WO2018229439A1 - Rapid predictive method for characterising the radiosensitivity and/or the risk of tissue toxicity of an individual to irradiation - Google Patents

Abstract

A method for characterising the radiosensitivity of a cell sample of an individual to ionising radiation, said cell sample having been obtained from cells removed from said individual, the method being characterised in that - the radiosensitivity is determined from the quantity of the phosphorylated ATM protein, pATM, in the basal state t0, - said method not including irradiation of said cell sample.

Description

 Rapid predictive method for characterizing the radiosensitivity and / or the risk of tissue toxicity of an individual to irradiation

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, tissue and clinical radiosensitivity based on the determination and cross-checking of a parameter and cellular and enzymatic criteria as well as a kit for predicting cell, tissue and clinical radiosensitivity. of an individual.

State of the art

Approximately 1 to 15% of patients treated by radiotherapy for cancer show a tissue reaction (such as a dermatitis or proctitis) that may jeopardize the smooth course of treatment as it may lead the physician to decide on discontinuation of radiotherapeutic treatment before the end of the planned protocol. Moreover, this tissue reaction is an indicator of a particularly high sensitivity of the patient to ionizing radiation. Thus, radiotherapy treatment, even if interrupted at the time of the appearance of the first visible tissue signs, may increase the morbidity or even the post-treatment mortality of the patients, not only because the cancer it was supposed to treat was not able to be completely eradicated due to the premature termination of treatment, but also because of the collateral damage of healthy tissue induced by the radiation itself.

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 organization has repair systems specific to each type of DNA damage. For CBD, mammals have two primary modes of repair: suture repair (ligation of strands) and recombinant repair (insertion of a homologous or non-homologous strand).

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 in 1981. Thus, various studies on the therapeutic effects and side effects of radiotherapy have shown that there are individuals who enjoy radioresistance particularly high, and individuals that show instead a radiosensitivity that can range from a clinically recognized side effect but without consequences to a lethal effect. Even apart from some rare cases of extreme radiosensitivity, whose genetic origin seems to be proven, radiosensitivity seems to follow generally from a genetic predisposition: it is therefore specific to an individual. It would therefore be desirable to have a predictive test method to determine the maximum cumulative dose that a given individual such as a given patient can safely receive. This question arises first of all in radiotherapy in a context of high ionizing doses. However, this question is also likely to arise for any other exposure to high ionizing doses, equivalent to those used in radiotherapy.

Two proteins of the kinase family, commonly known as ATM and ATR, are known to be involved in the detection, repair and signaling of CBD; their action requires at least the presence of a protein known as the BRCA1 designation and an ordered phosphorylation cascade of the different ATM substrates (see the article by N. Foray et al., "A subset of ATM- and ATR-dependent phosphorylation events requires BRCA1 protein ", published in The EMBO Journal 22 (1 1), 2860-2871 (2003)). The test was undertaken to use the ATM enzyme in an explanatory model of cellular radiosensitivity (see Joubert et al., "DNA double-strand break repair defects in syndromes associated with acute radiation response" At least two different assays to predict intrinsic radiosensitivity? ", published in Int.J. Radiat Biol., vol 84 (2), pp. 107-125 (2008)), and this allowed identification of three types of radiosensitivity: radioresistant cells ( so-called Group I radiosensitivity), moderately radiosensitive cells (so-called Group II radiosensitivity), and extremely radiosensitive cells (so-called Group III radiosensitivity).

Numerous documents describe the conditions under which ΓΑΤΜ can contribute to the detection and repair of DNA damage (Gately et al., "Characterization of ATM Expression, Localization, and Associated DNA-dependent Protein Kinase Activity" Molecular biology of the cell 1998; Khalil et al. "ATM in focus: A damage sensor and cancer target" biodiscovery, 2012; Bhatti et al. kinase: the cell line of cellular defenses to stress "Cellular and Molecular Life Science, 201 1; Khoronenkova and Dianov, "ATM prevents DSB training by coordinating SSB repair and eye cycle progression," PNAS 2014).

These latter documents therefore describe radiation-induced CBD repair pathways but offer no correlation to establish a link with observations and clinical diagnoses. The application WO 2015/121 596 A1 describes a predictive method for characterizing the radiosensitivity and the tissue reaction of a patient to therapeutic ionizing radiation. More particularly, this method includes the irradiation of a biological sample, the detection and quantification of radioinduced nuclear foci on fibroblastic-type connective tissue by the use of at least two detection markers among pH2AX, pATM and MRE1 1.

It is the only method of the state of the art that quantifies individual radiosensitivity, assesses the risk of post-radiotherapy tissue reactions, and can be used for any patient and any type of ionizing radiation that may be present. induce CBD, and that is predictive. However, this method requires specific equipment for the irradiation of cell samples and the quantification of radioinduced nuclear foci. In particular, the analysis of foci in lymphocyte-like cells is quite difficult because of the small size of their nucleus and makes this method complex and time-consuming. The problem that the present invention seeks to solve is to propose a predictive method for characterizing the radiosensitivity and the tissue reaction of an individual such as a patient to ionizing radiation, which is easier to use than that described in the document. WO 2015/121 596 A1.

In particular, the present invention aims to propose a new rapid, simple and reliable predictive method of individual radiosensitivity making it possible to screen individuals such as patients and guide them if the test is positive towards a more complete method of Tissue and clinical radiosensitivity.

OBJECTS OF THE INVENTION The inventors have found that, according to the method according to the invention, the pATM marker provides information on activation of the suture pathway by phosphorylation of H2AX and inhibition of the MRE1 pathway. dependent.

The inventors have also found that the amount of pATM present in the basal state in the cytoplasm and the nucleus of a cell is a function of the cell type studied. Of surprisingly, the inventors have also found that the radiosensitivity status of an individual can be easily determined as a function of the amount of pATM present in the basal state in a cell of a given cell type, and this without this cell undergoes stress related to ionizing radiation inducing CBD within the cell. It is 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.

According to the invention the problem is solved by a method based on: 1) detecting the amount of total, cytoplasmic and nuclear ATM protein; 2) a mechanistic model valid for human cells, especially blood cells; 3) functional tests regardless of the therapeutic modality.

Surprisingly, the inventors have discovered that the radiosensitivity of an individual can be predicted from non-irradiated cells.

Surprisingly, the inventors have discovered that the radiosensitivity of an individual can be predicted from cells taken from the blood of that individual.

Surprisingly, the inventors have discovered that the radiosensitivity of an individual can also be predicted from mucosal cells, epithelial cells, fibroblasts such as those present in the capillary bulbs, the epithelium of the inner face of the cheeks or from skin, said individual. Surprisingly, the inventors have discovered that the radiosensitivity of an individual can be predicted by a simple ELISA test without using foci counting techniques.

The present method is applicable to the determination of radiosensitivity in relation to high doses as usually used in the treatment of cancers by ionizing radiation (electrons, photons, protons, particles) such as proton therapy or radiotherapy. . In radiotherapy, the absorbed doses are generally between 0.5 Gy and 6 Gy. This dose range typically corresponds to an individual session of radiotherapeutic treatment, the number of sessions depending on the location, type and stage of advancement. of the tumor. The dose for a radiotherapeutic treatment session is often of the order of 2 Gy.

A first subject of the invention is a method for characterizing the radiosensitivity of an individual cell sample to ionizing radiation, said cell sample having been obtained from cells taken from said individual, said method being characterized in that the radiosensitivity is determined from the amount of ATM protein phosphorylated pATM in the basal state t ₀ ,

 said method does not include irradiating said cell sample. Advantageously, for the determination of the amount of the pATM protein, an immunoenzymatic method, such as an ELISA test, is used.

In this advantageous embodiment, the amount of the activated pATM protein can be detected in the cytoplasm and the cell nucleus by an ELISA assay.

These embodiments are suitable for any type of cell sample, but advantageously, the cell sample is selected from: a blood sample comprising lymphocytes or lymphoblasts, and a sample from a biopsy comprising fibroblasts.

A second subject of the invention is a method for characterizing the radiosensitivity of a cellular sample of an individual to ionizing radiation said cellular sample having been obtained from cells taken from said individual, in which process

(a) optionally, target nucleated cells are isolated from the cell sample;

(b) total fractions or cytoplasmic and nuclear fractions or nuclear fractions of the cell sample are extracted;

(c) determining on said fractions of said cell sample, the amount of ATM protein phosphorylated pATM in basal state t ₀ ;

(d) the radiosensitivity of the cell sample is determined using at least one parameter selected from the group consisting of the average number pATM to (normalized naked), the average number pATM to (standard cyto + nuc), the average number pATM to (normalized total), and the average number pATM L _t o (normalized total)!

 and or

^• pATM to (standard naked), expressed in ppm, denotes the average amount of pATM present in the nuclear compartment in the basal state t ₀ expressed in ng divided by the total mass of cellular proteins extracted from said nuclear compartment expressed in μg;

^• to Patm (cyto + nuc normalized), expressed in ppm, is the average amount of Patm present in the cytoplasmic and nuclear compartments basal condition t ₀ expressed in ng divided by the total weight of cellular proteins extracted from said cytoplasmic compartments and nuclear expressed in μg; ^• pATM to (normalized total), expressed in ppm, denotes the average total quantity of pATM extracted from the cells expressed in ng divided by the total mass of proteins extracted from said cells expressed in μg;

^• Patm l_to (normalized total), expressed as (ng / ml) / million cells refers average concentration Patm present in the cells in the basal condition t ₀ expressed in ng / ml divided by the total number of cells.

The total fractions or the cytoplasmic and nuclear fractions or the nuclear fractions of the cell sample are chosen in step (b) according to the parameter that it is desired to use in step (d) to determine the radiosensitivity of the cell. cell sample.

In one embodiment, between step (c) and step (d), the amount of the pATM phosphorylated ATM protein at base state t ₀ determined in step (c) is calculated from , a parameter selected from the group consisting of the average number pATM to (normalized nude), the average number pATM _t o (standard cyto + nuc), the average number pATM _t o (normalized total), and the average number pATM L _{t0 (to} standard tai) -

Advantageously, in step (d) the radiosensitivity of the cell sample is determined by comparing at least one parameter selected from the group formed by the average number pATM _t o (normalized naked), the average number pATM _t o (cyto + normalized nuc), the average number pATM to (normalized total), and the average number pATM L _t0 (normalized total) to a threshold value Z, and we consider that the sample is: radioresistant if the selected parameter is greater than or equal to Z

 radiosensitive if the selected parameter is less than Z,

and or

Z is a decimal or integer threshold value expressed in the same unit as that of the selected parameter.

Advantageously, said target nucleated cells are chosen from: mucosal cells, epithelial cells, lymphoblasts, lymphocytes and fibroblasts, and even more preferentially from lymphoblasts, lymphocytes and fibroblasts.

In another embodiment, the radiosensitivity of a cell sample of an individual to ionizing radiation is determined on fibroblasts derived from biopsy of said individual and in which the sample is considered radio-resistant if pATM to (normalized naked) ≥ B, it is considered that the sample is radiosensitive if pATM to (normalized nude) <B, where

pATM to (standard naked), expressed in ppm, has the meaning indicated above, and B is an integer or decimal value between 1 x 10 ^-2 and 1 x 10 ² ppm.

In another embodiment, the radiosensitivity of a cell sample of an individual to ionizing radiation is determined on fibroblasts derived from biopsy of said individual and in which the sample is considered radio-resistant if pATM to (cyto + nuc normalized) ≥ C, - the sample is considered radiosensitive if pATM to (cyto + normalized nuc) <C, where

pATM to (standard cyto + nuc), expressed in ppm, has the meaning indicated above, and C is an integer or decimal value between 1 x 10 ^-2 and 1 x 10 ² ppm In another embodiment, radiosensitivity of a cell sample of an individual to ionizing radiation is determined on biopsy fibroblasts of said individual and in which it is considered that the sample is radioresistant if pATM to (normalized total) ≥ D, it is considered that the sample is radiosensitive if pATM to (normalized total) <D, where

pATM to (standard total), expressed in ppm, has the meaning indicated above, and D is an integer or decimal value between 0.1 x 10 ^-1 and 5 x 10 ² ppm.

In another embodiment, the radiosensitivity of an individual's cell sample to ionizing radiation is determined on lymphoblasts and / or lymphocytes derived from blood cells of said individual and wherein the sample is considered radio-resistant if pATM to (normalized naked) ≥ A, the sample is considered radiosensitive if pATM to (normalized naked) <A, where

 - pATM to (standard naked), expressed in ppm, has the meaning indicated above, and

A represents an integer or decimal value of between 1 x 10 ^-2 and 5 x 10 ² ppm.

In another embodiment, the radiosensitivity of an individual cell sample to ionizing radiation is determined on lymphoblasts and / or lymphocytes derived from blood cells of said individual, preferably on lymphocytes derived from blood cells of said individual. and in which the sample is considered radio-resistant if pATM l_to (normalized total) ≥ E, the sample is considered radiosensitive if pATM l_to (normalized total) <E, where

 pATM l_to (normalized total), expressed in (ng / ml) / million cells, has the meaning indicated above, and

E represents an integer or decimal value of between 1 x 10 ^-2 and 5 x 10 ² (ng / ml) / million cells.

A third object of the invention is a kit for characterizing the radiosensitivity of a cell sample of an individual to ionizing radiation, comprising:

 a) means for extracting the total fractions of the cell sample,

 b) means for determining, on said fractions of said cell sample, the amount of ATM protein phosphorylated pATM in the basal state to.

A fourth object of the invention is the use of this kit for determining the radiosensitivity, preferably the radiosensitivity status of a cellular sample of an individual to ionizing radiation.

DESCRIPTION OF THE FIGURES FIGS. 1 and 2 illustrate certain aspects of the invention, but do not limit its scope.

Figure 1 shows a sensitivity / specificity curve (also called a ROC diagram of the English Receiver Operating Characteristics). This diagram makes it possible to evaluate the performance of a test by representing the rate of true positives (fraction of the individuals detected positive that are detected correctly by this test) as a function of the false positive rate (fraction of the individuals detected positive by this test then that they are actually negative). This diagram shows the performance of the prediction of the radiosensitivity of an individual evaluated from fibroblasts according to the ELISA method explained later where the sample is considered radio-resistant if pATM to (normalized normal) ≥ B ppm or radiosensitive if pATM _t o (normalized normal) <B ppm, with pATM _t o (naked normalized), expressed in ppm, denoting the average amount of pATM present in the nuclear compartment in the basal state t ₀ expressed in ng divided by the total mass of proteins cells extracted from said nuclear compartment expressed in μg and detected by ELISA.

This B decimal value is thus between 1 x 10 ^-2 and 1 x 10 ² ppm.This threshold value B is fixed and is determined by the ROC curve (statistical tests in binary). depending on the number of true or false tolerated positives, ie depending on the desired sensitivity and specificity.

For a sensitivity of 0.68 and a specificity of 0.95, B is 7.6 ppm. Figure 2 shows the classification tree of the tested cell samples as a function of the pATM parameter _t0 (normalized _nuc ) and the threshold value B of 7.6 ppm. The classification approach used is based on the binary decision tree method (L. Breiman, J. Friedman, R. Olshen and C. Stone, "Classification and regression trees", Wadsworth & Brooks, 1984.). When for a given cell sample, the amount of pATM to (normalized nuc) is greater than or equal to the threshold value B (here 7.6 ppm), it is then radioresistant and is classified in category 0. When the amount of pATM _t o (normalized nuc) is below threshold value B, the sample is considered radiosensitive and is classified in category 1.

 Of 40 tested cell samples, 26 are categorized as 0, i.e. radio-resistant and 14 are classified as radio-sensitive, i.e.

 Of the 26 samples in category 0, 20 are true positives (fraction of individuals detected positive that are correctly detected by this test) and 6 are false positives (fraction of individuals detected positive by this test when they are actually negative).

Of the 14 samples in Category 1, 13 are true positives and 1 is a false positive.

FIG. 3 represents the histogram distribution of the tested cell samples as a function of the amount of pATM _t0 (normalized _nuc ) measured on the sample in ppm (ie average amount of pATM present in the nuclear compartment in the basal state). t ₀ expressed in ng divided by the total mass of cellular proteins extracted from said nuclear compartment expressed in μg). The threshold value B for classifying the samples according to their radiosensitivity status as a function of the amount of pATM _t o (normalized nuc) is represented on the histogram by a vertical bar at 7.6 ppm. FIG. 4 represents, according to the Wilcoxon test (also called the Mann-Whitney U test or the Wilcoxon rank sum test), the amount of pATM to (standardized cyto + nuc) measured on the cellular samples in ppm (ie from Patm present in the cytoplasmic and nuclear compartments basal condition t ₀ expressed in ng divided by the total weight of cellular proteins extracted from said cytoplasmic and nuclear compartments expressed in g) as a function of their status of radiosensitivity. The Wilcoxon test is a non-parametric statistical test to test the hypothesis that the distribution of data is the same for two groups, here for the two radiosensitivity states. The results of this test are characterized by the probability p according to which the difference observed between the two groups, ie two radiosensitivity status is only due to chance. The lower the value p, the greater the probability of having two groups of data that are significantly different. The probability p associated with this test is 0.001 and is relatively low indicating the existence of two distinct groups of data, ie two groups of radiosensitivity.

 The points present outside the uncertainty bars (°) are aberrant points.

Figure 5 shows the variations in the proportion of pATM to (normalized naked) present in the cell nucleus of lymphoblasts from blood samples of patients GM03798, GM03380, GM07078, 37792 and 40479.

FIG. 6 represents a matrix illustrating the correlations existing between the quantities of pATM present in the nucleus and the cell cytoplasm of lymphoblasts originating from blood samples of individuals in the basal state (0 Gy - non-irradiated state), after 5 min. and 10 min post irradiation at 2 Gy.

0Gy_nuc denotes the average amount of nuclear pATM present in the basal state t ₀ , expressed in ppm.

 5mnucu refers to the average amount of nuclear pATM observed in the nucleus 5 minutes after exposure of the cell sample to irradiation with a dose of 2 Gy. 5mnucu is expressed in ppm.

 10mnucu means the average amount of nuclear pATM observed in the nucleus 10 minutes after exposure of the cell sample to irradiation with a dose of 2 Gy.

 10mnucu is expressed in ppm.

Gy_cyto refers to the average amount of pATM present in the basal state t ₀ in the cytoplasm of lymphoblasts, expressed in ppm.

 5mn_cyto refers to the average amount of pATM observed in the cytoplasm of lymphoblasts 5 minutes after exposure of the cell sample to irradiation with a dose of 2 Gy.

5mn_cyto is expressed in ppm.

 10mn_cyto refers to the average amount of pATM observed in the cytoplasm of lymphoblasts 10 minutes after exposure of the cell sample to irradiation with a dose of 2 Gy.

 10mn_cyto is expressed in ppm.

These amounts expressed in ppm represent the average amount of pATM observed in a given compartment in ng divided by the total mass of proteins present in said compartment in μg. Groups A, B and C, shown in FIG. 6, show the correlations existing between the aforementioned variables.

FIG. 7 represents a sensitivity / specificity curve (also called a Receiver Operating Characteristics (ROC) diagram). This diagram makes it possible to evaluate the performance of a test by representing the rate of true positives (fraction of the individuals detected positive that are detected correctly by this test) as a function of the false positive rate (fraction of the individuals detected positive by this test then that they are actually negative). This diagram shows the performance of the prediction of the radiosensitivity of an individual evaluated from lymphocytes according to the ELISA method subsequently clarified where the sample is considered radioresistant if pATM Lto (total normalized) ≥ E (ng / ml) / million cells or radiosensitive if pATM Lto (normalized total) <E (ng / ml) / million cells, with pATM Lto (total normalized), expressed in (ng / ml) / million cells, denoting the average concentration of pATM present in the cells at baseline t ₀ expressed in ng / ml divided by the total number of cells.

This E decimal value is thus 1 x 10 ^-2 and 5 x 10 ² (ng / ml) / million cells This threshold value E was fixed and determined by the ROC curve (statistical tests in binary) as a function of the number of true or tolerated false positives, ie, depending on the sensitivity and specificity desired, for an area under the curve (AUC of the English Area Under Curve) at 81% with a negative predictive value (abbreviated NPV). English Negative Predictive Value) at 87% and a positive predictive value (PPV) of 62%, the E value is 3.86 (ng / ml) / million cells.

Description of the invention

A. General Definitions

The terms "radioinduced damage", "radioinduced", "radiosensitivity", "radioresistance", "radiotoxicity", "radiotherapy" all refer to ionizing radiation (as defined by IUPAC, known to those skilled in the art). ), and in particular to a particle-type radiation, such as consisting of particles of the proton, alpha (a) or beta (β) type, or else to a high energy electromagnetic radiation, in particular of the gamma (γ) or X type. .

The term "basal state" of a protein means the level of expression of said protein in the absence of activation or repression, especially in the absence of damage induced by oxidative stress, especially in the absence radiation damage. The term "nuclear compartment" means the set of spaces of a nucleated cell delimited by a nuclear membrane and constituting the nucleus.

The term "cytoplasmic compartment" means the set of spaces of a nucleated cell delimited by a cytoplasmic membrane and constituting the cytoplasm excluding the nuclear compartment.

The term "total fraction" is understood to mean an extract of the cell sample obtained after total lysis containing all of the proteins of the cell, including nuclear and cytoplasmic proteins and more particularly pATM proteins.

The term "cytoplasmic fractions", an extract of the cytoplasmic compartment of the cell sample obtained after subcellular fractionation, this extract containing cytoplasmic proteins including pATM proteins.

The term "nuclear fractions" means an extract from the nuclear compartment of the cell sample obtained after subcellular fractionation, this extract containing nuclear proteins including pATM proteins. Biological parameters pATM to (nude), pATM to (naked normalized), pATM tO (cyto + naked), pATM tO

(standardized cyto + nuc), pATM tO (total), pATM tO (normalized total), pATM l_to (normalized total), Used in the context of this description, are defined in section 4b below.

B. Detailed Description Here we describe an embodiment with several variants that is suitable for an individual such as a human patient. The method according to the invention uses at least one sample of healthy tissue, preferably fibroblasts or lymphoblasts / lymphocytes. The first are preferably taken from the connective tissue of an individual and the second from the blood. This sample can be taken by biopsy or blood sample.

1. Preparation of the test

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

Then, the operator takes from said individual a cell sample such as a sample blood cell comprising lymphoblasts or lymphocytes or a skin sample comprising fibroblasts.

Cellular skin sample

The skin cell sample is generally taken by biopsy via the method of "dermatological punch" known to those skilled in the art. The cell sample is then placed in DMEM medium + 20% sterile fetal calf serum and then transferred without delay to a specialized laboratory, so that the sample does not remain for more than 38 hours at room temperature and is not altered.

The cell sample from a biopsy can be used as is, upon receipt. It can also be established in the form of an amplifiable cell line according to a procedure that is well known in the culture laboratories and to those skilled in the art: by using in particular the tryptic dispersion (trypsination), the cells are again diluted in medium renewed and so on until the desired number of cells is obtained. After obtaining a sufficient number of cells (usually after one to three weeks), the first experiments are carried out using the method according to the invention. The cells are seeded on glass slides in petri dishes. These basal state fibroblasts thus obtained are then used as they are, without being irradiated (absorbed dose 0 Gy). - Blood sample

 The blood sample includes lymphoblasts or lymphocytes.

The lymphocytes and / or lymphoblasts can be isolated by any appropriate means. The lymphocytes and / or lymphoblasts are preferably isolated from fresh whole blood via the technique of Ficoll ^® known to those skilled in the art for more than 30 years and in particular described in the article by B0yum published in 1968 entitled "Isolation of mononuclear cells and granulocytes from human blood. "(Paper IV). Scand. J. Clin. Lab. Invest. 21 Suppl. 97, p.77-89.

The separation of the blood components may be carried out by centrifugation on a cushion consisting of a copolymer of sucrose (sucrose) and epichlorohydrin, Ficoll ^®. After centrifugation under appropriate conditions known to those skilled in the art, the constituents of the blood are separated by density. After removal of the supernatant containing the plasma and platelets, the lymphocytes and / or lymphoblasts are extracted from the cell sample and are then washed with at least one centrifugation / redispersion cycle in order to remove all traces of Ficoll ^® , which is a toxic polymer for the cells. After removing the supernatant containing the Ficoll ^®, the thus purified lymphocytes are redispersed in a suitable culture medium, preferably in a RPMI 1640 culture medium provided by Gibco and comprising 10% fetal calf serum and 1% penicillin / streptomycin. These basal cell lymphocytes thus obtained are then used as they are, without being irradiated (absorbed dose 0 Gy). The biological material on which the experiments are carried out may be in particular lymphoblasts, isolated lymphocytes or cells isolated from tissue.

2. Extraction of total fractions or cytoplasmic and nuclear fractions or nuclear fractions of the cell sample

The biological material is subjected to total lysis by any appropriate means. Preferably, the lysis of the cells is carried out by the use of a RIPA lysis buffer (Thermofisher) according to the methodology described in the article by Kirby et al., "Adult hippocampal neural stem and progenitor cells regulate the neurogenic niche by VEGF secreting »Proc Natl Acad Sci USA 2015; (1 12): 13 p.4128-4133 or subcellular fractionation to recover total proteins or to extract and separate cytoplasmic proteins from nuclear proteins in the basal state. In one embodiment, this fractionation is carried out by means of a kit (PIERCE) according to the methodology described for example in the article by Trotter and Archer, "Reconstitution of glucocorticoid receptor-dependent transcription in vivo", Molecular and Cellular Biology (2004).

In the case of subcellular fractionation, the cytoplasmic and nuclear fractions can be detected by immunoblotting tests also called "Western blot". Antibodies for detecting the presence of target proteins specific for each compartment are employed, such as anti-tubulin or anti-actin for the cytoplasmic compartment and anti-topoisomerase for the nuclear compartment. After isolation of the cytoplasmic and nuclear compartments, immunoblotting tests can then be performed on the protein mixtures contained in each of the isolated cytoplasmic and nuclear fractions in order to detect the presence of phosphorylated ATM proteins in each of the fractions. The phosphorylated ATM proteins are present in each of the cytoplasmic and nuclear fractions.

The isolation and the capture of the phosphorylated ATM proteins in each of the total, cytoplasmic and nuclear fractions are carried out by any appropriate means, preferably by an ELISA test. 3. Quantification of pATM Proteins by ELISA Test:

The isolation and the capture of the phosphorylated (and therefore active) ATM proteins in each of the total, cytoplasmic and / or nuclear fractions is carried out by TEST ELISA (Kit R & D Systems and Novateinbio) thanks to the use of plates coated with a specific antibody. anti-pATM (R & D Systems and Novateinbio). In each fraction, the amount of pATM present is then analyzed by luminescence at 450 nm using a plate reader and the total amount of protein present is determined by a Bradford test known to those skilled in the art.

 In each well of each plate making it possible to carry out this ELISA test, were determined among the total quantity of proteins present in μg, the quantity of pATM present in ng.

 This method has been implemented and validated on five lines of lymphoblasts, on human lymphocytes of 122 patients and 40 human fibroblast lines.

4. Determination of biological and clinical parameters a. Overview

 The method according to the invention is based on the analysis of the kinase activity of the ATM protein present in the cell sample in the basal state studied, and more particularly on the quantification of the active ATM protein, ie the pATM marker. present in the nucleated cells of said sample, ie in the total, cytoplasmic and nuclear fractions of these cells in the basal state, ie unexposed (spontaneous state).

 The averages obtained for each point are computed with standard deviation errors of the mean (called "SEM") over a number of replicates greater than or equal to 2. When the number of replicates is less than 30, the Gaussian standard deviation standard error type

SE "can not be applied. b. Biological parameters

The parameters used according to the invention are presented below.

pATM to (naked) denotes the average amount of nuclear pATM present in the basal state t ₀ , expressed in ng. pATM to (standard naked) designates the average amount of pATM present in the nuclear compartment in the basal state t ₀ expressed in ng detected by ELISA divided by the total mass of cellular proteins extracted from said nuclear compartment expressed in μg. pATM _t o (normalized naked) is expressed in ppm. pATM to (cyto + nuc) refers to the average amount of pATM present in cytoplasmic and nuclear compartments in the basal state t ₀ , expressed in ng. Patm to (cyto + nuc standardized) means the average amount of Patm present in the cytoplasmic and nuclear compartments basal condition t ₀ expressed in ng detected by ELISA divided by the total weight of cellular proteins extracted from said cytoplasmic and nuclear compartments expressed in micrograms. pATM to (standard cyto + nuc) is expressed in ppm. Patm to (total) is the total average amount of Patm present either basal t ₀ extracted from cells present in the analyzed sample, expressed in ng. pATM to (standard total) refers to the total average amount of pATM extracted from the cells expressed in ng divided by the total mass of proteins extracted from said cells expressed in μg. pATM _t o (normalized total) is expressed in ppm. pATM l_to (normalized total) refers to the average concentration of pATM present in basal cells t ₀ expressed in ng / ml divided by the total number of cells. pATM Uo (normalized total) is expressed in (ng / ml) / million cells.

In the context of the present invention, the parameters used are measured in basal condition t ₀ and, preferably, by an ELISA.

In the context of the present invention, normalized parameters are calculated from the quantity of the phosphorylated protein ATM Patm to basal condition t ₀ present in the cell sample. The normalized parameter pATM to (normalized normal), (respectively pATM to (standardized cyto + nuc)) is calculated from the amount of ATM phosphorylated pATM protein in the basal state t ₀ present in the nuclear fractions (respectively in the cytoplasmic and nuclear fractions) of the cell sample. The normalized pATM to (normalized total) parameter is calculated from the amount of ATM phosphorylated pATM protein in the basal state t ₀ present in the total fractions of the cell sample. The standardized pATM Lto parameter (normalized total) is calculated from the quantity (ie concentration) of the pATM phosphorylated ATM protein at basal state t ₀ present in the total fractions of the cell sample. vs. Predictive evaluation

The method according to the invention aims at the prediction of clinical parameters such as the radiosensitivity status from measured biological data, ie the amount of the active forms of the ATM protein in the cytoplasmic and nuclear compartments of nucleated cells given. This predictive evaluation results from a correlation between clinical data observed on cell lines such as the CTCAE grade and measured biological data.

The so-called CTCAE (Common Terminology Criteria for Adverse Events) classification, published in 2006 by the National Cancer Institute of the United States of America, is a descriptive terminology of adverse events (particularly side effects) in cancer therapy.

An adverse event is any adverse and unintended sign, symptom or illness temporally associated with the use of a medical treatment or procedure that may or may not be considered to be related to the treatment or medical procedure. An adverse event is a unique representation of a specific event used for medical documentation and scientific analysis.

The so-called CTCAE classification provides a brief definition of each adverse event to clarify the meaning of the adverse event. This scale, valid for other genotoxic stress (eg fire injury) is particularly used in radiotherapy.

The grade refers to the severity of the adverse event. The CTCAE displays 5 severity grades (1 to 5) with unique clinical descriptions of severity for each adverse event and described in Table 1 below. Each grade of severity is defined by specific tissue reactions.

Table 1: latest version of the CTCAE scale published by the National Cancer Institute of the United States of America on June 14, 2010 grade 1 Slight severity; asymptomatic or mild symptoms; clinical or diagnostic observations alone; intervention not indicated grade 2 Moderate severity; minimal intervention, local or noninvasive intervention indicated; event limiting the activities of daily life instrumented and adapted to the age (preparation of the meal, to do the shopping, use of the telephone ...) grade 3 Serious severity or medically significant, but not immediately putting in danger; hospitalization or prolongation of the indicated hospitalization; disabling event; event limiting activities of care of daily life (baths, dressing, feeding, toilet use, taking medication, and not being bedridden) grade 4 Potentially life-threatening consequences; urgent intervention indicated grade 5 Death related to the adverse event

To these 5 grades is added a grade 0 corresponding to an absence of tissue effect.

The method according to the invention allows a qualitative diagnosis, which is directly derived from the mathematical value of scores or mathematical formulas using these scores.

According to the invention, the radiosensitivity status can be determined on any type of nucleated cells and preferably on lymphocytes, lymphoblasts or fibroblasts.

The method according to the invention makes it possible to characterize the radiosensitivity of a cellular sample of an individual to an ionizing radiation by comparing with a threshold value the amount of ATM protein phosphorylated pATM in the basal state t ₀ . This method is binary insofar as it leads to the storage of cellular samples in two classes: "radiosensitive" and "radioresistant". It is simple, easy to use and without irradiation of said cell sample; this significantly facilitates its use.

Predictive Evaluation of the Radiosensitivity Status from a Blood Sample In an advantageous embodiment, the radiosensitivity of an individual cell sample to ionizing radiation is determined on lymphoblasts and / or lymphocytes derived from blood cells. of said individual as follows: the sample is considered radio-resistant if pATM to (normalized normal) ≥ A is considered to be radiosensitive if pATM to (normalized nuci) <A where

 pATM to (standardized naked) has the meaning given above in section 4b, and

A is an integer or decimal value between 1 x 10 ^-2 and 5 x 10 ² ppm.

In another advantageous embodiment, the radiosensitivity of a cellular sample of an individual to ionizing radiation is determined on lymphoblasts and / or lymphocytes, preferably lymphocytes derived from blood cells of said individual, as follows: considers that the sample is radio-resistant if pATM Lto (normalized total) ≥ It is considered that the sample is radiosensitive if pATM Lto (normalized total) <E where

pATM Lto (normalized total) has the meaning given above in section 4b, and E is an integer or decimal value between 1 x 10 ^-2 and 5 x 10 ² (ng / ml) / million cells.

The radiosensitivity status can be determined according to pATM to (standard nude) or pATM Lto (normalized total) - In a preferred embodiment, with a blood sample, only one test is performed, ie that based on pATM to (normalized nude), that is based on pATM Lto (normalized total) -

These results are based on a correlation analysis of the discriminant variables (statistical technique allowing to assign to an individual his radiosensitivity status based on his physical characteristics). The threshold value A was determined by a correlation analysis of the discriminant values. The threshold value E was determined by a correlation analysis of the discriminant values.

Predictive evaluation of radiosensitivity status from a skin sample

The radiosensitivity status of an individual is determined from a skin sample of said individual based on a threshold value expressed in relative value in parts per million (ppm).

In an advantageous embodiment, the radiosensitivity of a cellular sample of an individual to an ionizing radiation is determined on fibroblasts resulting from a skin biopsy of said individual in the following manner: it is considered that the sample is radioresistant if pATM to (normalized N) It is considered that the sample is radiosensitive if pATM to (normalized normal) ^< B

 OR

 pATM to (standardized naked) has the meaning given above in section 4b, and

B is an integer or decimal value between 1 x 10 ^-2 and 1 x 10 ² ppm.

This threshold value B is fixed and is determined by the ROC curve (statistical tests in binary) according to the number of true or false positive tolerated, ie according to the desired sensitivity and specificity. For a sensitivity of 0.68 and a specificity of 0.95, B is 7.6 ppm (see Figures 1 and 2). Figure 2 shows the classification tree for 40 cell samples tested as a function of the pATM to (normalized nude) parameter and the B threshold value of 7.6 ppm (see Figure 3). In another advantageous embodiment, the radiosensitivity of a cellular sample of an individual to an ionizing radiation is determined on fibroblasts resulting from a skin biopsy of said individual in the following manner: the sample is considered to be radio-resistant if pATM to (cyto + normalized nuc) ≥ C - the sample is considered radiosensitive if pATM to (cyto + normalized nuc) <C where

pATM to (standard cyto + nuc) has the meaning given above in section 4b, and C is an integer or decimal value between 1 x10 ^"2 and 1 x 10 ² ppm Determination of the radiosensitivity of a cell sample of an individual to ionizing radiation according to the latter method was validated by the Wilcoxon test with a probability of 0.001 indicating the existence of two distinct groups of data, ie two groups of radiosensitivity (see Figure 4).

In another advantageous embodiment, the radiosensitivity of a cellular sample of an individual to an ionizing radiation is determined on fibroblasts resulting from a skin biopsy of said individual in the following manner: the sample is considered to be radio-resistant if pATM to (normalized total) ≥ D is considered to be radiosensitive if pATM to (normalized total) <D where

 pATM to (normalized total) has the meaning given above in section 4b, and

D is an integer or decimal value between 0.1 x 10 ^-1 and 5 x 10 ² ppm.

The threshold values B and C are fixed and are determined by the curve ROC (statistical tests in binary) (Figure 1) which makes it possible to evaluate the performance of this test by representing the rate of true positives (fraction of the individuals detected positive which are detected correctly by this test) according to the rate of false positives (fraction of the individuals detected positive by this test whereas they are actually negative) (Figure 2). The threshold value D is determined by deducting the other two threshold values B and C.

These formulas are derived from clinical data observed on the fibroblast lines presented in the following tables 2 and 3 and correlated with the amount of pATM to (normalized naked) OR pATM _t 0 (standardized cyto + nuc) - the quantities of pATM tO (normalized naked) and pATM to (standardized cyto + nuc) have the meaning given above in section 4b. 2: Relative amounts of pATM protein in the nucleus in ppm (pATM to (normalized normal)) in the basal state in fibroblasts pATM to (naked normalized) Grade CTCAE Clinically determined radiosensitivity status

Lineage (ppm) observed

 clinically

 "+" = Radioresistant (integer)

 "-" = radiosensitive

03OLL 24.76 1 +

 HDF 5,04 0+

 HFF2 10.87 0 +

 I MR90 16.95 +

 THORS 2 TS1 10,53 1 +

 1 BR3 6.91 0 +

 DIJ002 43.44 1 +

 DIJ003 27.27 1 +

 DIJ006 35.46 1 +

 DIJ004 1 1, 24 1 +

 DIJ005 10.05 1 +

 NeoGre002 19.8 1 +

 NeoGre003 9, 7 1 +

 NeoGre004 64.54 1 +

 02CUR 5.94 2 -

1603946-CAR-AI 3.08 2 -

1306835 6.77 2 -

01 CJP 7.54 3 -

01 DAX 33.8 3 -

02CAV 1, 25 3 -

02CLB 5.64 4 -

02MTZ 3,52 3 -

03HLS 2.45 3 -

03HM 3.44 2 -

04CAV 2.91 4 -

05HM 5.53 3 -

07HM 10.54 3 -

08H NG 0.21 3 -

09HM 9.71 2 -

10HM 4.02 2 -

1 1 HM 3,23 2 -

04 CLB 25, 1 2 -

07 CLB 31, 66 3 -

14CLB 0.08 3 -

17CLB 3.99 3 -

21 CLB 4.96 2 -

34CLB 17.49 3 - Table 3: Relative Quantities in ppm of the pATM Protein in the Cytoplasmic and Nuclear Compartments (pATM to (Standardized Cyto + Nuc)) Present in Fibroblasts in the Basal State

 pATM tO (Cyto + nuc Grade CTCAE Standardized radiosensitivity status) observed clinically determined

Clinically lineage

 (ppm) "+" = radioresistant

 (number

 integer) "-" = radiosensitive

03OLL 26,13 1 +

 HDF 7.82 0+

 HFF2 14.09 0 +

 IMR90 24.26 0 +

 THORS 2 TS1 13,16 1 +

 HFL1 18.5 0 +

 Wi38 18.91 0 +

 1 Br3 8.48 0 +

 DIJ002 45.63 1 +

 DIJ003 31, 07 1 +

 DIJ006 38.01 1 +

 DIJ004 1 1, 96 1 +

 DIJ005 1 1, 96 1 +

 NeoGre002 21, 67 1 +

 NeoGre003 1 1, 02 1 +

 NeoGre004 66.21 1 +

 02CUR 7.96 2 -

1603946-CAR-AI 3.81 2 -

1306835 7.8 2 -

01 CJP 8.38 3 -

01 DAX 38.9 3 -

02CAV 1, 94 3 -

02CLB 7.5 4 -

02MTZ 4,21 3 -

03HLS 3.25 3 -

03HM 4.65 2 -

04CAV 3.68 4 -

05HM 8.99 3 -

07HM 12.43 3 -

08HNG 0.26 3 -

09HM 1 1, 22 2 -

10HM 5.12 2 -

1 1 HM 4.14 2 -

04 CLB 28,24 2 -

07 CLB 34.58 3 - 14CLB 0.1 3 -

17CLB 6.78 3 -

21 CLB 6.44 2 -

34CLB 18.86 3 -

d. Levels and methods of analysis

In one embodiment, all the parameters measured experimentally enter into the determination of the scores. In case of conflict between scores, some measurements or determination are repeated, or other experimental tests are performed in order to add other measures involved in the determination of the scores. The level of some single parameters such as pATM _t o (nude normalized) or pATM L _t0 (normalized total) is sufficient to establish a score and a diagnosis. 5. Predictive Evaluation of Radiosensitivity Status from a Cell Sample Using a Kit According to the Invention

A kit according to the invention can be used to characterize the radiosensitivity of a cellular sample of an individual to ionizing radiation. This kit comprises a) means for extracting the total fractions of the cell sample, i.e. reagents such as a lysis buffer known to those skilled in the art,

 b) means for determining, on said fractions of said cell sample, the amount of ATM protein phosphorylated pATM in the basal state, such as a plate coated with an anti-pATM specific antibody making it possible to carry out an ELISA test.

Examples

The invention is illustrated below by examples which, however, do not limit the invention. These examples relate to the analysis of cell lines of individuals for determining the radiosensitivity status to which the individual belongs.

1. Preparation of the cell sample

A blood sample from an individual with lymphocytes or lymphoblasts was taken. Lymphocytes were isolated from fresh whole blood via the Ficoll ^® technique described previously. The separation of the constituents of the blood was obtained by centrifugation on a cushion of Ficoll ^® (carbohydrate polymer of high molar mass). After centrifugation for 20 min at 400 g at room temperature, the blood constituents were separated by density. After removing the supernatant containing the plasma and platelets, lymphocytes were extracted from the cell sample, and then were washed in 1 x PBS and recentrifuged under the same conditions, so as to remove all traces of Ficoll ^®. After removing the supernatant containing the Ficoll ^®, the thus purified lymphocytes were redispersed in 1X PBS.

Lymphocytes in the basal state were thus obtained and were then used as such, without being irradiated (absorbed dose 0 Gy).

A cell sample of skin of an individual was taken by biopsy via the method of "dermatological punch". The cell sample was then placed in DMEM medium + 20% sterile fetal calf serum. The cell sample was then established as an amplifiable cell line. Using in particular the tryptic dispersion, the cells were then diluted again in renewed medium and so on until the desired number of cells was obtained. After one week of culture, the first experiments were carried out using the method according to the invention. The cells thus obtained, i.e. the fibroblasts were then used as such, without being irradiated (absorbed dose 0 Gy).

Separation of cytoplasmic and nuclear compartments from nucleated cells by subcellular fractionation

 Subcellular fractionation to separate cytoplasmic pATM proteins from nuclear pATM proteins was performed on cultured fibroblasts from the individual skin cell sample and lymphocytes from the above-mentioned individual blood sample. This fractionation was carried out using the PIERCE NE-PER nuclear and cytoplasmic extraction kit according to the manufacturer's protocol and the methodology described in the article by Trotter, KW and Archer, TK, Molecular and Cellular Biology (2004). Reconstitution of glucocorticoid receptor- depend on in vivo transcription.

The presence of the proteins in each of the cytoplasmic and nuclear fractions was verified by a "Western blot" test known to those skilled in the art with antibodies specific for each cytoplasmic compartment (anti-tubulin or anti-actin) and nuclear (anti- topoisomerase). 2. Determination of the amount of pATM present in the nucleated cells in the basal state by an ELISA test (Kit R & D Systems and Novateinbio)

The amount of phosphorylated forms of ATM proteins (pATM) was measured on the cells cultured and fractionated, depending on the cell type studied. The amount of pATM protein was thus measured in each of the cytoplasmic and nuclear fractions of cultured fibroblasts resulting from the skin cell sample of the individual and in each of the cytoplasmic and nuclear fractions of the lymphocytes derived from the blood sample of the individual.

Quantification of phosphorylated forms of ATM proteins (pATM) in each of the cytoplasmic and nuclear fractions was performed by ELISA (Kit).

For each of the cytoplasmic and nuclear fractions, 50 μί were collected and placed in a well of a plate coated with a specific anti-pATM antibody (R & D Systems and Novateinbio). In each well, the amount of pATM present was then analyzed by luminescence at 450 nm using a plate reader and the total amount of protein was determined by a Bradford test known to those skilled in the art.

3. Determination of the radiosensitivity of individuals from lymphoblasts derived from these same individuals and the relative amount of standardized pATM to rmc proteins) obtained by Elisa test

Several cell samples from patients were analyzed. In order to obtain results of sufficient statistical reliability, 3 sets of independent analyzes were performed per sample. The results obtained are presented in the table below (see Table 4), and this, for different lines of lymphoblasts of patients.

Table 4: Quantities of the nuclear pATM protein (pATM to (normalized naked)) present in lymphoblasts from patients in the basal state

Grade CTCAE Status Status of pATM tO (nude

 observed radiosensitivity radiosensitivity

Standardized line)

 clinically determined determined according to

(integer) clinically (ppm) the invention

GM03798 0 radio-resistant 10,640 radio-resistant

GM03380 1 radiosensitive 6,219 radiosensitive

GM07078 1 radiosensitive 4,133 radiosensitive

37792 1 radiosensitive 6,328 radiosensitive

40479 1 radiosensitive 9,330 radiosensitive In Table 4, pATM to (standard naked) has the meaning given above in section 4b. pATM to (normalized naked) is expressed in ppm.

 Patients 40479 and 37792 show mutations in the BRCA1 gene inducing a tendency to develop breast or ovarian cancers. The lymphoblast lines GM07078, 40479 and 37792 correspond to patients for whom the CTCAE grades are known.

For the 5 cell lines (lymphoblasts) presented in Table 4, the clinically determined radiosensitivity status corresponds to that determined by the method according to the invention as a function of the relative amount of pATM to (normalized naked) and the threshold value A .

According to the invention, the sample is considered to be:

- radioresistant if pATM _t o (normalized naked) ≥ A

radiosensitive if pATM _t o (normalized nude) <A.

or

 - pATM to (standard naked), expressed in ppm, has the meaning given above in section 4b, and

- A is an integer or decimal value between 1x10 ^"2 and 5x10 ² ppm Considering these threshold values, patient GM03798 is considered radioresistant and patients GM03380, GM07078, 37792 and 40479 are considered radiosensitive. shows the variations in the proportions of pATM in the nucleus in the basal state for these patients.

 Similarly, the method has been applied to a series of lymphocytes from 122 patients shown in Table 5. The clinically determined radiosensitivity status corresponds to that determined by the method according to the invention as a function of the relative amount of pATM LTO. (normalized total) and threshold value E.

According to the invention, the sample is considered to be:

- radioresistant if pATM L _t o (normalized total) ≥ E

radiosensitive if pATM L _t o (total normalized) <E.

or

 - pATM Lto (normalized total) expressed in (ng / ml) / million cells, has the meaning given above in section 4b, and

E is an integer or decimal value between 1 × 10 ^-2 and 5 × 10 ² (ng / ml) / million cells). Table 5:

Mean concentrations of pATM protein / million cells (pATM l_to (normalized total)) in lymphocytes of 122 individuals (patients) in the basal state

PATM line L _t0 Grade CTCAE Location Radiosensitivity status

 (normalized total) observed determined

(ng / ml) / clinically clinically:

 million (whole number) "+" radioresistant;

 "-" radiosensitive cells

 CRM-00207 27.5 1 prostate +

 CRM-00208 23.95 1 prostate +

 CRM-00209 19.22 -I prostate +

 CRM-00210 21 .5 1 prostate +

 CRM-0021 1 16.99 1 prostate +

 CRM-00212 23 1 prostate +

 CRM-00213 41 .5 1 prostate +

 CRM-00214 25.1 1 prostate +

 CRM-00216 9.37 1 prostate +

 CRM-00217 10 1 prostate +

 CRM-00218 9.33 1 prostate +

 CRM-00220 12.82 2 ENT

 CRM-00221 54 4 ENT

 CRM-00222 23 1 ENT +

 CRM-00223 32.7 2 ENT

 CRM-00224 29.8 1 ORL +

 CRM-00225 1 1 .6 1 ENT +

 CRM-00226 24.37 1 prostate +

 CRM-00227 25.6 1 prostate +

 CRM-00228 36 1 prostate +

 CRM-00229 43 1 prostate +

 CRM-00230 40.3 1 prostate +

 CRM-00231 24.3 1 prostate +

 CRM-00233 58.2 1 prostate +

 CRM-00238 39.25 1 prostate +

 CRM-00241 25.3 1 ORL +

 CRM-00242 39.26 -I ENT +

CRM-00244 4.28 1 prostate + CRM-00246 1 1.6 2 ENT

 CRM-00247 1 1.8 1 ORL +

CRM-00248 2.65 2 ENT

 CRM-00249 8.2 2 breast

 CRM-00250 6.53 3 brain

 CRM-00251 10.16 2 anal channel

CRM-00252 14.7 2 rectum

 CRM-00253 5.26 2 breast

 CRM-00254 7.74 2 prostate

 CRM-00255 1 1.2 2 breast

 CRM-00256 5.3 1 right orbit +

CRM-00258 1 1.87 2 breast

 CRM-00260 10.23 1 breast +

CRM-00261 7.58 1 breast +

CRM-00262 9.25 1 anal channel +

CRM-00263 12.7 1 breast +

CRM-00264 12.63 2 breast

 CRM-00265 18.5 1 breast +

CRM-00266 12 1 breast +

CRM-00267 10 1 pelvis +

CRM-00268 9.2 1 breast +

CRM-00269 5.35 2 ENT

 CRM-00270 10.64 2 breast

 CRM-00271 8.47 2 breast

 CRM-00272 13.4 1 breast +

CRM-00273 5.9 2 Anal Channel

CRM-00274 4.43 2 ENT

 CRM-00275 8.9 1 prostate +

CRM-00276 9.9 2 ENT

 CRM-00277 19.25 1 breast +

CRM-00278 4.1 1 prostate +

CRM-00279 14.62 2 prostate

 CRM-00280 14 2 ENT

 CRM-00281 5 2 esophagus

CRM-00282 15.37 3 ENT

 CRM-00283 9.1 1 3 ENT

CRM-00284 18.7 2 breast CRM-00285 23.6 1 lung +

CRM-00286 6.33 1 breast +

CRM-00287 9 2 Prostate

 CRM-00288 9.22 1 prostate +

CRM-00289 14.59 1 prostate +

CRM-00292 6.6 2 prostate

 CRM-00293 2.48 1 breast +

CRM-00294 7.2 1 prostate +

CRM-00295 13.55 1 prostate +

CRM-00296 4.66 2 prostate

 CRM-00301 3.9 1 ORL +

CRM-00302 5.44 1 ORL +

CRM-00303 6.2 2 ENT

 CRM-00304 5 2 ENT

 CRM-00305 3 1 ENT +

CRM-00307 3.27 1 ORL +

CRM-00308 4.4 2 ENT

 CRM-00309 6 3 ENT

 CRM-00310 5.25 3 ENT

 CRM-00312 5.1 1 prostate +

CRM-00313 5.3 2 prostate

 CRM-00314 5.49 1 prostate +

CRM-00315 6.1 2 prostate

 CRM-00316 5.55 1 prostate +

CRM-00317 5.24 3 Prostate

 CRM-00318 4.22 1 breast +

CRM-00319 6.59 2 prostate

 CRM-00320 8.35 1 prostate +

CRM-00325 10.8 2 ENT

 CRM-00326 10.3 2 ENT

 CRM-00327 20.8 2 ENT

 CRM-00328 1 1.59 2 ENT

 CRM-00329 8.57 4 ENT

 CRM-00330 1 1.14 1 ORL +

CRM-0031 1 10.03 2 ENT

 CRM-00331 25.8 2 ENT

CRM-00333 1 .46 4 ENT CRM-00337 0.45 4 ENT

 CRM-00338 0.61 1 ORL +

 CRM-00339 2.39 3 ENT

 CRM-00341 4.17 2 ENT

 CRM-00342 7.32 1 ORL +

 CRM-00343 6.5 3 ENT

 CRM-00344 6.5 2 ENT

 CRM-00345 5.6 1 ORL +

 CRM-00346 6.6 3 ENT

 CRM-00348 29.8 1 ORL +

 CRM-00349 2.79 3 ENT

 CRM-00350 1 .26 4 ENT

 CRM-00352 8.9 1 ORL +

 CRM-00356 1 1 .75 1 ENT +

 CRM-00361 16.49 3 ENT

 CRM-00365 1 1 .98 1 ENT +

 CRM-00372 14.6 2 ENT

 CRM-00378 21 2 ENT

 CRM-00383 19.5 2 ENT

 CRM-00385 18 1 ENT +

 "ENT" means a location within the Otorhinolaryngology sector.

4. Validation of the method for determining the radiosensitivity of an individual according to the invention

A blood sample from an individual with lymphoblasts was taken. Lymphoblasts were isolated from fresh whole blood via the Ficoll ^® technique described previously. Lymphocytes in the basal state were thus obtained. A part of these lymphoblasts was then used as is, without the lymphoblasts being irradiated (absorbed dose 0 Gy - basal state) and a part of these lymphoblasts was irradiated on a medical irradiator according to a certified dosimetry with an absorbed dose D of 2 Gy. 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, the cells remain in the culture incubator at 37 ° C, and the amount of pATM present in the nucleus and cell cytoplasm of the radio-induced lymphoblasts is determined after 5 minutes and 10 minutes of repair (post-irradiation repair time). FIG. 6 represents a matrix illustrating the correlations existing between the quantities of pATM present in the nucleus and the cellular cytoplasm of lymphoblasts originating from blood samples of individuals in the basal state (0 Gy - non-irradiated state), after 5 minutes and 10 minutes of repair (post-irradiation repair time).

The correlation coefficient takes a value between 0 and 1. 0 means that there is no linear correlation between the two variables tested while 1 indicates that there is a relationship of proportionality between the two. According to FIG. 6, the proportion of pATM proteins in the nucleus before irradiation and those after irradiation can not be correlated (see group A). The proportion of pATM proteins in the nucleus before irradiation is not correlated with the proportions of pATM proteins present in the cytoplasmic compartment, either in the non-irradiated basal state or at 5 or 10 minutes post irradiation. In this case, the correlation coefficient between 0Gy_nuc and 0Gy_cyto or 5mn_cyto or 10mn_cyto is close to 0. The quantities of pATM present in the nucleus at 5 min and 10 min post irradiation can be correlated with each other (see group B with a coefficient correlation close to 1). The amounts of pATM present in the cytoplasm in the basal state (0 Gy) at 5 min and 10 min post irradiation can be correlated together (see group C with a correlation coefficient close to 1).

In the context of the present invention, the threshold values Z, A, B, C, D and / or E can be defined according to the number of true or false positives tolerated, in particular by the practitioner, ie according to the sensitivity and the specificity required for the test for characterizing radiosensitivity, in particular for the test defining the radiosensitivity status.

Claims

A method for characterizing the radiosensitivity of an individual cell sample to ionizing radiation, said cell sample having been obtained from cells taken from said individual,

 said method being characterized in that

the radiosensitivity is determined from the amount of ATM protein phosphorylated pATM in the basal state t ₀ ,

 said method does not include irradiating said cell sample.

2. Method according to claim 1, wherein for the determination of the amount of the pATM protein an immunoenzymatic method is used.

The method of any one of claims 1 or 2, wherein the amount of ATM phosphorylated pATM protein is detected by an ELISA assay.

The method of any one of claims 1 to 3, wherein the cell sample is selected from: a blood sample comprising lymphocytes or lymphoblasts and a biopsy sample comprising fibroblasts.

A process according to any one of claims 1 to 4, wherein

 (a) optionally, target nucleated cells are isolated from the cell sample;

(b) total fractions or cytoplasmic and nuclear fractions or nuclear fractions of the cell sample are extracted;

(c) determining on said fractions of said cell sample, the amount of ATM protein phosphorylated pATM in basal state t ₀ ;

(d) the radiosensitivity of the cell sample is determined using at least one parameter selected from the group consisting of the average number pATM to (normalized nude), the average number pATM _t o (standard cyto + nuc), the average number pATM _t o (normalized total), and the average number pATM L _t o (normalized total);

 and or

^• pATM to (standard naked), expressed in ppm, is the average amount of pATM present in the nuclear compartment at basal state t ₀ expressed in ng divided by the total mass of cellular proteins extracted from said nuclear compartment expressed in μς;

^• to Patm (cyto + nuc normalized), expressed in ppm, is the average amount of Patm present in the cytoplasmic and nuclear compartments basal condition t ₀ expressed in ng divided by the total weight of cellular proteins extracted from said cytoplasmic compartments and nuclear expressed in μg;

^• pATM to (normalized total), expressed in ppm, denotes the total average amount of pATM extracted from the cells expressed in ng divided by the total mass of proteins extracted from said cells expressed in μg

^• Patm l_to (normalized total), expressed as (ng / ml) / million cells, means the average concentration of Patm present in the cells in the basal condition t ₀ expressed in ng / ml divided by the total number of cells.

6. Method according to any one of claims 1 to 5, wherein said target nucleated cells are selected from: mucosal cells, epithelial cells, lymphoblasts, lymphocytes and fibroblasts, and even more preferably from lymphoblasts, lymphocytes and fibroblasts.

The method according to any one of claims 1 to 6, wherein the radiosensitivity of a cell sample of an individual to ionizing radiation is determined on fibroblasts derived from biopsy of said individual and wherein

 the sample is considered radio-resistant if pATM to (normalized normal) ≥ B, it is considered that the sample is radiosensitive if pATM to (normalized naked) <B, where

pATM to (standard naked), expressed in ppm, denotes the average amount of pATM present in the nuclear compartment in the basal state t ₀ expressed in ng divided by the total mass of cellular proteins extracted from said nuclear compartment expressed in μg, and

- B is an integer or decimal value between 1 x10 ^"2 and 1 x10 ² .

The method according to any one of claims 1 to 6, wherein the radiosensitivity of a cell sample of an individual to ionizing radiation is determined on fibroblasts derived from biopsy of said individual and wherein the sample is considered radio-resistant if pATM to (normalized cyto + nuc) ≥ C, the sample is considered radiosensitive if pATM to (cyto + normalized nuc) <C, where

Patm to (cyto + nuc normalized), expressed in ppm, is the average amount of Patm present in the cytoplasmic and nuclear compartments basal condition t ₀ expressed in ng divided by the total weight of cellular proteins extracted from said cytoplasmic and nuclear compartments expressed in μg, and

C is an integer or decimal value between 1 x10 ^"2 and 1 x10 ² .

A method according to any one of claims 1 to 6, wherein the radiosensitivity of a cell sample of an individual to ionizing radiation is determined on fibroblasts derived from biopsy of said individual and wherein

 the sample is considered radio-resistant if pATM to (normalized total) ≥ D, the sample is considered radiosensitive if pATM to (normalized total) <D, where

 pATM to (normalized total), expressed in ppm, denotes the average total quantity of pATM extracted from the cells expressed in ng divided by the total mass of proteins extracted from said cells expressed in μg, and

D is an integer or decimal value between 0.1 x 10 ^-1 and 5 x 10 ² .

The method according to any one of claims 1 to 6, wherein the radiosensitivity of an individual cell sample to ionizing radiation is determined on lymphoblasts and / or lymphocytes derived from blood cells of said individual and wherein

 it is considered that the sample is radio-resistant if pATM to (normalized normal) ≥ A, the sample is considered radiosensitive if pATM to (normalized naked) <A, where

pATM to (standard naked), expressed in ppm, denotes the average amount of pATM present in the nuclear compartment in the basal state t ₀ expressed in ng divided by the total mass of cellular proteins extracted from said nuclear compartment expressed in μg,

A represents an integer or decimal value between 1 x 10 ^-2 and 5 x 10 ² . A method according to any one of claims 1 to 6, wherein the radiosensitivity of a cell sample of an individual to ionizing radiation is determined on lymphoblasts and / or lymphocytes derived from blood cells of said individual and wherein

 the sample is considered radio-resistant if pATM l_to (normalized total) ≥ E, the sample is considered radiosensitive if pATM l_to (normalized total ^ E, where

 pATM l_to (normalized total), expressed in (ng / ml) / million cells, refers to the average concentration of pATM in the cells expressed in ng / ml divided by the total number of cells;

E represents an integer or decimal value between 1 x 10 ^-2 and 5 x 10 ² .

A kit for characterizing the radiosensitivity of an individual cell sample to ionizing radiation, comprising:

 a) means for extracting the total fractions of the cell sample,

 b) means for determining, on said fractions of said cell sample, the amount of ATM protein phosphorylated pATM in the basal state to.

13. Use of a kit according to claim 12 for determining the radiosensitivity of a cellular sample of an individual to ionizing radiation.