Changes in the concentration of freely circulating mutant DNA and wild-type DNA of the H3F3А (K27M) gene in the blood and cerebrospinal fluid of children with diffuse midline gliomas during a course of radiation therapy

Olga S. Regentova; Vladimir K. Bozhenko; Elena A. Kudinova; Tatyana M. Kulinich; Elena L. Dzhikiya; Valeriy V. Kaminskiy; Fedor F. Antonenko; Roman A. Parkhomenko; Natalya I. Zelinskaya; Nelly Sidibe; Pavel V. Polushkin; Andrey I. Shevtsov; Maria A. Bliznichenko; Vladimir A. Solodkiy

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South Russian

Journal of Cancer..

Vol. 5

No. 3, 2024

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South Russian

Journal of Cancer..

Vol. 5

No. 3, 2024

South Russian Journal of Cancer. 2024. Vol. 5, No. 3. P. 64-75

https://doi.org/10.37748/2686-9039-2024-5-3-6

https://elibrary.ru/mzrjgd

ORIGINAL ARTICLE

Changes in the concentration of freely circulating mutant DNA and

wild-type DNA of the H3F3� (K27M) gene in the blood and

cerebrospinal fluid of children with diffuse midline gliomas during

a course of radiation therapy..

O. S. Regentova1 , V. K. Bozhenko1, E. A. Kudinova1, T. M. Kulinich1, E. L. Dzhikiya1, V. V. Kaminskiy1,

F. F. Antonenko1, R. A. Parkhomenko1,2, N. I. Zelinskaya1, N. Sidibe1, P. V. Polushkin1, A. I. Shevtsov1,

M. A. Bliznichenko1, V. A. Solodkiy1

1 Russian Scientific Center of Roentgenoradiology, Moscow, Russian Federation

2 RUDN University, Moscow, Russian Federation

olgagraudensh@mail.ru

ABSTRACT

Purpose of the study. To study the possibility of detecting freely circulating DNA of the H3F3A (K27M) gene in blood plasma and

cerebrospinal fluid in the lumbar spine in children with diffuse midline gliomas (DMG) during a course of radiation therapy (RT).

Materials and methods. Molecular genetic studies were carried out by digital PCR. 96 samples of lumbar cerebrospinal fluid

and 288 samples of peripheral blood plasma from 96 pediatric patients were analyzed. The concentration of circulating tumor

(ctDNA) mutant DNA and wild-type DNA of the H3F3A (K27M) gene was determined in the studied material against the

background of a course of RT. Lumbar cerebrospinal fluid sampling was performed once at the beginning of therapy, blood

sampling was

performed three times: The 1st

test

before the start

of RT, the 2nd against

the background of a total

dose 10�15

Gy, and the 3rd after the completion of the RT

course. Patients are divided into the following groups: patients with stabilization

of brain tumor

growth during early magnetic

resonance (MR) control 3 months after

completion of the course of RT;

patients

with disease progression during the same follow-up period who underwent radiation or chemoradiotherapy.

Results. When the disease stabilized after a RT

course during treatment, the concentration level of both the mutant variant

of

ctDNA and wild-type ctDNA significantly decreased in the third blood fraction. The absence of changes or an increase in the

concentration of mutant ctDNA and wild-type ctDNA of the H3F3A (K27M) gene by the end of the course of radiation therapy

was typical for patients with disease progression in the form of the appearance of metastatic foci in the central nervous

system or continued tumor growth. At the same time, the concentration of wild-type DNA of the H3F3A (K27M) gene in the

group of patients

with

progression

was

higher both

in

the

lumbar cerebrospinal

fluid and in

the

first

fraction

of blood plasma.

Connclusion. Determination of the concentration and dynamics of circulating tumor DNA of the mutant and wild type of the

H3F3A (K27M)

gene in blood plasma and lumbar cerebrospinal fluid in children with diffuse median gliomas

of the brain during

radiation therapy is promising from the point of view of predicting the effectiveness of therapy.

Keywords: glioma, diffuse median glioma, digital drip PCR, H3F3A, K27M, circulating tumor DNA (ctDNA)

For citation: Regentova O. S., Bozhenko V. K., Kudinova E. A., Kulinich T. M., Dzhikiya E. L., Kaminskiy V. V., Antonenko F. F., Parkhomenko R. A.,

Zelinskaya N. I., Sidibe N., Polushkin P. V., Shevtsov A. I., Bliznichenko M. A., Solodkiy V. A. Changes in the concentration of freely circulating mutant DNA and

wild-type DNA of the H3F3� (K27M) gene in the blood and cerebrospinal fluid of children with diffuse midline gliomas during a course of radiation therapy.

South Russian Journal of Cancer. 2024; 5(3):64-75. https://doi.org/10.37748/2686-9039-2024-5-3-6, https://elibrary.ru/mzrjgd

For correspondence: Olga S. Regentova � Cand. Sci. (Med.), MD, head of pediatric radiation oncology department with beds for oncology patients, Russian

Scientific Center of Roentgen Radiology, Moscow, Russian Federation

Address: 86 Profsoyuznaya Street, Moscow 117997, Russian Federation

E-mail: olgagraudensh@mail.ru

ORCID: https://orcid.org/0000-0002-0219-7260

SPIN: 9657-0598, AuthorID:1011228

Compliance with ethical standards: this research has been carried out in compliance with the ethical principles set forth by the World Medical Association

Declaration of Helsinki, 1964, ed. 2013. The study was discussed and approved at a meeting by the Scientific Council of the Russian Scientific Center of

Roentgenoradiology (Scientific Protocol No. 3/2022, 12/12/2022, Protocol No. 7). Informed consent was received from all the participants of the study

Funding: this work was not funded

Conflict of interest: the authors declare that there are no obvious and potential conflicts of interest associated with the publication of this article

The article was submitted 22.07.2024; approved after reviewing 19.08.2024; accepted for publication 25.08.2024

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Polushkin P. V., Shevtsov A. I., Bliznichenko M. A., Solodkiy V. A., 2024

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South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 64-75

Regentova O. S. , Bozhenko V. K., Kudinova E. A., Kulinich T. M., Dzhikiya E. L., Kaminskiy V. V., Antonenko F. F., Parkhomenko R. A., Zelinskaya N. I., Sidibe N.,

Polushkin

P.

V., Shevtsov

A.

I., Bliznichenko

M.

A., Solodkiy

V.

A.

Changes in the concentration of freely circulating mutant DNA and wild-type DNA of the

H3F3� (K27M) gene in the blood and cerebrospinal fluid of children with diffuse midline gliomas during a course of radiation therapy

INTRODUCTION

Over

the past 10 years, there has been a fundamental

paradigm shift in the field of diffuse midline

gliomas (DMG) diagnosis, where among the most

significant discoveries is the K27M mutation in the

H3F3A or HIST1H3B genes, which encode histone

variants H3, H3.3 and H3.1. The H3K27M mutation

gives odds in gliomagenesis a head start due to persistent

clonogenicity and aberrant differentiation

and determines the associated changes in histone

and DNA methylation

[1]. The

preservation

of proliferative

clonogenic states increases the likelihood

of acquiring additional mutations in nascent neomorphic

cells. In addition, aberrant differentiation

can change the organization of tissues and create

a microenvironment that promotes the development

of tumors. Both of them are potential consequences

of the H3K27M mutation, and may contribute to the

occurrence of DMG [1]. To date, the detection of the

K27M mutation in the H3F3A gene is recommended

in a number of foreign countries

to assess

the prognosis

of the disease and the choice of treatment tactics

[2�4]. The detection of a mutation in DMG is associated

with an extremely aggressive clinical course

and an unfavorable prognosis, [5�8] regardless of

histological examination data, therefore, when the

K27M mutation is detected in the H3F3A gene, the

tumor is classified as grade 4 malignancy [4, 6, 7].

When comparing adult and pediatric patients with

central nervous system (CNS) tumors, it was shown

that in adults, the K27M mutation occurs with the highest

frequency in high-grade gliomas (HGGs) of the

thalamus and spinal cord, and in children � with diffuse

median gliomas of the brain, while the frequency of the

K27M mutation in the H3F3A gene can reach 94

%

[6,

9, 10]. An important difference is that in children suffering

from DMG, the presence of the K27M mutation

is an extremely unfavorable prognostic factor, and for

supratentorial gliomas in adults, this gene change is

not

clinically significant. There

were

no significant

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differences

in survival and clinical course of the disease

for adult patients with and without the K27M mutation,

H3K27M may be present both in histologically verified

HGGs and in low-grade gliomas (LGGs) [9].

In children with HGG with the K27M mutation, they

have a more aggressive clinical course in comparison

with HGG of a different genetic nature. In this

regard, the identification of mutant forms has prog

nostic value and most studies are focused specifically

on the study of the mutant DNA of the H3F3A

gene in DMGs, especially among children [2]. At the

same time, there is practically no information on

the prognostic role of determining changes in the

concentration of wild-type DNA during treatment

and the ratio of concentrations of mutant DNA and

wild-type H3F3A gene

[11] among diffuse

midline

gliomas, although for tumors of other localizations,

an increase in the concentration of wild-type DNA is

a poor prognostic factor [12, 13].

In DMGs, due to the peculiarities of the anatomical

location of tumors,

it is difficult to

obtain histological

material using surgical intervention. Unfortunately,

the use of targeted stereotactic biopsy does not always

allow to obtain an adequate amount of mate

rial for histological and molecular analysis [14, 15].

When a diffuse tumor is biopsied, several samples

are taken from different points, which sometimes

does not allow to identify intra-tumor heterogeneity

for an accurate diagnosis

[16]. The use of a liquid biopsy

method aimed at identifying biological markers

by analyzing circulating tumor DNA (ctDNA) in blood

plasma and lumbar liquor samples makes it possible

to determine the molecular profile of a tumor without

using traumatic invasive techniques. Modern

approaches to monitoring the course of the disease

that meet international standards use radiographic

imaging � magnetic resonance imaging (MRI) to

determine how the tumor reacts to treatment. It is

worth noting that performing an MRI examination

is an expensive procedure, and often, in the case of

pediatric patients, requires the use of an anesthetic

aid, which is difficult to access in the regions. In the

case of DMG H3K27M, diffuse tumor growth and

radiation-induced edema complicate the interpretation

of images under dynamic observation. Studies

have shown that the levels of tumor biomarkers in

biological media, such as blood or cerebrospinal

fluid, correlate

with

the

course

of the

disease. Thus,

consistent

quantification

of these biomarkers

can

help identify disease progression in advance. The

diagnostic potential of using liquid biopsy in children

with DMG has not been fully disclosed, although re

search in this direction is actively underway [7, 17]. In

addition, the use of liquid biopsy in pediatric neurooncology

lags behind similar methods in adults, however,

these studies show that the technology has

significant potential [7, 17�19].

��-�� 2024. �. 5, � 3. �. 64-75

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H3F3� (K27M) � ��

The most accessible material for the liquid biopsy

method is blood plasma, and for many solid tumors,

the determination of ctDNA in plasma is an important

diagnostic method [6, 7]. However, in

DMG, the

bloodbrain

barrier (BBB) significantly restricts the flow

of

ctDNA into the blood [20], therefore, an alternative

source of ctDNA

is the lumbar

cerebrospinal fluid [7,

21]. In CNS tumors in children, molecular examination

of the lumbar

cerebrospinal fluid can also

be a significant

alternative to

morphological verification at high

risks or

inability to

obtain biopsy material [19].

To date, the most sensitive method for evaluating

ctDNA, which allows obtaining adequate results with

a small amount of

material,

iНе можете найти то, что вам нужно? Попробуйте сервис подбора литературы.

is the digital drip PCR

method [7].

In recent years,

the number

of

studies

conducted by this method has increased many times,

including in CNS tumors [7, 11].

An integrated approach in liquid biopsy studies,

especially in DMG, should include a combination of

the choice of research material and molecular analy

sis

methods

[6, 7]. In recent

years, the gold standard

has become the study of ctDNA in both plasma and

cerebrospinal

fluid, which

allows

us

to obtain

the

most accurate molecular data necessary for the diagnosis

and prognosis of the course of the disease.

In this work, we examined the ctDNA of the H3F3A

(K27M) gene both in blood plasma and in lumbar

cerebrospinal

fluid in

children

with

diffuse

midline

gliomas

during radiation therapy. Special attention was

paid not only to the detectability of mutant ctDNA,

but also to the ratio of the amount of mutant ctDNA

to wild-type ctDNA � variant allele fraction (VAF), the

change in H3.3K27M VAF over time ("delta VAF"), as

well as its correlation with various clinical parameters

[22]. In

our opinion, the study of patients

without

the H3.3K27M mutation is important, but poorly

studied. To date, we have not found information in

the available literature on changes in the concentration

of wild-type DNA of the H3F3A gene against the

background of radiation therapy in children, which

confirms

the

relevance

of our work and the need for

further development of molecular diagnostics and

personalized therapy of DMG.

MATERIALS AND METHODS

The study included 96 children with diffuse midline

gliomas of the brain who underwent radiation and

chemoradiotherapy at the Department of Russian

Scientific Center of Roentgenoradiology in the period

from 2022 to 2024. The study cohort consisted of

53 (55 %) boys and 43 (45 %) girls aged 18 months

to 18 years, the average age at the time of diagnosis

was 8 years. Clinical indicators included gender, age,

and the nature of disease progression � the appearance

of metastatic dropouts in the central nervous

system or continued tumor growth, but they had no

significant

differences

in

the

study groups

and associations

with DNA concentrations in blood plasma

and lumbar liquor (p >

0.05).

When conducting an

instrumental examination of patients based on the

results of MRI of the brain natively and with contrast

enhancement before the start of therapy, it was found

that in all patients the tumors had diffuse growth and

median location. Histological examination of tumors

in 18 cases showed that HGG prevailed mainly, 6 of

them had a K27M mutation

in

the

H3F3A gene. The

assessment of groups with continued growth and stabilization

of the disease was carried out on the basis

of MRI data of the brain without and with contrast

enhancement (CE), performed within 3�4 months

after completion of the course of RT.

The scheme of radiation therapy

Radiation therapy was performed using Varian

Clinac 2100 linear accelerators, True Beam, and the

Varian Eclipse dosimetric calculation system. During

therapy, the traditional version of fractionation of

the

dose

of 1.8�2

Gy was

used, with

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a

total

focal

dose of up to 54 Gy. In the presence of pronounced

perifocal edema and symptoms of developing intracranial

hypertension, treatment began in the mode

of multifractionation in single doses of 1.0�1.1 Gy

2

times

a day with an interval

between

fractions

of 4�6

hours with a gradual transition to the usual

fractionation mode as the condition stabilizes, but

with

a correction

of the

total

dose

over the

period

of multifractionation in the direction of its increase

equivalent to 54

Gy.

In patients with a histologically

confirmed diagnosis of

HGG,

a course of

RT

was

performed with

parallel

radio modification

with temozolomide,

75 mg/m2, daily against the background

of the entire course of RT.

Obtaining research material

During the study, we received samples of periph

eral

blood plasma and lumbar cerebrospinal

fluid

from 97 patients. Samples of lumbar cerebrospinal

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 64-75

Regentova O. S. , Bozhenko V. K., Kudinova E. A., Kulinich T. M., Dzhikiya E. L., Kaminskiy V. V., Antonenko F. F., Parkhomenko R. A., Zelinskaya N. I., Sidibe N.,

Polushkin

P.

V., Shevtsov

A.

I., Bliznichenko

M.

A., Solodkiy

V.

A.

Changes in the concentration of freely circulating mutant DNA and wild-type DNA of the

H3F3� (K27M) gene in the blood and cerebrospinal fluid of children with diffuse midline gliomas during a course of radiation therapy

fluid were

taken

once

against

the

background of

radiation therapy. Blood plasma was taken at three

stages: before the start of therapy,

during radiation

therapy and after completion of the course of radiation

therapy.

Isolation of circulating DNA from lumbar

cerebrospinal fluid

To isolate ctDNA from the lumbar liquor, we used

Sileks kits, which are based on the use of SileksMagNA-

Direct particles (particles for selective binding of

nucleic acids). The extraction procedure was carried

out according to the protocol provided by the manufacturer.

The collection of cerebrospinal fluid and the

beginning of the procedure for isolation of circulating

tumor DNA did not exceed 30 minutes. The lumbar

liquor was centrifuged at 1,500 revolutions per minute

for 5

minutes, and

a superabsorbent

fraction

with

a volume of 0.7 to 2

ml was used to isolate ctDNA.

In our work, mutant ctDNA of the H3F3A gene was

isolated from 96

cerebrospinal fluid samples in 33,

and wild-type ctDNA of the H3F3A gene was isolated

in all 96 cerebrospinal fluid samples (Fig. 1).

Isolation of circulating DNA from blood plasma

Plasma preparation. Plasma was separated im

mediately after receiving a blood sample. Sileks

kits

based on SileksMagNA-Direct particles were used

to isolate circulating DNA from blood plasma. The

isolation procedure was carried out according to

the manufacturer's protocol. Of the 288 peripheral

blood plasma samples obtained, mutant ctDNA of

the H3F3A gene was isolated in 29, wild-type ctD-

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NA of the H3F3A gene was isolated in all studied

samples.

Determination of the K27M mutation in the

H3F3A gene by digital droplet PCR (ddPCR)

Highly sensitive screening of the H3F3A (K27M)

mutation using Digital Droplet PCR (ddPCR) technology

using the H3F3A (K28M) Screening Kit (Bio-Rad,

USA) and the QX100 Droplet Digital PCR System (Bio-

Rad, USA) was used.

For ddPCR formulation, BioRad reagents were

used according to the research protocol. The DNA

probes

used to detect the amplification products

of

the studied and normalizing genes were labeled FAM

and HEX. The PCR mixture was placed in a droplet

generator, where a water-oil

emulsion was

created

from 20 .l of the sample in which the amount of

DNA under study was to be determined, and up to

20,000 drops of 1 nl were formed in each tube. In

this case, the genetic material is randomly distributed

into droplets: both target

DNA and background

DNA fall into them. The process of distributing the

target DNA by droplets is purely random and obeys

the law of distribution of small Poisson numbers.

Before dividing the sample into drops, it is not nec-

Cerebrospinal fluid

draw (30 min)

ctDNA isolation

(2 hours)

ctDNA

concentration

assessment

(2�2.5 hours)

Digital drop

PCR (2 hours)

Data analysis

(30 min)

Fig. 1. Isolation of circulating tumor DNA by drip PCR from lumbar cerebrospinal fluid

Radiation

therapy course

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Brain

MRI

No.1

Brain

MRI

No. 2

Brain

MRI

No. 3

Blood draw

No. 1

Cerebrospinal

fluid

Blood draw

No. 2

(10�15 Gy)

Blood draw

No. 3

(50�54 Gy)

weeks

Fig. 2. Study design

��-�� 2024. �. 5, � 3. �. 64-75

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H3F3� (K27M) � ��

essary to dilute it to a concentration so that each

drop contains either 0

or 1

copy of the target DNA:

when analyzing the results, situations are taken into

account when there is more than one copy of the

target in one drop. According to the Poisson distribution,

either one matrix chain gets into the drop,

or none gets into it. Samples were transferred from

the droplet generator to the applicator. The ampli

fication was carried out in "real time" mode. After

amplification, the tablet was placed in a "BIO-RAD QX

100TM DROPLET READER" device, where the signal

from the fluorescent labels was read. In a drop with

a matrix, amplification is many times more efficient

than with other types of PCR, which is due to the

presence of all components of the PCR mixture in

the nanoscale.

During amplification,

enzymatic

cleavage

of TaqMan probes occurs, as a result of which

the fluorescence efficiency of the droplet increases

many times. The product accumulated during the

amplification is detected in each drop separately, at

a

rate

of 1500

drops/s. Based

on

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the

ratio of the

total

number of microdrops and the number of microdrops

in which the fluorescence level exceeds the background,

the reader calculates the absolute amount

of DNA in one microliter of the sample. The results

were recorded in the "Quanta Self 16" program.

The database of clinical cases was formed using

electronic databases of Microsoft Excel tables. Statistical

processing was carried out using the SPSS

software for Windows, version 26.0 (SPSS, Chicago,

Illinois, USA) and Statistica, version 13.

The normality of the sample distribution was

checked using the Kolmogorov-Smirnov criterion.

The reliability of the differences was determined using

the Mann-Whitney criterion. The exact one-sided

Fisher criterion was used to evaluate qualitative

features. The results of comparing quantitative data

were considered statistically significant at p < 0.05.

STUDY RESULTS AND DISCUSSION

1. Mutation analysis

In recent years, scientists have focused on

H3.3K27M mutant DMG

[19],

since the H3F3A

(K27M) mutation is more common than G34V/R

mutations in children with highly malignant diffuse

astrocytomas [23�25]. This mutation is considered

as a potential diagnostic marker for the identification

of these tumors, similar to the use of IDH1/2 mutations

for the diagnosis of diffuse gliomas in adults.

All H3K27� mutations described in DMG in most

cases have the same epigenomic consequences

for the PRC2 complex (PRC2 � Polycomb repressive

complex 2 � conservative protein complex) as

a whole [26, 27], despite the different functions and

genomic distribution of many variants. It is important

to note that the life expectancy of patients largely

depends on the type of histone where the K27M mutation

is present. Back in 2014, Wu et al. It was found

that

patients

with a mutation in histone H3.1

respond

better to radiation

therapy, have

a less

aggressive

course and are less likely to have metastases [28].

Therefore, the assessment of the type of histone

mutation

can

be

used as

a predictive

stratification

factor in future prospective studies [4].

In this study, variants of allelic fractions (VAF)

H3.3K27M were evaluated � the ratio of the concentration

of freely circulating mutant DNA to the

wild-type DNA of the H3F3A (K27M) gene in samples

of lumbar liquor, as well as in blood plasma. At the

same time, blood plasma samples were taken before

the start of radiation therapy (sample 1), during

radiation therapy

(sample 2) and after

the end of

radiation therapy (sample 3)

(Fig. 2). A total of 8

parameters were evaluated. Thus, we determined

the concentration and assessed the dynamics of

changes, against the background of radiation therapy,

not only mutant DNA, but also wild-type DNA of

the H3F3A (K27M) gene. It is necessary to understand

the molecular features of the development of

DMG from various angles in order to make it possible

to improve and create new therapeutic strategies.

2. Correlation status of the H3F3A (K27M)

gene

and clinical and pathological characteristics

Previous studies have shown that K27M-mutant

DSGs are associated with significantly shorter-term

survival [28]. Moreover, in a multivariate analysis that

also took into account the effect of treatment, the

type of histone H3 mutation was a more accurate

predictor of survival duration than the assessment

of the clinical and radiological risk of DMG [11, 30].

When analyzing the data we obtained, we established

the presence of a significant correlation between

progression and mutant ctDNA of the H3F3A

(K27M) gene obtained from the lumbar liquor fraction

(Table 1).

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 64-75

Regentova O. S. , Bozhenko V. K., Kudinova E. A., Kulinich T. M., Dzhikiya E. L., Kaminskiy V. V., Antonenko F. F., Parkhomenko R. A., Zelinskaya N. I., Sidibe N.,

Polushkin

P.

V., Shevtsov

A.

I., Bliznichenko

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M.

A., Solodkiy

V.

A.

Changes in the concentration of freely circulating mutant DNA and wild-type DNA of the

H3F3� (K27M) gene in the blood and cerebrospinal fluid of children with diffuse midline gliomas during a course of radiation therapy

In a recent preclinical study, Grasso et al., investigating

the

efficacy of panobinostat

in

DMG, established

its effectiveness against cells containing both

mutant DNA and wild-type DNA for the H3F3A (K27M)

gene in vitro, although cells with the H3K27M mutation

developed resistance to panobinostat within

a few

weeks after exposure to low

doses of the drug.

It

is

worth noting that

panobinostat

treatment

significantly

prolongs the survival of mice with tumors without

mutation in the H3F3A gene

[31]. These

results

led to the initiation of NCT02717455 (clinaltrials.gov),

a clinical

trial

of panobinostat

(Phase

I�LBH589)

conducted by the Pediatric Brain Tumor Consortium

(PBTC) for the treatment of children with recurrent

or progressive HGG.

In this regard, we paid special attention to the

change in the concentration of wild-type DNA

for the H3F3A (K27M) gene, suggesting that this

phenomenon may become one of the effective

prognostic markers of tumor progression and

the effectiveness of therapy. The analysis of the

mutual correlations of the concentrations of wild

and mutant DNA of this gene in different fractions

of blood

plasma

showed

a

number of interesting

dependencies, for example, the concentration of

mutant DNA K27M in the lumbar liquor had highly

reliable correlations with the concentration of the

same

mutant

DNA in

the first

and second fractions

of blood plasma (Table 2).

There was also a high correlation of the concentration

of mutant DNA of the H3F3A (K27M) gene in

the second fraction of blood with cerebrospinal fluid,

with the first fraction and the third, as well as with

the concentration level of the wild-type gene in the

third blood sample (Table 3).

Evaluation of the results of the study of the mutation

status in blood plasma and lumbar cerebrospinal

fluid of patients using ddPCR showed high

Table 1. Correlation between mutant DNA of the H3F3A gene (K27M) and disease progression

Value

Correlations are significant when p < 0.05Progression

K27Mmut in

0.271440

cerebrospinal fluid

Table 2. Correlation between the mutant DNA of the H3F3A gene (K27M) in cerebrospinal fluid and the first two blood

samples on the background of RT

Value

Correlations are significant when p < 0.05mut(K27M cerebrospinal fluid)

mut(K27M draw 1) 0.555019

mut(K27M draw 2) 0.384082

Table

3. Correlation between the

mutant

DNA

of the

H3F3A

(K27M)

gene

in the

second blood sample

and cerebrospinal fluid

draws,

the

first

and third blood samples against

the

background of RT

mutant

variant

of the

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gene,

as well as the

wild-type

gene

in the

third sample

Value

Correlations are significant when p < 0.05mut(K27M draw 2)

mut(K27M cerebrospinal fluid) 0.384082

mut(K27M draw 1) 0.211165

mut(K27M draw 3) 0.360417

wt(K27M draw 3) 0.390472

��-�� 2024. �. 5, � 3. �. 64-75

�� . �. , �� . �., �� . �., �� . �., �� . �., �� . �., �� . �., �� . �., �� . �.,

�� ., �� . �., �� . �., �� . �. ��

H3F3� (K27M) � ��

informativeness of both blood plasma and lumbar

cerebrospinal

fluid, which

is

confirmed

in

studies

where ctDNA are found in the blood and lumbar

cerebrospinal

fluid in

the

blood and lumbar cerebrospinal

fluid,

which are more sensitive and can

reflect various types of

mutations in glioma cells

[11, 32, 33]. At

the

same

time, it

was

previously

stated that the presence of BBB means that the

cerebrospinal fluid can provide a more detailed

characteristic of the tumor than blood plasma, and

contains certain biomarkers that are unlikely to be

detected in plasma [34]. However, in our study, we

found highly reliable correlations of the studied DNA

in blood plasma and lumbar

cerebrospinal fluid. At

the same time, the concentration of mutant DNA of

the H3F3A (K27M) gene in the lumbar cerebrospinal

fluid also had highly significant

correlations

with

the concentration of wild-type DNA of the H3F3A

(K27M) gene in the first fraction of blood plasma.

Thus, the assessment of VAF in blood plasma has

a high prognostic significance in assessing the effectiveness

of therapy.

3. Analysis of the dynamics of changes in the

concentration of freely circulating DNA of the

H3F3A (K27M) gene: wild and mutant type in

the group with and without progression on the

background of radiation therapy

Radiation therapy, as a standard strategy for the

treatment of DMG, improves the quality of life of

patients, after which 70�80 % of patients experience

temporary relief of symptoms, as well as increased

survival [24, 35, 36]. However, within 4�9

months,

the disease progresses again. Ionizing radiation

(AI) used in RT can inhibit tumor growth by inducing

DNA damage directly or through reactive oxygen

species (ROS) [37].

So

far,

there are no

predictors for

assessing the early effect of RT during treatment for

children with DMG, only MR control brain monitoring

after 1.5�3

months will allow to exclude or confirm

the progression of the disease. Accordingly, it is impossible

to personalize anti-relapse therapy in time.

During the ongoing study, the concentration level

and VAF of mutant and wild ctDNA of the H3F3A

(K27M) gene were studied for groups with stabiliza-

VAF and delta-VAF DURING radiation therapy of VAF and delta-VAF DURING radiation therapy of

mutant ctDNA of the H3F3A gene mutant ctDNA of the H3F34 gene

Average; Interval: SE

0,11

0,10

0,09

0,08

0,07

0,06

0,05

0,04

0,03

0,02

0,01

28

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26

24

22

20

18

16

14

12

10

8

10

MRI stability

mut(K27M draw 1)

mut(K27M draw 2)

mut(K27M draw 3)

Fig. 3. VAF and delta-VAF against the background of radiation therapy

of mutant ctDNA of the H3F3A (K27M) gene in blood plasma in a

group of patients with continued growth (group 1) and stabilization

(group 0), depending on the data of an MR brain study 3 months after

completion of treatment

MRI stability

wt(K27M cerebrospinal fluid) wt(K27M draw 1)

wt(K27M draw 2) wt(K27M draw 3)

Fig. 4. VAF and delta VAF against the background of radiation therapy

of wild-type ctDNA of the H3F3A (K27M) gene in cerebrospinal fluid

and blood plasma in a group of patients with continued growth (group

1)and stabilization (group 0), depending on the data of an MRI study

of the brain 3 months after completion of treatment

South Russian Journal of Cancer 2024. Vol. 5, No. 3. P. 64-75

Regentova O. S. , Bozhenko V. K., Kudinova E. A., Kulinich T. M., Dzhikiya E. L., Kaminskiy V. V., Antonenko F. F., Parkhomenko R. A., Zelinskaya N. I., Sidibe N.,

Polushkin

P.

V., Shevtsov

A.

I., Bliznichenko

M.

A., Solodkiy

V.

A.

Changes in the concentration of freely circulating mutant DNA and wild-type DNA of the

H3F3� (K27M) gene in the blood and cerebrospinal fluid of children with diffuse midline gliomas during a course of radiation therapy

tion and progression against the background of RT.

In the group of patients with stabilization, the concentration

of mutant DNA of the H3F3A (K27M) gene

in the blood was lower in three blood plasma samples

compared with the concentration of mutant ctD-

NA in the group with early progression. VAF did not

tend

to significantly increase

against

the

background

of RT with stabilization of the disease. Whereas in

children with early progression, the concentration of

VAF in three plasma samples was 2�3 times higher

compared to the group with a favorable prognosis,

and the delta of VAF increased 2 times with each

subsequent measurement against the background

of RT (Fig. 3).

When analyzing the VAF and delta VAF wild-type

ctDNA of the H3F3A (K27M) gene in cerebrospinal

fluid and blood plasma, the following patterns were

revealed in the group of patients with progression

(group 1)

and

without

progression

(group

0), depending

on the data of the MR brain examination

3 months after completion. The concentration level

of wild-type ctDNA of the H3F3A (K27M) gene in the

cerebrospinal

fluid at

the

beginning of the

course

of

RT was identical in both groups of patients. At the

same time, in the group of patients with early progression

(Fig. 4), the wild-type VAF ctDNA had the

following pattern: a decrease in the RT

process in the

second plasma sample and an increase in the third

sample against the background of completion of

the RT course. Whereas in the group of patients with

stabilization of the disease, the VAF of the wild-type

ctDNA of the H3F3A (K27M) gene differed, namely:

an increase in concentration in the second plasma

sample and a significant decrease in it at the end of

the course of RT. The delta of VAF in blood plasma,

first of all,

indicates the presence of a significant dependence

between these indicators and the course

of the disease.

The data obtained prove the diagnostic value of

the wild-type ctDNA of the H3F3A (K27M) gene, allowing

us

to significantly expand the

possibilities

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of

molecular diagnostics and monitoring the effectiveness

of treatment of DMG. In addition, based on the

analysis of delta VAF during treatment, it is possible

to predict early tumor recurrence after radiation therapy

and timely initiation of personalized therapy.

CONCLUSION

1. Studies of VAF in blood plasma and lumbar

cerebrospinal

fluid of children

with tumors

diffuse

midline gliomas before the start of radiation therapy

are comparable and have equal diagnostic value.

2. High plasma concentrations of wild-type DNA

of the H3F3A (K27M) gene correlate with early progression,

which also affects survival rates.

3. Dynamic control of the DNA concentration of

both mutant and wild-type H3F3A (K27M) gene in

blood plasma and lumbar cerebrospinal fluid in children

with diffuse midline gliomas during radiation

therapy can be used to predict the effectiveness of

RT. In addition, based on the analysis of the dynamics

of the concentration levels of the wild-type DNA

of the H3F3A (K27M) gene during treatment, it is

possible to predict early tumor recurrence after radiation

therapy.

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Information about authors:

Olga S. Regentova � Cand. Sci. (Med.), MD, head of pediatric radiation oncology

department with beds

for oncology

patients, Russian Scientific

Center of Roentgen Radiology, Moscow, Russian Federation

ORCID: https://orcid.org/0000-0002-0219-7260, SPIN: 9657-0598, AuthorID: 1011228

Vladimir K. Bozhenko � MD, Professor, Head of the Department of Molecular Biology

and Experimental Therapy

of Tumors, Russian Scientific Center

of Roentgen Radiology, Moscow, Russian Federation

ORCID: https://orcid.org/0000-0001-8351-8152, SPIN: 8380-6617, AuthorID: 97295

Elena A. Kudinova � Dr. Sci. (Med.), MD, Head of the Clinical Diagnostic Laboratory, Russian Scientific Center of Roentgen Radiology, Moscow,

Russian Federation

ORCID: https://orcid.org/0000-0002-5530-0591, SPIN: 8380-6617, AuthorID: 97295

Tatyana M. Kulinich � Cand.Sc. (Med), MD, Head of the Laboratory

of Immunology

and Oncocytology, Russian Scientific Center of Roentgen

Radiology, Moscow, Russian Federation

ORCID: https://orcid.org/0000-0003-2331-5753, SPIN: 4697-5143, AuthorID: 171802

��-�� 2024. �. 5, � 3. �. 64-75

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Elena L. Dzhikiya � Cand. Sci. (Biol.), Researcher at the Laboratory

of Immunology, Oncocytology

and Cell Technologies

in Oncology

of the Research

Department �f Molecular Biology and Experimental Tumor Therapy, Russian Scientific Center of Roentgen Radiology, Moscow, Russian Federation

ORCID: https://orcid.org/0000-0001-8369-2011, SPIN: 1423-4712, AuthorID: 146408

Valeriy V. Kaminskiy � Junior Researcher of the Laboratory of Cell and Gene Therapy, Russian Scientific Center of Roentgenoradiology, Moscow,

Russian Federation

ORCID: https://orcid.org/0000-0001-5702-6090, SPIN: 8709-6269, AuthorID: 1028900

Fedor F. Antonenko � Dr. Sci. (Med.), MD, Professor, corresponding member of RAS, Head of the Laboratory

of Radiation Therapy

and complex

methods of cancer treatment, Russian Scientific Center of Roentgen Radiology, Moscow, Russian Federation

ORCID: https://orcid.org/0000-0001-5900-6755, SPIN: 6582-8081, AuthorID: 261007

Roman A. Parkhomenko � Dr. Sci. (Med.), MD, leading researcher at the Laboratory

of Radiation Therapy

and complex methods

of cancer treatment,

Russian Scientific Center of Roentgen Radiology, Moscow, Russian Federation; Professor of the Department of Oncology

and Radiology, RUDN

Medical Institute, Moscow, Russian Federation

ORCID: https://orcid.org/0000-0001-9249-9272, SPIN: 9902-4244, AuthorID: 702112

Natalya I. Zelinskaya � Cand. Sci. (Med.), MD, senior researcher of the Laboratory

of Radiation Therapy

and complex methods of cancer treatment,

Russian Scientific Center of Roentgen Radiology, Moscow, Russian Federation

ORCID: https://orcid.org/0009-0000-5380-2056, SPIN: 4092-4845, AuthorID: 123005

Nelly

Sidibe � Cand. Sci. (Med.), MD, radiation oncologist of pediatric radiation oncology

department with beds for oncology

patients, Russian

Scientific Center of Roentgen Radiology, Moscow, Russian Federation

ORCID: https://orcid.org/0000-0002-5556-0166, SPIN: 3660-6207, AuthorID: 1108540

Pavel V. Polushkin � Cand. Sci. (Med.), MD, researcher of the Laboratory

of Radiation Therapy

and complex methods

of cancer treatment, radiation

oncologist of pediatric radiation oncology department with beds for oncology patients, Russian Scientific Center of Roentgen Radiology, Moscow,

Russian Federation

ORCID: https://orcid.org/0000-0001-6661-0280, SPIN: 7600-7304, AuthorID: 1099115

Andrey

I. Shevtsov � Cand. Sci. (Med.), MD, radiation oncologist of pediatric radiation oncology

department with beds for oncology

patients,

Russian Scientific Center of Roentgen Radiology, Moscow, Russian Federation

ORCID: https://orcid.org/0000-0002-4539-5187, SPIN: 5605-6768, AuthorID: 996411

Maria A. Bliznichenko � MD, clinical resident of pediatric radiation oncology department with beds for oncology patients, Russian Scientific Center

of Roentgen Radiology, Moscow, Russian Federation

ORCID: https://orcid.org/0009-0007-4300-5759

Vladimir A. Solodkiy

� Dr. Sci. (Med.), MD, Professor, Academician of RAS, Director, Russian Scientific Center of Roentgen Radiology, Moscow,

Russian Federation

ORCID: https://orcid.org/0000-0002-1641-6452, SPIN: 9556-6556, AuthorID: 440543

Contribution of the authors:

Regentova O. S. � development of research design, review of publications on the topic of the article, interpretation of the results, final approval of

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the published version of the manuscript, writing the text of the manuscript;

Bozhenko V. K. � development of the research design, analysis of the data obtained, writing the text of the manuscript, interpretation of the results;

Kudinova E. A. � development of the research design, analysis of the data obtained, writing the text of the manuscript, interpretation of the results;

Kulinich T. M. � development of the research design, analysis of the data obtained, writing the text of the manuscript, interpretation of the results;

Dzhikiya E. L. � development of the research design, analysis of the data obtained, writing the text of the manuscript, interpretation of the results;

Kaminskiy V. V. � review of publications on the topic of the article, a set of clinical material, interpretation of the results;

Antonenko F. F. � review of publications on the topic of the article;

Parkhomenko R. A. � research design development;

Zelinskaya N. I. � review of publications on the topic of the article;

Sidibe N. � review of publications on the topic of the article, technical editing;

Polushkin P. V. � review of publications on the topic of the article, technical editing;

Shevtsov A. I. � review of publications on the topic of the article;

Bliznichenko M. A. � review of publications on the topic of the article;

Solodkiy V. A. � development of the research design, final approval of the published version of the manuscript.

Аннотация научной статьи по клинической медицине, автор научной работы — Olga S. Regentova, Vladimir K. Bozhenko, Elena A. Kudinova, Tatyana M. Kulinich, Elena L. Dzhikiya

Похожие темы научных работ по клинической медицине , автор научной работы — Olga S. Regentova, Vladimir K. Bozhenko, Elena A. Kudinova, Tatyana M. Kulinich, Elena L. Dzhikiya

Текст научной работы на тему «Changes in the concentration of freely circulating mutant DNA and wild-type DNA of the H3F3А (K27M) gene in the blood and cerebrospinal fluid of children with diffuse midline gliomas during a course of radiation therapy»