WO2017029451A1

WO2017029451A1 - Method for predicting and evaluating the excess of dose due to iodinated contrast products injected during radiodiagnostic examinations

Info

Publication number: WO2017029451A1
Application number: PCT/FR2016/052084
Authority: WO
Inventors: Nicolas FORAY; Adeline GRANZOTTO; Clément DEVIC; Mélanie FERLAZZO; Larry BODGI; Sandrine PERREIRA
Original assignee: Neolys Diagnostics; Institut National de la Santé et de la Recherche Médicale; Centre Leon Berard; Centre National De La Recherche Scientifique; Universite Claude Bernard Lyon 1
Priority date: 2015-08-19
Filing date: 2016-08-16
Publication date: 2017-02-23
Also published as: EP3338091A1; FR3040176A1; FR3040180A1

Abstract

A process for evaluating the additional DNA-breaking effect of an iodinated contrast agent injected into an individual in the context of a medical imaging examination using X‑rays, on a tissue taken from said individual, in which process: cells of a tissue to be evaluated which has been sampled (preferably by biopsy) from an individual are supplied; said cells are dispersed and/or amplified so as to obtain a cell sample; said cell sample is brought into contact with a test medium, said test medium comprising said iodinated contrast agent and/or iodide ions; said cell sample in contact with said test medium is subjected to a determined absorbed dose Dtherap OR Dscan of ionizing radiation, in the knowledge that: o Dscan represents the dose of X‑rays used for a medical imaging examination, o Dtherap represents the dose of ionizing radiation administered during a radiotherapy session which follows a medical imaging examination using X‑rays; the number of double-strand breaks of the DNA, and/or a biomarker representing this number, and/or the number of micronuclei, is determined on the cell sample.

Description

Predictive and evaluation method of excess dose due to iodinated contrast media injected during radiodiagnostic examinations

Technical field of the invention

The invention relates to the field of medical radiobiology, and more particularly to the field of radiobiological laboratory methods. The present invention relates to a novel predictive method of cellular and clinical radiosensitivity in the field of radiodiagnostics involving iodinated contrast agents and which is based on the determination and cross-checking of several cellular and enzymatic parameters and criteria. The invention also relates to a novel method for evaluating the combined effect of injecting iodinated contrast material and X-radiation administered in a radiodiagnostic examination.

State of the art

Radiodiagnosis represents the greatest single cause of exposure to ionizing radiation and is one of the greatest sources of radiation-induced cancer risk (according to a report "Biological Effects of Low Radiation Doses", Vol I Annex G published in 2000 by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR). Generally consisting of exposure to a low dose of x-rays ranging from 2 to 40 mSv for mammograms, scans or angiography and of the order of μ5ν for radiology, radiodiagnostic examinations sometimes require an increase in contrast to to better visualize the organ to be studied (Perez, Devic et al., 2015). Since the 1920s, iodine (administered in the form of iodine compounds) remains the best compromise to increase the low-energy X-ray contrast between sufficiently heavy elements and easy-to-produce, non-toxic, easily removable chemicals. (see Bae, "Intraveneous contrasts medium administration and scanning timing at CT: considerations and approaches," Radiology 256 (1), p.32-61 (2010)).

Currently available iodinated contrast agents can be divided into four classes: 1) ionic monomers (the oldest, developed in the 1950s); 2) nonionic monomers, 3) ionic dimers; 4) non-ionic dimers (the most recent ones). The basic structure of these iodinated contrast agents is a benzene ring, on which three iodine atoms are attached. Contrast agents are injected before and during treatment and accumulate mainly in tumors, more vascularized. Iodine diffuses easily into the intercellular space and there is a significant natural gradient of concentration between the pathological zone and the healthy zone. The concentration of iodine in healthy tissues remains low (see the aforementioned article by Bae). This concentration gradient results in a gradient in the sections effective absorption. Radiating with x-ray diagnostic radiology (120-140 kV) with a mean energy of about 65 keV implies a strengthening of the ionizing radiation dose deposition by the photoelectric effect in the vicinity of the iodine atoms of the contrast agent.

By the 1970s, Norman et al. ("Cytogenetic effects of contrast media and triiodobenzoic derivatives in human lymphocytes", Radiology 129 (1), p.19-203 (1978)) observed chromosomal breaks and aberrations in circulating lymphocytes of patients undergoing CT scans. iodinated contrast media. Such observations multiplied without a true mechanistic explanation.

In 2005, Joubert et al. showed that the irradiation by scanner emits a maximum of energy enter 50 and 60 keV which corresponds to the maximum of energy absorption of the iodine in the water ("Irradiation in the presence of iodinated contrast agent results in radiozensitization of endothelial cells: Consequences for coped tomography therapy ", Int J Radiation Oncology Biol Phys 62 (5), p.1486-1496 (2005)). This publication shows that such energy causes the radiolysis of iodinated contrast media which results in the production of ions that turn into sodium or potassium iodide. Such molecules, unlike iodinated contrast media molecules, can enter the cells and bind to the DNA. At this stage, double-strand breaks in DNA that are induced by irradiation will have limited or faulty repair due to the presence of iodides in the DNA by inhibition of DNA-PK and ATM kinases that regulate repair. and the signaling of DNA damage. It is also known that the question of tissue sensitivity to ionizing radiation is inseparable from that of DNA damage repair mechanisms. Indeed, at the cellular level, ionizing radiation can break certain types of chemical bonds by generating free radicals (in particular by peroxidation) and other reactive species that cause DNA damage. Damage to DNA by endogenous or exogenous aggressions (such as ionizing radiation and free radicals) can lead to different types of DNA damage depending in particular on the deposited energy: base damage, single-strand breaks and double-strand breaks (DSBs). Unrepaired CBD is associated with cell death, toxicity and more specifically radiosensitivity. Badly repaired CBD is associated with genomic instability, mutagenicity, and susceptibility to cancer. The body has specific repair systems for each type of DNA damage. In the case of CBD, mammals have two main modes of repair: repair by suture (ligation of strands) and repair by recombination (insertion of a homologous or non-homologous strand).

In 2013, Bodgi et al. have proposed a formula that can model the kinetics of recognition and repair of CDB in DNA (Bodgi et al, "A single formula to describe radiation-induced protein relocalization: towards a mathematical definition of individual radiosensitivity", J Theor Biol , vol.333, p135-145 (2013)):

1 1

N _pH 2Axtt, D) - _Therapy ID (l - _{1 + x + h} ^ _{t _ _¾) )

With:

I: DNA-induced CDB number equivalent to 40 CBD per Gy for X-rays

Dthérap ^'■ the irradiation dose b _rec: inverse time at which 50% of CBD are recognized min ^"1 _boundary representation: inverse time at which 50% of recognized CBD are repaired ^{min" 1} t _0: average gap between CBD recognition and repair.

It is known that the sensitivity of tissues to ionizing radiation is very variable from one organ to another and from one individual to another; the idea of "intrinsic radiosensitivity" was conceptualized by Fertil and Malaise ("Inherent cellular radiosensitivity as a basic concept for human tumor radiotherapy", Int.J. Radiation Oncology Biol.Ph. 7, p.621 - 629 ( 1981), "Intrinsic radiosensitivity of human cells", Int.J. Radiation Oncology Biol Physics 11, p.1699-1707 (1985). Thus, the various studies on the therapeutic effects and side effects of radiotherapy have shown that there are individuals with particularly high radioresistance, and individuals who show a radiosensitivity that can be a side effect. recognized clinically but without consequences until a lethal effect. Even apart from some rare cases of extreme radiosensitivity, whose genetic origin seems to be proven, it is thought that radiosensitivity generally results from a genetic predisposition: it is therefore specific to an individual. What is true for radiosensitivity is also true for susceptibility to cancer, and more particularly to radiation-induced cancer. Thus, any excess dose increases both the risk of radiosensitivity tissue reaction and the risk of radiation-induced cancer. However, at low doses used in radiodiagnosis, radiation-induced tissue reactions (burns) are not observed: only the excess risk of cancer due to overdose is to be considered. It would therefore be desirable to have a predictive test method to determine the risk and excess dose due to radiolysis of the iodinated contrast agent caused by radiodiagnostic exposure.

Despite this broad state of the art, the applicant finds that the patents described above do not describe a method for quantifying the excess dose and risk associated with the use of iodinated contrast products in radiodiagnostic examination. The problem of providing a predictive method of individual radiosensitivity therefore remains without an operational solution. The present invention aims at providing a novel predictive method of individual tissue and clinical radiosensitivity following radiodiagnostic examination involving iodinated contrast agents.

Objects of the invention

The inventors have found, and this is the first starting point of the present invention, that the emission lines of the X-ray spectrum produced by a standard tube of radiodiagnostic devices, which correspond to K lines of tungsten (K line). _Q at 59.3 keV, line K _p at 67.23 keV) composing the anode of the tubes, and which are superimposed on the continuous polychromatic spectrum (spreading, according to the acceleration voltage, of approximately 20 keV up to typically at about 80, 100 or 120 keV), are very close to the maximum energy absorption of the iodine atoms in water (maximum ratio between the relative coefficients of mass energy absorption of iodine and water) is between 40 and 60 keV, knowing that the absorption threshold K of the iodine atoms is about 33,2 keV: at this energy the photoelectric effect is predominant. These findings are summarized in Table 1 below.

It should therefore be expected that with a tungsten anode the radiolytic effect of X-radiation on an iodinated contrast agent is maximal. The inventors have found that the radiolysis of an iodinated contrast agent during a radiodiagnostic examination leads to the release of iodine, this free iodine having a DNA-breaking effect which adds to the brittle effect. X-ray dose DNA from radiodiagnostic examination that causes said radiolysis. This additional DNA-binding-radiolysis-related effect of the iodinated contrast agent is referred to herein as "excess biological dose" or "biological dose overload".

The doses not to be exceeded for the general public, subjected to X-ray and fluoroscopic examinations, are identical to those of the exposed workers: exposure is limited to 50 mSv in children and 100 mSv in adults. However, it is suspected that the cumulative doses linked to the repetition of such examinations would be likely to induce a true excess of cancers, even if precise epidemiological data are not available. do not seem to exist. The measurement of a possible excess of dose related to iodine is therefore of interest because it would make it possible to better know, for each individual and for given irradiation conditions, the dose and the cumulative thresholds not to be exceeded.

The inventors have furthermore found, and this is the second starting point of the present invention, that the double-strand breaks (DSBs) of the DNA are the most radioinduced radiation-induced damage of radiosensitivity when they are unrepaired. on the one hand, and genomic instability when they are poorly repaired on the other. The inventors have discovered that the CBDs are supported by the majority mode of suture repair, and / or by the minority mode of faulty repair, referred to as MRE1 1-dependent recombination. The balance between these two modes of repair is controlled by the ATM protein. The pH2AX marker indicates a CBD site recognized by the suture repair mode. The marker MRE1 1 indicates a CBD site that was supported by the MRE1 1 -dependent fault repair. The pATM marker provides information on activation of the histone H2AX phosphorylation suture pathway and inhibition of the 1 - dependent MRE1 pathway.

The inventors have also observed a transfer of the cytoplasmic forms of the ATM protein into the cell nucleus following an oxidative-type stress, and in particular following a stress linked to an ionizing radiation inducing CBD.

Finally, the inventors have shown that it is the specific radiolysis products of the iodinated contrast product that are responsible for the excess of undamped CBD after radiodiagnostic examination: this is particularly the case of sodium iodides.

To note the damage of a DNA by an exogenous aggression due to the specific radiolysis products of the iodinated contrast product, it is necessary to take into account:

on the one hand, the spontaneous state of DNA,

on the other hand, from its radioinduced state,

- finally, its radioinduced state after the action of radiolysis products of

iodinated contrast media, especially sodium iodide.

Furthermore, after irradiation, the DNA repair must be taken into account, the kinetics of which depends on the repair mechanism and therefore on the type of radiation induced damage. It is also known that the efficiency and speed of DNA repair varies from one individual to another, and that there are also particular genetic conditions that lead to exceptional radiosensitivity. The effect of iodinated contrast media radiolysis products adds to the radioinduced effects.

According to the invention the problem is solved by a method in which

(1) amplification of untransformed cells from a cell sample, in particular cells from skin biopsies;

(2) a mechanistic model valid for quiescent human cells is established;

(3) Functional testing of CBD recognition, repair and signaling is performed;

(4) radiolysis products of the iodinated contrast media or an iodide of an alkaline element (preferably sodium iodide) are applied.

This method forms a first object of the present invention. The two embodiments of step (4) are equivalent but are not used for the same predictive purpose.

The use of radiolysis products of iodinated contrast media is adapted to the equipment available to the radiotherapist, ie a high energy gamma irradiator for radiotherapy. The radiotherapist is made to perform imaging by scanning to better situate the tumor before, during and after radiotherapy. An X-ray dose D _scan equivalent to a CT medical imaging session is used. This dose D _scan is advantageously between 5 mGy and 50 mGy, and preferably between 10 mGy and 40 mGy depending on the organs to be imaged.

The embodiment called "radiation therapist" of the method according to the invention consists in measuring the number of unrepaired CDB after a dose D _th mmo equivalent to a radiation therapy session, in the presence of sodium iodide, the final product of radiolysis iodinated contrast media. The dose D _th mmo is typically between 0.5 Gy and 4 Gy, preferably between 1 Gy and 3 Gy, and more preferably between 1, 7 Gy and 2.3 Gy.

According to a preferred embodiment of the invention, the D _scan parameters are standardized and according to the embodiment of the radiologist (D _sca n) or the radiotherapist (Dthérap) (and preferably both at the same time) to a given value. fixed to generate comparable data for different tissues and patients; these standardized values are: D _th = RAPA 2 Gy and / or D _sca n = 20 mGy. The use of an iodide of an alkaline element is adapted to the equipment available to the radiologist, that is to say a low energy X-ray scanner.

The so-called "radiologist" embodiment of the method according to the invention consists in measuring the number of unrepaired CBDs after 20 mGy (dose equivalent to a scanner imaging session) in the presence or absence of iodinated contrast medium. .

For X-ray scanner imaging, tubes generating X-rays with a tungsten anode are generally used. Another object of the invention is a method of evaluating the additional DNA-breaking effect of an iodinated contrast agent injected into an individual as part of a medical imaging examination using X-rays, on a tissue taken from said individual, in which process:

(a) Cells of an evaluable tissue that has been removed (preferably by biopsy) are delivered to an individual;

(b) dispersing and / or amplifying said cells to obtain a cell sample;

(c) contacting said cell sample with a test medium, said test medium comprising said iodinated contrast agent and / or iodide ions;

(d) said cell sample is subjected in contact with said test medium to absorbed dose D _th era OR _sca n determined ionizing radiation, whereby:

D _scan represents the X-ray dose used for a medical imaging examination,

Dthera represents the dose of ionizing radiation administered during a radiation therapy session that follows a medical imaging examination using X-rays; (e) The number of double-strand breaks of the DNA, and / or a biomarker representing this number, and / or the number of micronuclei are determined on the cell sample at the end of step (d).

These X-rays are typically generated with a tungsten anode.

In one embodiment of this process: (a) The said cell sample is subjected after step (d) to contact with a medium to be tested (which advantageously comprises iodide ions, but no agent of iodinated contrast) at a given dose of gamma rays mmo D _th (D héra _t being preferably between 0.5 Gy and 4 Gy, more preferably between 1 Gy and 3 Gy, still more preferably between 1, 2 and 7 Gy , 3 Gy, and typically 2 Gy); and then (β) The number of double-strand breaks of the DNA is detected on the cell sample, and / or a biomarker representing this number, and / or the number of micronuclei.

In one embodiment of this method, the average number of nuclear foci obtained with the pH2AX and pATM markers at observation times t is determined on said cell sample which has not yet been placed in contact with the medium to be tested. average numbers being respectively called N _pH 2Ax (t) and N _pAT M (t)), obtained in a condition without irradiation (0 Gy) and also at time t = t4 (where t4 is a fixed value which represents the time for which the rate of DNA breaks reaches its residual value, and which must be at least 12 hours, and preferably between 12 and 48 hours, and which is even more preferably approximately 24 hours); and then

the average number of nuclear foci obtained with the pH2AX and pATM markers at observation times t and for the dose D _sca n) is determined on said cell sample at the end of step (d) (these average numbers being called respectively N _pH 2Ax (t, D _sca n)) and N _pA TM (t, D _SC an)), said observation time being t = t4.

Advantageously, it is determined on said sample cell at the end of step (a) the mean number of foci obtained with the nuclear marker pH2AX and patm the observation time t and the D _th mmo dose (Gy) (these average numbers being called N _pH 2AX respectively (t, Erap D _th)) and N _pA TM (t, Erap D _th)), said observation time being t = t4. The determinations in step (a) and / or step (β) can be made at additional observation times t1 and / or t2 and / or t3, given that

t4 is advantageously chosen between 6 times t3 and 8 times t3, but must be at least 12 hours, and preferably between 12h and 48h, and still more preferably about 24 hours;

t3 is a fixed value which represents the time after which about 25% of the double-strand breaks (DSBs) are repaired in control cells from radiation-resistant patients, and which is advantageously chosen between 3 times t2 and 5 times t2, but must in this case be at least 2.5 hours and at most 6 hours, and is preferably between 3 hours and 5 hours, and is even more preferably about 4 hours;

t2 is a fixed value which represents the time after which approximately 50% of the CBDs are repaired in control cells from radioresistant patients, and which is advantageously chosen between 5 times t1 and 7 times t1, but which must in this case be less than 35 minutes and not more than 90 minutes, and is preferably between 45 minutes and 75 minutes, and is even more preferably about 60 minutes;

t1 is a fixed value which represents the time after which the number of recognized CBDs reaches its maximum in control cells from radioresistant patients, and which is advantageously chosen between 5 minutes and

15 minutes after stopping the irradiation, preferably between 7.5 minutes and 12.5 minutes, and even more preferably at about 10 minutes.

For example, t1 is between 8 minutes and 12 minutes, t2 is between 50 minutes and 70 minutes, t3 is between 3.5 hours and 4.5 hours, and t4 is between 22 hours and 26 hours, or t1 is 10 minutes, t2 is 60 minutes, t3 is 4 hours, t4 is 24 hours; preferably, D _t hera is 2 Gy.

In one embodiment, in step (c) said test medium comprises iodine ions. In this case: - SI N | pH2Ax (t4 Dthérap) <2 and N | P atm (t1, D _th ERAP)> N | _pAT M (t2, D _th ERAP) and N | _pA TM (t1, D _th RAPA)> 30, then it is considered that the cellular specimen is radioresistant iodine effect: it is called "Group I";

if (N | _P H2Ax (t4,

> 24), then we consider that the cell sample is radiosensitive to the iodine effect: it is called "Group III";

- for all other conditions, the risk is considered to be moderate: the cell sample is called "Group II",

or :

N | _P H2AX is the number of foci of pH2AX obtained with irradiation in the presence of iodine ions,

NipATM is the number of pATM foci obtained with irradiation in the presence of iodine ions,

NIMN is the average number of micronuclei observed per 100 cells with irradiation in the presence of iodine ions. In another embodiment, in step (c) said test medium comprises an iodinated contrast agent. In that case :

- if N | _P H2Ax (t4, Dscan) - N _pH 2Ax (t4, D _sca n)) <2, then it is considered that the cell sample is radio-resistant to the iodine effect: it is called "Group I";

- if N | _P H2Ax (t4, D _scan )) - N _pH 2Ax (t4, D _scan ))> 8, then the sample is considered

cell is radiosensitive to the iodine effect: it is called "Group III"; For all other conditions, the risk is considered moderate: the cell sample is called "Group II",

N | _P H2AX is the number of pH2AX foci obtained with irradiation in the presence of an iodinated contrast agent,

N _P H2AX is the number of foci of pH2AX obtained with irradiation without iodinated contrast agent.

In another embodiment (hereinafter referred to as the "radiotherapist method") of the method according to the invention, the additional effect of breaking the DNA of iodide ions is determined, said method being characterized in that:

(i) said test medium comprises iodine ions (preferably a solution containing 10 μΜ of sodium iodide) but no iodinated contrast agent,

(ii) the additional dose is determined solely on the basis of the number of pH2AX foci at time t4, and preferably using the following formula:

_ Ni _p H2AX (t4, Dffter) ~~ N _pH2 AX (t4, D _Therapy )

'-'sup Z r. \ X ^ Therapy

^ pH2Ax (, t4, L ' _t / _lera p) or:

N i _P H2Ax is the number of foci of pH2AX obtained with irradiation in the presence of iodine ions, N _P H2Ax is the number of foci of pH2AX obtained with irradiation without iodine ions.

Moreover, the additional effect breaking the DNA of iodide ions (in the "radiotherapist method") can be determined also in the following way:

(ii) the additional dose is determined on the basis of the number of pH2AX foci obtained in a condition without irradiation (0 Gy) and also at times t1, t2, t3 and t4, and preferably according to the following method:

in a first step, the numbers of pH2AX foci obtained in a condition without irradiation (0 Gy) and also at times t-ι, t _2, t ₃ and t ₄ for the sample of irradiated cells without iodinated contrast medium are determined. ,

in a second step, the parameters b _rec , b _rep and t ₀ are calculated in the formula (\ -

N _V H2AX t> Dthérap) - ID _Therap (l- ^ ^ ^ t ^ l + Z (t ^~~ or?

I: number of CDBs induced by DNA equivalent to 40 CBD per Gy for X-rays, b _rec : inverse time after which 50% of CBD are recognized [min ^"1 ], b _rep : inverse of time at the end of which 50% of recognized CBDs are repaired [min ^"1 ], t ₀ : average time difference between CBD recognition and repair; in a third step, the numbers of pH2AX foci obtained in a condition without irradiation (0 Gy) and also at times t _1, t _2, t ₃ and t _{4 are determined} for the sample of cells irradiated with the iodinated contrast product, in a fourth step the equivalent dose D-ι of the irradiation with the iodinated contrast product is calculated by using the parameters b _re c, b _rep and t ₀ determined in the second step, using the formula: v 1 + b _rec t 1 + b _rep (t - ÎQ) in a fifth step the excess dose due to the iodinated contrast product D _{sup is determined} according to the formula:

In another embodiment (in the "radiologist" method) the number of foci pH2AX can be used only at time t4, and preferably using the following formula:

_ Ni _P H2Ax (t4, D _scan ) - Np _H2 Ax (t4, D _scan )

S ^U P ~ (tn ^ ^{x scan}

¹ ρΗ2ΑΧ ^ ^u scan)

or :

Ni _P H2Ax is the number of foci of pH2AX obtained with irradiation in the presence of an iodinated contrast agent,

N _P H2AX is the number of foci of pH2AX obtained with irradiation without iodinated contrast agent. In the context of the present invention is typically used between 5 and 25 mg of iodine per ml of contrast medium iodine, and preferably between 10 and 20 mg, but these values are not limiting.

We give here details of the two main modes of use of the method according to the invention.

1) The so-called "radiotherapist" embodiment

A first embodiment of the invention is a method for predicting cellular radiosensitivity of a cell sample to ionizing radiation associated with the presence of contrast media containing iodine atoms, said cell sample having been obtained from cells taken from a patient in a non-irradiated or poorly irradiated zone, in which process:

(i) isolating and / or amplifying said sampled cells, these isolated and / or amplified cells constituting the "cell sample"; (ii) determining on said cell sample the average number of nuclear foci obtained with the pH2AX and pATM markers at observation times t and at a dose of radiation D (this average number being called N H2Ax (t, D)) and N _p ATM (t, D)), obtained in a condition without irradiation (0 Gy) and also at the observation time t4 (and preferably in addition to the times t1, t2 and t3) after irradiation,

(iii) the average number of nuclear foci obtained with the pH2AX and pATM markers is determined on said cell sample at all observation times t and at a dose of radiation D in the presence (during the irradiation) of iodine ions ,

preferably at a rate of 2 to 20 μΜ of iodide (preferably at a rate of 5 to 15 μΜ, and even more preferably of about 10 μΜ), this embodiment being called the embodiment of "the radiation therapist"), knowing that the iodide can be added in the form of an iodide of an alkaline element, preferably Nal;

said average numbers of nuclear foci being respectively called N | H2AX (t, D)

or t4 is a fixed value which represents the time for which the rate of DNA breaks reaches its residual value, and which is advantageously chosen between 6 times t3 and 8 times t3, but in this case must be at least 12 hours, and preferably between 12h and 48h, and which is even more preferably about 24 hours;

t3 is a fixed value which represents the time after which approximately 25% of the CBDs are repaired in control cells from radioresistant patients, and which is advantageously chosen between 3 times t2 and 5 times t2, but in this case must be at least 2.5 hours and not more than 6 hours, and is preferably between 3 hours and 5 hours, and still more preferably about 4 hours;

t2 is a fixed value which represents the time after which approximately 50% of the CBDs are repaired in control cells from radioresistant patients, and which is advantageously chosen between 5 times t1 and 7 times t1, but which must in this case be less than 35 minutes and not more than 90 minutes, and is preferably between 45 minutes and 75 minutes, and even more preferably about 60 minutes;

For the use of the alkali metal iodide method, a solution containing 10 μl of sodium iodide is advantageously used, and if:

(l) N | pH2Ax (t4, Therapy) <2 and N | _pA TM (t1, D _th ERAP)> N | _pAT M (t2, D _th RAPA) and N, _pA T (t1, D _th RAPA)> 30, then it is considered that the sample is radioresistant iodine effect: it is called "Group I"; if

(ii) (N | _P H2Ax (t4,

)> 8 or N | MN (t4, D _th RAPA)> 24), then it is considered that the sample is radiosensitive iodine effect: it is called "group III"; and

(iii) for all other conditions the risk is considered to be moderate: the sample is "Group II",

or :

- N | _P H2AX is the number of foci of pH2AX obtained with irradiation in the presence of iodine ions,

NipATM is the number of pATM foci obtained with irradiation in the presence of iodine ions, N iMN is the average number of micronuclei observed per 100 cells with irradiation in the presence of iodine ions.

In this embodiment, the mathematical determination of the "excess dose" associated with irradiation with a dose D in the presence of iodine can be done according to two methods, depending on the number of available information: if it is available only time t4 then method 1 will be applied, if all the times are available (t1, t2, t3, t4) it is method 2 that will be applied.

Advantageously, an iodide solution of an alkali metal, preferably a solution containing 10 μl of sodium iodide, is used.

Method 1: using only the number of pH2AX foci at time t4

We use the following formula:

_ Ni _p H2AX (t4, Dffter) ~~ N _pH2 AX (t4, D _Therapy )

'-'sup Z r. \ X ^ Therapy

^ pH2Ax (, t4, L ' _t / _lera p) where:

N | _P H ₂ AX is the number of foci of pH2AX obtained with irradiation in the presence of iodine ions,

N _PH2A x is the number of foci of pH2AX obtained with irradiation without iodine ions.

Method 2: using the number of pH2AX foci in a condition without irradiation (0 Gy) and also at times t1, t2, t3 and t4

The second method to deduce the excess dose caused by the presence of iodinated contrast media will be in two steps:

Step 1 :

The numbers of pH2AX foci obtained (N _pH 2AX) in a condition without irradiation (0 Gy) and also at times t-ι, t _2, t ₃ and t ₄ for the sample of irradiated cells without the iodinated contrast media will be adjusted by the Bodgi formula (Bodgi et al (2013) cited above) and thanks to this adjustment, the parameters b _re c, b _{rep and} etto will be determined: _(λ _ _ί 1

N t> Therapy) ~ _Therapy ID (l- -) ( ^{~ ~} )

1 - | - - | - _re p \ l

With:

I: number of DSBs induced DNA equivalent to 40 Gy for CBD by X-ray Dthérap ^'■ the irradiation dose b _rec: inverse time at which 50% of CBD are recognized min ^"1 _boundary representation: inverse time after which 50% of recognized CBDs are repaired in min- ¹ t ₀ : average time difference between recognition and repair of CBDs. 2nd step :

The number of pH2AX foci obtained (N _PH 2AX) in a condition without irradiation (0 Gy) and also at times t _1, t _2, t ₃ and t ₄ for the sample of cells irradiated with the iodinated contrast media will be adjusted by . Bodgi the formula ((Bodgi et al (2013) cited above) by setting the parameters b _e c, _boundary representation and to determined in step 1: v 1 + b 1 + b _rec t _ref (t -

This formula makes it possible to determine the parameter D-ι, which is the equivalent dose of the irradiation with the iodinated contrast product.

The excess dose due to the iodinated contrast product D _su will therefore be given by the formula:

2) The so-called "radiologist" embodiment

A second embodiment of the invention is a method for predicting cellular radiosensitivity of a cell sample to ionizing radiation associated with the presence of contrast media containing iodine atoms, said cell sample having been obtained from cells taken from a patient in a non-irradiated or poorly irradiated zone, in which process: (i) said collected cells are isolated and / or amplified, these isolated and / or amplified cells constituting the "cell sample"; (ii) determining on said cell sample the average number of nuclear foci obtained with the pH2AX marker at observation times t and at a radiation dose D (this average number being called N H2Ax (t, D)), obtained in a condition without irradiation (0 Gy) and also at observation times t4 after irradiation,

(iii) determining on said cell sample the average number of nuclear foci obtained with the pH2AX marker at all observation times t and at a radiation dose D in the presence (during the irradiation) of iodine ions; preferably at a rate of 5 to 25 mg of iodine per ml of contrast medium (and preferably in a proportion of 10 to 20 mg, and still more preferably of approximately 15 mg), this embodiment being called the mode of realization of the "radiologist", these contrast products being for example: Noméron ™ comprising as active ingredient Noméprol (CAS #: 78649-41 -9), Xenetix ™ comprising as active ingredient Nobitridol (CAS #: 136949-58- 1) or Ultravist ™ comprising as active ingredient Nopromide (CAS No. 73334-07-3)).

For the use of the method with radiolysis products of contrast media containing iodine atoms, 15 mg of iodine per ml of iodinated contrast medium are advantageously used, and if: (a) N | _P H2AX (t4, D _scan) - N _pH 2AX (t4, _scan D) <2, then it is considered that the sample is radioresistant iodine effect: it is called "Group I";

(b) N | _P H2AX (t4, D _scan) - N _pH 2AX (t4, D _scan)> 8, then it is considered that the sample is radiosensitive iodine effect: it is called "group III";

(c) For all other conditions the risk is considered to be moderate: the sample is called "Group II",

or :

N _P H2Ax is the number of pH2AX foci obtained with irradiation without iodinated contrast agent.

In this embodiment of the invention, the mathematical determination of the "excess dose" associated with irradiation with a dose D in the presence of iodine can be determined according to the following method:

In the so-called "radiologist" embodiment, 15 mg of iodine per ml of iodinated contrast medium is advantageously used. The additional dose equivalent is determined using only the number of pH2AX foci at time t4 from the following formula:

N IpH2AX (t4, A scan) - N pH2AX (t4, A scan)

D s, up X D,

N (t4, A scan

pH2AX scan

or :

N | _P H ₂ AX is the number of pH2AX foci obtained with irradiation in the presence of an iodinated contrast agent,

N _PH2A x is the number of pH2AX foci obtained with irradiation without iodinated contrast agent.

detailed description

The terms "radioinduced damage", "radioinduced", "radiosensitivity", "radioresistance", "radiotoxicity", "radiotherapy" all refer to ionizing radiation, particularly to particle-type radiation, such as alpha-type particles. (a) or beta (β), or to high energy electromagnetic radiation, especially gamma (γ) or X.

The term "ATM cyto-nuclear transit" describes the translocation of the ATM protein from the cytoplasm to the nucleus, especially after irradiation. The "DNA effect" of a contrast agent injected into an individual as part of a medical imaging examination using X-rays on a cell sample from a tissue sample is called an "iodine effect". on said individual. This iodine effect is in addition to the DNA-breaking effect of the ionizing radiation administered during said medical imaging examination. The inventors have realized that X-ray spectrum emission lines produced by an X-ray tube with a tungsten anode, of a type commonly used in three-dimensional imaging devices (X-ray tomographic scanner), and superimposed on the continuous spectrum (whose spectral distribution depends on the acceleration voltage, which is typically between 80 and 120 kV), are close to the maximum energy absorption of the iodine atoms in the water . These data are collated in Table 1. Table 1

X-ray properties generated with a tungsten anode of water and iodine

1. Preparation of the test

Before any collection of cells and before any manipulation of the collected cells, the respective operators (belonging for example to a cytological analysis laboratory) are informed (typically by the doctor) of the possible infection status of the patient by HIV or hepatitis C for operators can take appropriate measures of increased biosecurity when collecting, handling and managing cell culture.

Then, the operator takes the patient a cell sample. Preferably he biopsy samples a skin sample; this sampling can be advantageously done according to a method known as the "dermatological punch". The cell sample is placed in DMEM medium + 20% sterile fetal calf serum. The sample is transferred without delay to a specialized laboratory, knowing that the sample should not remain more than 38 hours at room temperature.

In an advantageous embodiment, said control cells from radioresistant patients were selected as cells exhibiting an in vitro clonogenic survival rate of greater than 55% after irradiation with an absorbed dose of 2 Gy.

In another embodiment, said control cells from radio-resistant patients were selected as cells taken from patients who did not show significant tissue reactions during or following a CT scan or radiotherapeutic treatment.

The method according to the invention uses at least one sample of healthy tissue, preferably fibroblasts. These are preferably taken from the connective tissue of the patient. This sampling can be done by biopsy. Thus, in an advantageous embodiment, said cells are fibroblastic cells derived from a skin biopsy of a patient (typically taken by a method known as "dermatological punch"). The tissue sample is grown in a suitable culture medium.

The next step is the isolation and / or amplification of the sampled tissue. In one embodiment, upon receipt, the cell sample (typically the biopsy) is established as an amplifiable cell line without a viral or chemical transformation agent following an ancillary procedure and well known to culture laboratories, such as the underlines the publication of EIkin M. et al. "The radiobiology of cultured mammalian cells", Gordon and Breach (1967). As soon as the number of cells is sufficient (1 - 3 weeks), the first experiments are carried out using the method according to the invention. The cells are seeded on glass slides in petri dishes. The cells are placed in the presence of iodine ions, preferably in the proportion of 5 to 25 mg of iodine per ml of contrast media (and preferably in the proportion of 10 to 20 mg, and even more preferably of approximately 15 mg). this embodiment being referred to as the "radiologist" embodiment), such contrast media being, for example: lomeron ™, Xenetix ™ or Ultravist ™), or preferably 2 to 20 μΜ iodide (preferably at a rate of 5 to 15 μΜ, and even more preferably about 10 μΜ), this embodiment being called the "radiotherapist" embodiment), knowing that the iodide can be added in the form of an iodide of an alkaline element, preferably Nal. Part of these lamellae are irradiated on a medical irradiator according to a certified dosimetry with an absorbed dose D (for example 2 Gy). Another part is not irradiated; it represents the spontaneous state (absorbed dose 0 Gy).

Said ionizing radiation is defined by its absorbed dose (parameter called D _therapy and Ds _can expressed in Gray). In the context of the present invention the absorbed D _scan dose is between 0.01 Gy and 1 Gy, preferably between 0.01 Gy and 0.05 Gy, and is even more preferably 0.02 Gy. scope of the present invention absorbed dose D _th mmo is between 0.5 Gy and 4 Gy, preferably between 1, 7 Gy and 2.3 Gy, and still more preferably 2 Gy.

These areas typically correspond to an individual session of CT or radiotherapeutic treatment for which the number of sessions depends on the location, type and stage of advancement of the tumor. After irradiation and to undergo the repair times mentioned below, the cells remain in the culture incubator at 37 ° C.

For the irradiated cells, characteristics corresponding to the radio-induced state are acquired after several repair times (post-irradiation repair time). In the so-called radiotherapist method, at least two and even more preferably at least three points are preferably acquired, namely: t1, t2, t3 and t4. Said characteristics are represented by the foci corresponding to the pH2AX and pATM marker. In the so-called radiologist method, a point at t4 is acquired for the pH2AX marker.

The cells on glass slides are then fixed, lysed and hybridized. The following procedure, known per se (see the cited publication by Bodgi et al (2013)), can be used: the cells were fixed in 3% paraformaldehyde and 2% sucrose for 15 minutes at room temperature and permeabilized in 20 mM HEPES buffer solution (4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid) at pH 7.4, 50 mM NaCl, 3 mM MgCl ₂ , 300 mM sucrose, 0.5% Triton X-100 (one nonionic surfactant of formula t-Oct- ₆ H4- (OCH ₂ Cl-12) xOH with x = 9-10, CAS No. 9002-93-1, supplied by Sigma Aldrich) for 3 minutes. The coverslips were then washed in phosphate buffered saline (known as PBS) prior to immunostaining. Incubation was carried out for 40 min at 37 ° C in PBS supplemented with 2% bovine serum albumin (known as BSA or fraction V, provided by Sigma Aldrich) and followed by PBS washing. . The primary anti-pH2AX antibodies were used at a concentration of 1: 800, the other antibodies at 1: 100. Incubations with secondary antibodies FITC anti-mouse or TRITC anti-rabbit (1: 100, provided by Sigma Aldrich) were performed at 37 ° C in 2% BSA for 20 minutes. Glass slides were treated with Vectashield ™ containing DAPI (4,6-Diamidino-2-phenylindole) to mark the core. The staining with DAPI also makes it possible, indirectly, to determine the number of cells in the Gi phase (nuclei with homogeneous DAPI staining), in the S phase (nuclei with many pH2AX foci), in the G ₂ phase (nuclei with DAPI staining). heterogeneous) and metaphases (visible chromosomes).

The results are acquired from these slides on an immunofluorescence microscope (Olympus model, for example). The reading can be direct (typically by counting the foci on at least 50 cells in G ₀ / Gi for each point) or by dedicated image analysis software, or on an automated microscope; preferably the software or automated microscope methods are calibrated with manual determinations.

In order to obtain results of sufficient statistical reliability to serve as the basis of a diagnosis, at least 3 sets of independent (irradiation) experiments are performed and the average of each of the foci numbers for the defined times is calculated.

2. Determination of biological and clinical parameters

2.1 Generalities and markers used

The invention is based inter alia on the use of data acquired for at least two pH2AX, pATM markers on non-irradiated cells (spontaneous state) and irradiated (radioinduced state). The method is based on the kinetic study of the labeling by this marker as a function of the duration of the repair: the samples are marked after a determined lapse of time from the end of the irradiation, and their immunofluorescence is studied. It is possible to measure the complete kinetic curves, for example represented by 4 points advantageously located at t1 (preferably 10 minutes), t2 (preferably 1 h), t3 (preferably 4h) and t4 (preferably 24h), and before irradiation (spontaneous state). But the plaintiff realized that some points (corresponding to certain times of repair) are more important than others, and that some points are not predictive. Thanks to the judicious selection of the parameters determined at given times, it is thus possible to reduce the number of measurements and thus reduce the overall cost of the diagnosis, without reducing the predictive power of the method. It is this simplified method that forms the basis of the predictive method according to the invention. The averages of each point and each dose with each marker are calculated with standard deviation errors of the mean (called "SEM") since the sampling is n = 3 (no Gaussian standard deviation of "standard error SE ").

(i) pH2AX denotes the phosphorylated forms in serine 439 of the histone H2AX X variant which marks, according to the findings of the applicant, the number of double-strand breaks in the DNA (CDB) which are recognized by the method of Majority and faithful repair, the suture. The pH2AX marker is essentially nuclear in the form of only nuclear foci and only the number and size of the foci will be analyzed.

(ii) pATM refers to the phosphorylated forms in 1981 serine of the ATM protein kinase. According to the findings of the applicant, ATM passes cytoplasm to the nucleus after irradiation under normal conditions (radio-resistant status). The pATM forms are mainly concentrated in the cytoplasm and then mark CBD sites. The pATM marker is distinguished by a location that can be homogeneous cytoplasmic (no cytoplasmic foci) without nuclear foci, only nuclear in the form of nuclear foci (no homogeneous nuclear localization), or cytoplasmic and nuclear foci. Counterpoloration with DAPI (a DNA marker known to those skilled in the art) makes it possible to locate the nucleus to locate the cytoplasmic or nuclear localization (this distribution being modified pATM under the influence of ionizing radiation)

2.2 Biological parameters

We define and determine as indicated:

- N _P H2Ax (t), NpATM (t) the average numbers of nuclear foci obtained with the markers pH2AX, pATM, obtained in a condition without irradiation (0 Gy) and also at the observation times t1, t2, t3, t4 after irradiation (absorbed dose: 2 Gy or 20 mGy), knowing that the determination of the parameter N _pH 2Ax (t) is obligatory within the framework of the method according to the invention, whereas that of the other parameters N _pA TM (t) is optional but advantageous;

2.3 Predictive Evaluation It aims at predicting clinical or radiotherapeutic parameters based on measured biological data. Two levels of diagnosis are proposed: A) a quantitative diagnosis directly derived from the mathematical value of the scores or mathematical formulas linking the scores; this one concerns the two following criteria:

2.3.1 The classification of the patient in a group I, II or III (criterion called GROUP):

For the use of the so-called radiotherapist method with iodides of an alkali metal, a solution containing 10 μl of sodium iodide is advantageously used, and if:

(a) N | pH2Ax (t4, Therapy) <2 and N | pATM (t1, Therapy)> N | _pAT M (t2, D _th ERAP) and N | _pA T (t1, D _th RAPA)>

30, then it is considered that the sample is radioresistant to the iodine effect: it is called "Group I";

(b) (N | _P H2Ax (t4,

)> 8 or N, MN (t4, D _th RAPA)> 24), then it is considered that

the sample is radiosensitive to the iodine effect: it is called "Group III"; (c) for all other conditions the risk is considered moderate:

the sample is called "group II", where:

NIMN is the average number of micronuclei observed per 100 cells with irradiation in the presence of iodine ions.

For the use of the so-called radiologist method with radiolysis products of contrast media containing iodine atoms, 15 mg of iodine per ml of iodinated contrast medium is advantageously used, and if:

- N | _P H2AX (t4, D _scan) - N _pH 2AX (t4, _scan D) <2, then it is considered that the sample is radioresistant iodine effect: it is called "Group I";

- If N | _P H2AX (t4, D _scan) - N _pH 2AX (t4, D _scan)> 8, then it is considered that the sample is radiosensitive iodine effect: it is called "group III";

For all other conditions, the risk is considered to be moderate: the sample is called "Group II", Ni _P H2Ax is the number of foci of pH2AX obtained with irradiation in the presence of an iodinated contrast agent,

2.3.2 Determination of the additional dose

For the use of the radiotherapist method with iodides of an alkali metal, a solution containing 10 μl of sodium iodide is advantageously used.

Two methods are proposed depending on the number of information available:

Method 1: using only the number of foci pH2AX at time t4 We use the following formula:

_ Ni _p H2AX (t4, Dffter) ~~ N _pH2 AX (t4, D _Therapy )

'-'sup Z r. \ X ^ Therapy

^ pH2Ax (t4, L _'t / _léra p)

or :

Method 2: using the number of pH2AX foci obtained in a condition without irradiation (0 Gy) and also at times t1, t2, t3 and t4

Step 1 :

The numbers of pH2AX foci obtained obtained in a condition without irradiation (0 Gy) and also at times t _1, t _2, t ₃ and t ₄ for the sample of irradiated cells without the iodinated contrast media will be adjusted by the Bodgi formula. (Bodgi et al., JTB 2013) and thanks to this adjustment, the parameters b _rec , b _rep and to will be determined. The parameters b _rec , b _rep and t ₀ are numbers or decimal or integer numbers. The parameters b _rec , b _rep are preferably decimal numbers or numbers rounded to two significant digits after the decimal point, preferably three significant digits after the decimal point. This rounding corresponds to the arithmetic rounding of the value obtained by calculation. The parameters b _rec and b _rep are preferably between 0.001 and 5 min ^-1 The parameter t ₀ is preferably between 0 minutes and 120 minutes.

The parameters b _re c, b _rep and to are specific to the patient considered.

_ 1

N _V H2AX t _> Therapy) ~ _Therapy ID (l -) (77 -r)

1 TD _REC L 1 - | - _Dr. p \ l LQJ

With:

I: number of induced CBD DNA equivalent to 40 Gy for CBD by X-ray Dthérap ^'■ the irradiation dose for the method of the radiation oncologist in Gy b _r ec: inverse time at which 50% of CBD are recognized in min ^"1 b _rep : inverse of the time after which 50% of recognized CBDs are repaired in min ^{" 1} t ₀ : average difference between CBD recognition and repair. 2nd step :

The numbers of pH2AX foci obtained in a condition without irradiation (0 Gy) and also at times t _1, t _2, t ₃ and t ₄ for the sample of cells irradiated with the iodinated contrast media will be adjusted by the Bodgi formula ( Bodgi et al., JTB 2013) by setting the parameters b _re c, b ^ etto determined in step 1: v 1 + b _rec t 1 + b _rep (t - ÎQ)

This formula will make it possible to determine the parameter D-ι, which is the equivalent dose of the irradiation with the iodinated contrast product. The use of this formula according to the invention for the determination of D-ι leads to obtaining a digit or a decimal or integer number.

The excess dose due to the iodinated contrast product D _sup will therefore be given by the formula

Dsup- D- | -Dtherap For the use of the so-called radiologist method with products of radiolysis of contrast media containing iodine atoms, 15 mg of iodine per ml of iodinated contrast medium is advantageously used, using only the number of pH2AX foci at the time. t4.

The additional dose equivalent is obtained using only the number of pH2AX foci at time t4 from the following formula:

N IpH2AX (t4, A scan) - N pH2AX (t4, A scan)

D s, up X D,

N (t4, A scan

pH2AX scan where:

The use of this formula according to the invention for determining the additional dose equivalent leads to obtaining a digit or a decimal or integer number. Preferably, the value of D _sup obtained is a digit or a decimal number corresponding to the arithmetic rounding to two significant digits after the decimal point of the value obtained by calculation.

B) In the context of the present invention is also proposed a more qualitative diagnosis, influenced by the quantitative diagnosis but taking into account any clinical elements brought to the knowledge of the practitioner.

EXAMPLE:

Commercial HMEC control vascular endothelial cells were amplified according to the supplier's recommendations (Thermo Fisher Scientific) until the desired number of cells was obtained. After obtaining a sufficient number of cells (generally after one to three weeks), the first experiments were carried out using the method according to the invention. The cells were seeded on glass slides in Petri dishes. Part of these lamellae was then brought into contact with the test medium comprising an iodinated contrast agent and / or iodide ions at a given concentration as shown in Table 2 below. Another part of these lamellae has not been brought into contact with this test medium comprising an iodinated contrast agent and / or iodide ions.

Table 2 Evaluation of excess dose D _su due to iodinated contrast media injected during radiodiagnostic examinations

In Table 2, the concentrations of the test media comprising an iodinated contrast agent and / or iodide ions are expressed as mg of iodinated compound per ml. Thus, a solution of Ultravist ™ 10 mg / ml contains 10 mg of Iopromide (CAS #: 73334- 07-3) per milliliter.

All of these slides were then immediately irradiated on a medical irradiator following a dosimetry certified with an absorbed dose D as shown in Table 2. A dose of 2 Gy was used as part of a therapeutic treatment. A dose of 0.1 Gy was used as part of a radiological treatment.

The irradiation was carried out with a medical accelerator which delivers 6 MV photons with an absorbed dose rate of 3 Gy min ^-1 After irradiation with an absorbed dose of 2 Gy or 0.1 Gy, the cells were stored in the culture incubator at 37 ° C. The irradiated samples were labeled 24 hours after stopping irradiation (t4), and the average number of nuclear foci obtained with the pH2AX marker was acquired at this post-irradiation repair time (24h). The irradiated cells were then fixed, lysed and hybridized on glass coverslips. The cells were fixed in 3% paraformaldehyde and 2% sucrose for 15 minutes at room temperature and permeabilized in 20 mM HEPES buffer solution (4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid) at pH 7.4, 50 mM NaCl, 3 mM MgCl ₂ , 300 mM sucrose, 0.5% Triton X-100 (a nonionic surfactant of formula t-Oct-C ₆ H ₄ - (OCH ₂ CH ₂ ) xOH with x = 9-10, CAS No. 9002-93-1, provided by Sigma Aldrich) for 3 minutes. The coverslips were then washed in phosphate buffered saline (known as PBS) prior to immunostaining. Incubation was carried out for 40 min at 37 ° C in PBS supplemented with 2% bovine serum albumin (known as BSA or fraction V, supplied by Sigma Aldrich) and was followed by PBS washing. . The primary anti-pH2AX antibodies were used at a concentration of 1: 800, the other primary antibodies at 1: 100. Incubations with FITC anti-mouse or anti-rabbit TRITC secondary antibodies (1: 100, provided by Sigma Aldrich) were performed at 37 ° C in 2% BSA for 20 minutes.

The results were acquired from these slides on an immunofluorescence microscope (Olympus model). The reading was carried out directly by counting the foci obtained with the pH2AX marker on at least 50 cells in G0 / G1 for each point and by dedicated image analysis software (imageJ).

In order to obtain results of sufficient statistical reliability to serve as a basis for a diagnosis, 3 sets of independent irradiations have been carried out. The average of each foci number after 24h (t4) of post-irradiation repair was calculated and presented in Table 2 for different HMEC cell lines.

The additional dose equivalent D _sup was then calculated using only the number of pH2AX foci at time t4, namely at 24 hours of post-irradiation repair from the following formula:

N IpH2AX (t4, D) - N pH2AX (t4, D)

D s, up X D

N pH2AX (t4, D) or:

Ni _P H2Ax is the number of foci of pH2AX obtained with irradiation in the presence of an iodinated contrast agent and / or iodide ions, N _P H2Ax is the number of foci of pH2AX obtained with irradiation without iodinated contrast agent or without iodide ions. and D corresponds to a determined absorbed dose of ionizing radiation D _sca n or D _th era expressed in Gray.

Claims

1. A method of evaluating the additional DNA-breaking effect of an iodinated contrast agent injected to an individual in the context of a medical imaging examination using X-rays, on a tissue taken from said individual, wherein Method: (a) cells of an evaluable tissue which has been removed (preferably by biopsy) from an individual are provided;

(b) dispersing and / or amplifying said cells to obtain a cell sample;

(c) contacting said cell sample with a test medium, said test medium comprising said iodinated contrast agent and / or iodide ions; (d) said cell sample is subjected in contact with said test medium to absorbed dose D _th era OR _sca n determined ionizing radiation, whereby:

D _scan represents the X-ray dose used for a medical imaging examination,

Dthera represents the dose of ionizing radiation administered during a radiation therapy session that follows a medical imaging examination using X-rays;

(e) The number of double-strand breaks of the DNA, and / or a biomarker representing this number, and / or the number of micronuclei are determined on the cell sample at the end of step (d). The method of claim 1, wherein said X-rays are generated with a tungsten anode.

3. The method of claim 1 or 2, wherein (a) is subjected said cell sample at the end of step (d) in contact with a test medium (which advantageously comprises iodide ions, but not iodinated contrast agent) to a determined dose of gamma rays mmo D _th (D héra _t being preferably between 0.5 Gy and 4 Gy, more preferably between 1 Gy and 3 Gy, still more preferably between 1, 7 Gy and 2.3 Gy, and typically 2 Gy); and then

(β) The number of double-strand breaks of the DNA is detected on the cell sample, and / or a biomarker representing this number, and / or the number of micronuclei. The method of any one of claims 1 to 3, wherein:

The average number of nuclear foci obtained with the pH2AX and pATM markers at the observation times t is determined on said cell sample which has not yet been contacted with the medium to be tested (these average numbers being respectively called N _pH 2Ax ( t) and Ν _ΡΑ ΤΜ (Ϊ)), obtained in a condition without irradiation (0 Gy) and also at time t = t4,

where t4 is a fixed value which represents the time for which the rate of DNA breaks reaches its residual value, and which must be at least 12 hours, and preferably between 12h and 48h, and which is even more preferentially about 24 hours ; and then

The average number of nuclear foci obtained with the markers pH2AX and pATM at observation times t and for dose D _sca n) is determined on said cell sample at the end of step (d) (these average numbers being called respectively N _pH 2Ax (t, D _scan )) and N _pAT M (t, D _scan )), said observation time being t = t4.

Process according to any one of Claims 3 to 4, in which the average number of nuclear foci obtained with the pH2AX and pATM markers at observation times is determined on said cell sample at the end of step (a). and the D _th mmo dose (Gy) (the average number being called N _pH 2AX respectively (t, Dthéra)) and N _pA TM (t, héra D _t)), said observation time being t = t4.

A method according to any one of claims 3 to 5, wherein the determinations in step (a) and / or step (β) are carried out at additional times of observations t1 and / or t2 and / or t3, knowing that

t2 is a fixed value which represents the time after which about 50% of the CBDs are repaired in control cells from radioresistant patients, and which is advantageously chosen between 5 times t1 and 7 times t1, but which must in this case be at least 35 minutes and at most 90 minutes, and is preferably between 45 minutes and 75 minutes, and is even more preferably about 60 minutes;

t1 is a fixed value which represents the time after which the number of recognized CBDs reaches its maximum in control cells from radioresistant patients, and which is advantageously chosen between 5 minutes and 15 minutes after stopping irradiation, preferably between 7.5 minutes and 12.5 minutes, and even more preferably at about 10 minutes. 7. The method of claim 6, wherein t1 is between 8 minutes and 12 minutes, t2 is between 50 minutes and 70 minutes, t3 is between 3.5 hours and 4.5 hours, and t4 is between 22 hours and 26 hours.

8. A process according to any one of claims 6 or 7 wherein t1 is 10 minutes, t2 is 60 minutes, t3 is 4 hours, t4 is 24 hours, and preferably D _t hera is 2 Gy.

9. A method according to any one of claims 1 to 8, wherein in step (c) said test medium comprises iodine ions.

The method of claim 9, wherein:

- SI N | pH2Ax (t4, Dthérap) <2 and N | Patm (t1, D _th RAPA)> Nl _pAT M (t2, D _th RAPA) and N | _pAT M (t1,

> 30, then we consider that the cell sample is radioresistant to the iodine effect: it is called "Group I";

if (N | _P H2Ax (t4,

> 24), then we consider that the cell sample is radiosensitive to the iodine effect: it is called "Group III" - for all other conditions we consider that the risk is moderate: the cell sample is said to Group II ',

or :

Ni _P H2Ax is the number of foci of pH2AX obtained with irradiation in the presence of iodine ions, NipATM is the number of foci of pATM obtained with irradiation in the presence of iodine ions and N | _M N is the average number of micronuclei observed per 100 cells with irradiation in the presence of iodine ions.

1 1. A method as claimed in any one of claims 1 to 10, wherein in step (c) said test medium comprises an iodinated contrast agent.

The method of claim 11, wherein

- if N | _P H2Ax (t4, D- _scan ) - N _pH 2Ax (t4, D- _scan )) <2, then it is considered that the cell sample is radioresistant to the iodine effect: it is called "Group I";

- if N | _P H2Ax (t4, D- _scan )) - N _pH 2Ax (t4, D- _scan ))> 8, then it is considered that the cell sample is radiosensitive to the iodine effect: it is called "Group II I";

For all other conditions the risk is considered moderate:

the cell sample is called "Group I I", where:

Ni _P H2Ax is the number of foci of pH2AX obtained with irradiation in the presence of an iodinated contrast agent and N _pH 2Ax is the number of foci of pH2AX obtained with irradiation without iodinated contrast agent.

The method according to any one of claims 1 to 12, wherein the additional DNA-bridging effect of an iodinated contrast agent is determined, said method being characterized in that:

(i) said test medium comprises iodine ions (preferably a solution containing 10 μl of sodium iodide) but no contrast agent,

or :

Ni _P H2Ax is the number of foci of pH2AX obtained with irradiation in the presence of iodine ions and N _P H2Ax is the number of foci of pH2AX obtained with irradiation without iodine ions.

(ii) the additional dose is determined on the basis of the number of pH2AX foci obtained in a condition without irradiation (0 Gy) and also at times t1, t2, t3 and t4, and preferably according to the following method: in a first step, the numbers of pH2AX foci obtained in a condition without irradiation (0 Gy) and also at times t _1, t _2, t ₃ and t _{4 are determined} for the sample of irradiated cells without iodinated contrast medium,

in a second step, the parameters b _rec , b _rep and t ₀ are calculated in the formula

_i 1

N _V H2AX t _> Therapy) ~ _Therapy ID (l -) (77 -r)

1 TD _REC L 1 - | - _Dr. p \ l LQJ where:

I: number of CDBs induced by DNA equivalent to 40 CBD per Gy for X-rays, b _rec : inverse time after which 50% of CBD are recognized [min ^"1 ], b _rep : inverse of time at the end of which 50% of recognized CBDs are repaired [min ^"1 ], t ₀ : average time difference between CBD recognition and repair; in a third step, the numbers of pH2AX foci obtained obtained in a condition without irradiation (0 Gy) and also at times t _1, t _2, t ₃ and for the sample of cells irradiated with the iodinated contrast product are determined in a fourth step calculates the equivalent dose D-ι of the irradiation with the iodinated contrast product by using the parameters b _rec , b _rep and t ₀ determined in the second step, by using the formula:

1 1

in a fifth step, the excess dose due to the iodinated contrast product D _{sup is determined} according to the formula:

Dsu _P ^- D- | -Dthera _P

15. A method according to any one of claims 1 to 14, wherein between 5 and 25 mg of iodine are used per ml of iodinated contrast medium, and preferably between 10 and 20 mg.

16. The method of claim 15, wherein the number of foci pH2AX is used only at time t4, and preferably using the following formula:

_ Ni _P H2Ax (t4, D _scan ) - Np _H2 Ax (t4, D _scan )

S ^U P ~ ¹ pH2AX (t ^, ^u nscan) ^{x scan}

where: N | _P H2AX is the number of pH2AX foci obtained with irradiation in the presence of iodinated contrast agent and N _pH 2AX is the number of pH2AX foci obtained with irradiation without iodinated contrast agent.

17. Process according to claims 1, 2, 4, 6, 7, 9, 11, 12 and 15 to 16, in which Dscan is between 5 mGy and 50 mGy, preferably between 10 mGy and 40 mGy, more preferably is 20 mGy.