

HER2-negative BC

and TNBC

ctDNA-negative status at 3 weeks after treatment initiation was

associated with achieving pCR in the TNBC patients;

correlation between the ctDNA levels during NACT and pCR

achievement was observed in TNBC, but not in the HR-positive BC;

ctDNA levels were also linked to clinicopathological features of BC

such as tumor size, lymph node metastasis, and histological grade;

among the patients who did not achieve pCR, the ctDNA positivity

after NACT was associated with higher risk of distant metastasis

(HR  =  5.89 for HR-positive BC and HR  =  3.79 for TNBC), whereas

the ctDNA negativity indicated a favorable prognosis even

in the patients with residual disease;

tumor mRNA analysis before treatment revealed correlation between

the ctDNA appearance in circulation, cell cycle, and immune-related

signaling pathways

[108]

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Table 1 (cont.)

Patients in the study Main results Reference

Stage II and III BC patients who achieved complete response exhibited

a more pronounced decrease in the ctDNA levels during

postoperativetherapy;

early changes in the ctDNA levels during treatment had significant

prognostic value for the BC patients;

correlation was observed between the early ctDNA decline

and longer DFS compared with the patients showing increase

in the ctDNA levels (HR  =  12.54; 95%  CI 2.084-75.42; p  =  0.0063)

[116]

Hormone

receptor-positive/

HER2-negative BC

and TNBC

a positive correlation between the ctDNA levels and RCB

was established;

in TNBC, a moderate negative correlation between the cfDNA

concentration and RCB was detected at 3 weeks after treatment

initiation;

at the same time, ctDNA concentration showed a significant

positive correlation with RCB at all time points (correlation

coefficient R  >  0.3; p  <  0.05);

in the HR-positive/HER2-negative BC, the cfDNA concentration

was not associated with the response to NACT; however, the patients

with high baseline cfDNA levels had significantly shorter distant

DFS compared to those with low cfDNA levels (HR = 2.12; p = 0.037);

in TNBC, survival differences between the patients with high and low

cfDNA levels at all time points were not statistically significant;

meanwhile, the ctDNA levels at all time points showed significant

correlation with the distant DFS in both subtypes

[109]

Early-stage BC positive correlation between the ctDNA levels and RCB was identified

detection of ctDNA at mid-therapy was significantly associated

with the higher RCB (OR =  0.062; 95%  CI 0.01-0.48; p  =  0.0077),

allowing identification of the patients who would not achieve pCR

and would be classified as RCB II/III

[1]

Early-stage TNBC

with residual

disease after NACT

presence of ctDNA after completion of NACT and prior

to surgery was associated with the shorter distant DFS (median

distant DFS, 32.5 months vs. not reached; HR  =  2.99;

95% CI  1.38–6.48; p  =  0.006);

detection of ctDNA was also associated with worse DFS

(HR  =  2.67; 95%  CI 1.28–5.57; p  =  0.009) and OS (HR  =  4.16;

95%  CI 1.66–10.42; p  =  0.002)

[76]

TNBC

and HER2-positive BC

in the BC patients carrying PIK3CA and/or TP53 mutations in the

tumor tissue, absence of these mutations in the ctDNA after NACT

corresponded to achievement of pCR in 93.33% of cases; moreover,

presence of mutations in the ctDNA allowed exclusion of several

patients from the pCR group with an accuracy of 89.47%

[111]

HER2-positive BC presence of mutations in the TP53 gene in the ctDNA

was evaluated as a potential marker of resistance to anti-HER2

monoclonal antibody therapy in the HER2-positive subtype of BC;

the patients harboring mutations in the TP53 gene who received

anti-HER2 therapy had significantly shorter DFS (p  =  0.004);

in the patients with mutations in the TP53 gene, a trend

toward poorer prognosis under the anti-HER2 antibody

treatment was observed compared to those with wild-type TP53

in the phase II study (p  =  0.15), and this trend was confirmed

in the combined analysis of the MutHER and SUMMIT

cohorts (p  =  0.01)

[112]

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Table 1 (cont.)

Patients in the study Main results Reference

HER2-positive BC significance of the PIK3CA and/or TP53 mutations detected

in the ctDNA as predictors of poor response to anti-HER2 therapy was

demonstrated;

ctDNA positivity, defined by the presence of point mutations

in PIK3CA and/or TP53, was associated with the lower rate of pCR,

particularly at the stage prior to initiation of anti-HER2 therapy;

detection of ctDNA before NACT was associated with the reduced

likelihood of achieving pCR (HR =  0.15; 95%  CI 0.034-0.7; p  =  0.0089),

but not with DFS;

notably, the patients with HER2-positive tumors and absence

of ctDNA prior to treatment exhibited the highest pCR rates;

whereas those with detectable ctDNA both before treatment

and at week 2 had the lowest pCR rates

[64]

High-risk BC in the patients, who remained ctDNA-positive after NACT

and surgery, earlier recurrence was reported (reflected

by decreased DFS);

the Invitae Personalized Cancer Monitoring™ (PCM) panel, used

for monitoring before and during neoadjuvant therapy, after

surgery, and throughout observation, demonstrated 100% specificity

and prognostic value;

all patients (10 of 61; 16%) with detectable ctDNA during

the monitoring period subsequently developed recurrence;

detection of ctDNA during monitoring was associated

with the markedly increased risk of future recurrence (HR =  37.2;

95%  CI  10.5-131.9; p  <  0.0001), with a median lead time from ctDNA

detection to clinical recurrence of 11.7 months

[113]

Early-stage BC the patients who remained ctDNA-positive after NACT and surgery

experienced earlier recurrence (reflected by the reduced DFS);

correlation between the ctDNA detection and disease recurrence

or progression was observed, including during post-NACT observation;

preoperative ctDNA detection was significantly associated

with the decreased DFS (adjusted HR  =  3.09; 95%  CI 2.65-80.0;

p  =  0.001);

after a median observation period consisted of 26.6 months among

11 patients, recurrence occurred in 5 cases, all of whom had detectable

ctDNA at the 2-4-week postoperative time point;

ctDNA clearance at this time point was associated with significantly

longer DFS (p  =  0.0009), whereas persistent ctDNA positivity

after adjuvant therapy was observed in 36.4% (4 of 11)

of the patients with stage III disease;

during monitoring, ctDNA detection demonstrated sensitivity

of 90.9% and specificity of 98.8% for predicting recurrence,

with the median lead time of 9.7 months;

the patients with detectable ctDNA exhibited significantly shorter

progression-free survival than those without detectable ctDNA

(adjusted HR  =  207.05; 95%  CI 41.38-1000; p  =  0.001);

therefore, the ctDNA status both before and after surgery could aid

in risk stratification for recurrence in breast cancer patients

[114]

Non-metastatic BC

with residual disease

monitoring of ctDNA in the BC patients with residual disease after

NACT demonstrated that persistent ctDNA positivity following NACT

and surgery was associated with the decreased DFS;

the 4-year DFS rate was 100% in the patients who were ctDNA-negative

before treatment, compared with only 67% in the ctDNA-positive

patients (p  =  0.032)

[115]

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Table 1 (cont.)

Patients in the study Main results Reference

Early-stage BC association between ctDNA and disease recurrence or progression

was also observed during the long-term monitoring after NACT;

in this study, only plasma samples were analyzed, without prior

identification of tumor-specific mutations from the biopsy samples;

to detect ctDNA, a bioinformatic classifier was developed to identify

tumor-associated somatic variants and methylation profiles

for the specific cancer types using the 5-Mb NGS panels;

ctDNA was detected during or before distant recurrence

in 11 of 14 (79%) patients, with sensitivity of 85% (11 of 13)

among the samples collected within 2 years prior to recurrence;

lead time was estimated in 4 of 6 (67%) of the ctDNA-positive

samples collected before distant recurrence and ranged

from 3.4 to 18.5 months;

no ctDNA was detected in the samples from the patients

without recurrence (n = 13)

[72]

Primary BC prediction of recurrence with the lead time of up to 38 months

(median, 10.5 months; range, 0-38 months) was demonstrated,

with ctDNA positivity being associated with shorter distant DFS

(p  <  0.0001) and OS (p  <  0.0001) during the long-term monitoring

over 12 years; the most significant results were observed

in the patients with TNBC;

among all patients with recurrent TNBC (n  =  7 of 23),

the ctDNA positivity was detected within a median lead time

of 8 months (range, 0-19 months), whereas in 16 TNBC patients

without recurrence, ctDNA remained undetectable throughout

a median observation period of 58 months (range, 8-99months)

[65]

Early-stage TNBC according to the results of the meta-analysis including

1202 patients with TNBC, ctDNA positivity detected after NACT,

either before or after surgery, was associated with the risk

of recurrence and OS (HR  =  3.26; 95%  CI 1.88-5.63)

in this BC subtype

[117]

Stage II-III early TNBC in the patients with early-stage TNBC, prognostic significance

of ctDNA was demonstrated, allowing identification

of the subgroups with high treatment sensitivity and increased risk

of disease recurrence;

a threshold value of the maximum allele frequency

of 1.1% at baseline stratifies patients according to recurrence risk,

as confirmed by both internal and external quality controls;

the systemic tumor burden model integrating baseline

and postoperative ctDNA represents an independent prognostic

model (p  =  0.022);

combination of the systemic tumor burden with pathological response

allows identification of a subgroup of the patients with high likelihood

of cure and a subgroup of the patients with high-risk early-stage TNBC;

long-term ctDNA monitoring enables detection of the patients

with high risk of recurrence

[118]

most reliable results were obtained for TNBC [65].

Magbanua et al. [108] reported that correlation be-

tween the ctDNA levels during NACT and achieve-

ment of pCR was observed in TNBC, but not in the

hormone receptor-positive BC. Several studies have

also shown that the ctDNA-negative status correlates

with improved RFS and distant metastasis-free sur-

vival in the patients with TNBC [108, 116]. Meta-

analysis including 1,202 TNBC patients further con-

firmed correlation between the ctDNA positivity and

increased risk of recurrence and decreased OS in this

BC subgroup [117]. In another recent study involving

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patients with early-stage TNBC, ctDNA demonstrated

prognostic relevance, enabling identification of the

patients highly sensitive to therapy as well as those

at high risk of disease recurrence [118].

The key findings from the studies evaluating

ctDNA analysis for NACT efficacy and disease progno-

sis are summarized in Table  1. The accumulated body

of evidence has formed the basis for the development

of the commercial Signatera assay for MRD detection,

based on ctDNA analysis in blood plasma (Natera) of

the BC patients. This assay employs whole-exome –

and potentially whole-genome – sequencing of tumor

tissue, followed by ultra-deep sequencing of plasma

ctDNA and bioinformatic analysis. The method is al-

ready being used in clinical trials for ctDNA analy-

sis [65, 81].

CONCLUSION

Thus, persistence of ctDNA during neoadjuvant

therapy and after primary treatment is associated

with an increased risk of recurrence and an unfavor-

able prognosis. In recent years, post-neoadjuvant ther-

apy approaches have been supplemented with sever-

al highly effective agents. In particular, the CDK4/6

inhibitors are used in the hormone receptor-positive

HER2-negative BC, TD-M1 in the HER2-positive BC,

capecitabine and immunotherapy in TNBC, while

the PARP inhibitor olaparib has proven effective in

the BRCA-associated BC. However, despite their high

efficacy, these agents do not achieve complete cure

in all patients and are associated with considerable

toxicity. Quantitative and qualitative assessment of

ctDNA is important not only from the therapeutic

and prognostic perspective for optimizing treatment

strategies, but also from an economic standpoint,

since personalized therapy can markedly reduce

treatment costs. Detection of cfDNA after neoadju-

vant therapy and surgical treatment could help iden-

tify patient subgroups, based on the ctDNA biologi-

cal characteristics, for whom appropriate subsequent

systemic therapy is indicated.

The ctDNA analysis also enables regular molec-

ular monitoring throughout treatment and subse-

quent observation. Integration of ctDNA analysis, as

a form of liquid biopsy, into routine clinical practice

will make it possible to track real-time tumor re-

sponse, predict disease progression, and guide deci-

sions regarding treatment duration. Compared with

the conventional tissue biopsy, the ctDNA testing is

suggested to be a more convenient and minimally

invasive biomarker, and could become an additional

tool for personalized therapy optimization.

At present, investigation of ctDNA as a marker

of response to neoadjuvant therapy in the BC pa-

tients is limited only to clinical trials. Nevertheless,

the success of ctDNA-based approaches in colorectal

cancer – for which the biomarker has already been

incorporated into the ASCO (American Society of Clin-

ical Oncology) recommendations – and the growing

body of evidence from the ctDNA studies in BC sug-

gest that clinical implementation of ctDNA as a sensi-

tive predictive and prognostic BC biomarker is likely

in the near future.

Abbreviations

95% CI 95% confidence interval

BC breast cancer

cfDNA cell-free DNA

ctDNA circulating tumor DNA

DFS disease-free survival

dPCR digital PCR

HR hazard ratio, risk ratio

MAF mutant allele fraction

MRD minimal residual disease

NACT neoadjuvant chemotherapy

NGS next-generation sequencing

OS overall survival

RCB residual cancer burden

pCR pathologic complete response

TNBC triple-negative breast cancer

Contributions

G.T.S. supervised the work and performed review

of the final manuscript with subsequent approval;

T.M.Z. came up with the concept of the work, car-

ried out the literature search, prepared the manu-

script including structuration and editing; I.V.P. pro-

vided the section “Methods for detecting circulating

tumor DNA” and edited the manuscript; P.S.M. pro-

vided the section “Methods for detecting circulating

tumor DNA” as well as technical preparation of the

manuscript; S.V.Kh. provided the section “Potential

of ctDNA analysis for evaluating the efficacy of neo-

adjuvant drug therapy in breast cancer patients”

and participated in manuscript editing.

Funding

This study was financially supported by the Min-

istry of Health of the Russian Federation (project

no. 125050605836-5) and by the Ministry of Science

and Higher Education of the Russian Federation

(project no.122041400080-0).

Ethics approval and consent to participate

This work does not contain any studies involving hu-

man and animal subjects.

Conflict of interest

The authors of this work declare that they have

noconflicts of interest.

ZAVARYKINA et al.1496

BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025

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