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    公开号:FR3017625A1
    申请号:FR1451216
    申请日:2014-02-17
    公开日:2015-08-21
    发明作者:Nicolas Foray；Adeline Granzotto；Clement Devic
    申请人:CT LEON BERARD；Centre National de la Recherche Scientifique CNRS；Universite Claude Bernard Lyon 1 UCBL；Institut National de la Sante et de la Recherche Medicale INSERM；Centre Leon Berard；
    IPC主号:
    专利说明:

    [0001] 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, which is based on the determination and cross-checking of several cellular and enzymatic parameters and criteria.
    [0002] STATE OF THE ART Approximately 1 to 15% of patients treated with radiotherapy for cancer show a tissue reaction (such as a dermatitis or proctitis) that may affect the smooth course of treatment as it may lead to the physician decide to stop the 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 to be eradicated completely due to premature discontinuation of treatment, but also because of the collateral damage of healthy tissue induced by the radiation itself.
    [0003] 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. 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, it is thought that radiosensitivity generally results 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 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 (11), 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 At least two different assays to predict intrinsic radiosensitivity ", published in Int.J. Radiat Biol., vol 84 (2), pp. 107-125 (2008)), and this has identified three types of radiosensitivity: radiation-resistant cells ( Group I radiosensitivity), moderately radiosensitive cells (so-called Group II radiosensitivity), and extremely radiosensitive cells (so-called Group III radiosensitivity), but no predictive model has been proposed on this basis. the conditions under which the ATM can contribute to the detection and repair of DNA damage The patent application WO 2004/013634 (KUDOS Pharmaceuticals Ltd) describes the identification of a component of e-ATM-dependent damage signaling that interacts with other DNA damage response factors, including the MRE11 / Rad51 / NBS1 complex. US Patent Application 2007/0072210 (Ouchi and Aglipay) provides a method of screening for potential therapeutic agents that promote a DNA damage response, in which a protein called BAAT1 (which is associated with a predisposition to breast cancer linked to the BRCA1 gene), an ATM protein and the candidate compound; if the phosphorylation of the ATM is increased relative to a control mixture not comprising the candidate compound, the latter is identified as a potential active ingredient promoting the repair of the DNA. Patent Application EP 2 466 310 A1 (Helmholtz Zentrum München) describes the repair of breaks in double-stranded DNA in the presence of the phosphorylated form of histone H2AX (called gamma-H2AX or g-H2AX). WO 00/47760 and US Pat. No. 7,279,290 (St. Jude's Children's Research Hospital) describe the role of ATM kinase function in DNA repair. These latter documents describe repair routes but offer no correlation to establish a link with the clinic. Patent EP 1 616 011 B1 (International Center for Genetic Engineering and Biotechnology) proposes a method for the diagnosis of a genetic defect in DNA repair based on three steps: the culture of cells isolated from a test sample , incubating these cells with a chimeric polypeptide, characterizing the cellular response. Said cellular response is the level of expression of a biochemical marker consisting of intracellular proteins of p53, ATM, Chk1, Chk2, BRCA1, BRCA2, Nbs1, MRE11, Rad50, Rad51 and histone type. However, the radioinduced expression may not be predictive of the functionality of these proteins (some syndromes have a normal expression level while the protein is mutated): these procedures are not functional tests.
    [0004] Patent applications WO 01/90408, WO 2004/059004 and WO 2006/136686 (Atomic Energy Commission) describe other methods for detecting damage to DNA following ionizing irradiation. The first document concerns the detection of incision activities of DNA lesions, it does not allow the quantification of enzymatic activities of excision and resynthesis of DNA or the repair of CBD. The other two documents describe the quantitative evaluation of the ability of a biological medium to repair its DNA using supercoiled circular double-stranded DNA (according to the third document: immobilized in a porous polyacrylamide hydrogel film). These methods do not directly concern CBD in their physiological environment in situ and no correlation exists to validate their clinical application. KR20030033519 proposes to deduce the radiation sensitivity of the activity of catalysis or superoxide dismutase, KR20030033518 uses glutathione peroxidase or glucose 6-phosphate dehydrogenase. Such methods do not detect markers directly related to DNA damage or repair. US Patent Application 2011/312514 (Dana Farber Cancer Institute) proposes to use detection of FANCD2 foci as a marker. US Patent Application 2007/0264648 (National Institute of Radiological Sciences) proposes the use of oligomers of DNA for predicting the occurrence of side effects in radiotherapy. However, some radiosensitivity can be observed while the FANCD2 foci rate is normal.
    [0005] US patent applications 2008/234946 and US 2012/041908 (University of South Florida et al.) Disclose a method for predicting radiosensitivity of cancer cells, not healthy cells; it is in addition based on genomic data and not on functional tests. US Patent No. 8,269,163 (New York University School of Medicine) describes a large number of proteins that can be used as markers to appreciate in a simple and rapid way the importance of the accidental exposure to ionizing radiation that a person has undergone, with a view to sorting patients and directing them to appropriate emergency treatment. The latter patent concerns biological dosimetry (determination of the accidental dose) while the detection of radiosensitivity is carried out starting from a known dose. Patent application WO 2010/88650 (University of Texas) describes methods and compositions for identifying cancer cells that are either sensitive or resistant to a particular radiotherapy treatment; they are therefore not applicable to any radiotherapeutic treatment. Patent application WO 2010/136942 (Philips) describes a global method for monitoring a patient during radiotherapy using biomarkers. The method comprises obtaining at least one descriptor derived from an image extracted from an image modality, in which the descriptor belongs to a tissue of interest for which radiotherapy or tissue is provided in the vicinity. of this target volume. The method further includes selecting at least one disease-specific biological marker for detecting or quantifying side effects of radiotherapy in the area of the tissues of interest. In addition, the method includes recovering at least one in vitro measurement value of the biomarker specific for the selected disease. In addition, the method comprises treating the at least one descriptor of the at least one in vitro biomarker value using a correlation technique, resulting in an output signal indicative of radiotoxicity in the region of the tissue of interest. However, the teaching of this patent only takes into account the tissue-dependent and not the individual-specific toxicity and is mainly based on image analysis.
    [0006] Patent application WO 2010/109357 describes a method and apparatus for adaptive radiotherapy protocol planning based on optimizing the probability of complication of normal tissues and the probability of tumor control according to markers specific to each patient. The normal tissue marker values include in vitro test values, protein spectrometric signatures, history data, and patient history. The in vitro test values can be of cellular, proteomic and genetic origin such as, but not limited to, various cell counts, HB, CRP, PSA, TNF-alpha, ferritin, transferrin, LDH, IL-1. 6, hepcidin, creatinine, glucose, HbAlc, and telomere length. Anamnesis and patient history markers include anterior abdominal surgery, hormonal medications or anticoagulants, diabetes, age, and measures related to tumor growth. Biomarkers unrelated to radiotoxicity are also contemplated, such as biomarkers associated with various forms of ablation or chemotherapeutic agents. However, individual radiosensitivity is not taken into account. Despite this broad state of the art, the applicant notes that the patents described above do not describe a method of quantifying the individual radiosensitivity to assess the risk of post-radiotherapy tissue reactions, which can be used for any patient and any type of ionizing radiation likely to induce CBD, and which is predictive. The problem of providing a predictive method of individual radiosensitivity therefore remains without an operational solution. The present invention aims to propose a new predictive method of tissue and clinical radiosensitivity.
    [0007] OBJECTS OF THE INVENTION The inventors have found, and the method according to the invention is based on this observation, that the double-strand breaks (DSBs) of the DNA are the most radioinduced radiation-induced damage of radiosensitivity when they are not repaired on the one hand, and genomic instability when they are poorly repaired on the other. The inventors have discovered that CBD are supported by the majority mode of repair called suture, and / or by the minority mode of faulty repair called MRE11-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 MRE11 marker indicates a CBD site that has been supported by the MRE11-dependent fault repair. The pATM marker provides information on activation of the suture pathway by phosphorylation of H2AX and inhibition of the 1-dependent RE1 M-pathway.
    [0008] The inventors have also observed a transfer of the cytoplasmic forms of the ATM protein into the celluloid nucleus following an oxidative-type stress, and in particular following a stress linked to an ionizing radiation inducing CBD.
    [0009] To observe the damage of a DNA by an exogenous aggression, one must take into account, on the one hand, the spontaneous state of the DNA, and on the other hand, its radioinduced state. 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. According to the invention the problem is solved by a method based on: 1) an amplification of non-transformed cells, in particular cells derived from skin biopsies; 2) a mechanistic model valid for quiescent human cells; 3) Functional tests for the recognition, repair and signaling of valid CBD regardless of the therapeutic modality.
    [0010] A first subject of the invention is a method for predicting the cellular radiosensitivity of a cell sample to ionizing radiation, said cell sample having been obtained from cells taken from a patient in a non-irradiated or poorly irradiated zone, in which method: (i) amplifying said sampled cells, said 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 (this average number being called NpH2Ax (t)), said observation times t being the time t = 0 min (called t0, non-irradiated state) and the observation time t4 (and preferably in addition to the times t1, t2 and t3) after irradiation with an absorbed dose D; (iii) determining the total dose not to be exceeded (DTNPD), expressed in Gray (Gy), using at least the parameter NpH2Ax (t4), and in which process - 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 must in this case be at least 12 hours, and preferably between 12h and 48h, and which is still more preferably about 24 hours; t3 is a fixed value which represents the time after which approximately 25% of the double-strand breaks (DSB) 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 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 originating 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 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 about 10 minutes.
    [0011] In an advantageous embodiment, the average number of nuclear foci obtained with the pH2AX marker at the observation times t1, t2 and t3 is also used to verify the shape of the kinetic curve of recognition and repair of CBD. The total dose not to be exceeded (DTNPD), expressed in Gray (Gy), is an important parameter for the radiation therapist, who can predict what maximum dose a given patient can absorb without experiencing a potentially lethal reaction; this parameter also makes it possible to exclude from radiation therapy patients who have a particularly strong radiosensitivity.
    [0012] According to the invention, the DTNPD can be determined according to the formula DTNPD = 60 / NpH2Ax (t4) if NpH2Ax (t0) 3, or according to the formula DTNPD = 60 / [NpH2Ax (t4) + Nel2Ax (t0)] if NpH2Ax ( In a variant of the method according to the invention, the mean number of micronuclei observed at time t per 100 cells [in%] is also determined on said cell sample (this average number being called NMN (t)), the time t being at least t0 (non-irradiated) and t4 after irradiation with an absorbed dose D, the parameter NNM (t4) is used in the determination of DTNPD.
    [0013] The DTNPD is then determined according to the formula DTNPD = 120 / [2 x NpH2Ax (t4) + NNM (t4)], and / or according to the formula DTNPD = 120 / [2 x NpH2Ax (t4) + NNM (t4)] if Nel2Ax (t0) 3, or according to the formula DTNPD = 120 / [2 x NpH2Ax (t4) + NpH2Ax (t0) + NNM (t4)] if Nel2Ax (t0)> 3. 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.
    [0014] The first step of the process according to the invention which follows the removal of the cells (namely in the preferred embodiment of the establishment of the fibroblastic line biopsy) consists in characterizing the spontaneous state of the DNA (state at t0). that is to say without irradiation. This step may include in particular the examination of the size of the nuclei, the presence of micronuclei, possible spontaneous apoptotic bodies and multilésées cells: the cells are observed under a fluorescence microscope. Using the DAPI dye (4'6'-diamidino-2-phenylindole, CAS RN 28718-90-3 for dihydrochloride) the micronucleus level per 100 cells is determined, which is an indicator of genomic instability. Apoptotic bodies are also determined.
    [0015] The population of abnormally large nuclei, whose presence indicates the decondensation of chromatin, is also determined. Said ionizing radiation is defined by its absorbed dose (parameter called D and expressed in Gray). In the context of the present invention, the absorbed dose D is between 0.5 Gy and 4 Gy, preferably between 1 Gy and 3 Gy, preferably between 1.7 and 2.3 Gy, and is even more preferably 2 Gy. These areas typically correspond to an individual session of radiotherapeutic treatment, the number of sessions depending on the location, type and stage of the tumor. It is essential for the method according to the invention that all the time values t1, t2, t3 and t4 are defined at the beginning of a series of tests (ie at least for a given patient, and preferably for a plurality of patients to calibrate the method against a set of statistically significant observations) and be the same for all determinations of all parameters that refer to these time intervals. In the process according to the invention, t1 is advantageously between 8 and 12 minutes, and / or t2 is advantageously between 50 and 70 minutes, and / or t3 is advantageously between 3.5 and 4.5 hours, and / or or t4 is advantageously between 22 and 26 hours; preferably all four conditions are met.
    [0016] In a variant of the process which is particularly interesting and easy to standardize, t1 is 10 minutes, t2 is 60 minutes, t3 is 4 hours, t4 is 24 hours, and D is 2 Gy. The determination of NpH2Ax (t) advantageously involves an immunofluorescence test. Control cells from radiation-resistant patients may be taken from patients selected from clinical examination as patients who have not shown significant tissue reactions during or following radiotherapeutic treatment. They can also be selected as cells exhibiting an in vitro clonogenic survival rate of greater than 55% after irradiation with an absorbed dose of 2 Gy. We describe here a typical embodiment.
    [0017] The cells are observed with the pH2AX marker. The observations with the markers pATM and / or MRE11 can be added to the observation times t (these average numbers being respectively called NpATm (t) and NmREi (t)) and at least one observation time selected from t = t1 , t2, t3 and t4 after irradiation with an absorbed dose D. In one embodiment, the number of foci with the pH2AX marker and the presence of multilosed cells are determined. The location of the pATM protein and that of the MRE11 protein (nuclear or cytoplasmic) are noted. This first step makes it possible to identify a possible genomic instability in the spontaneous state. The second step of the process according to the invention comprises irradiation with the desired absorbed dose D (for example 2 Gy) and evaluation of the cellular response to ionizing radiation. a) In a first embodiment, the repair of radiation-induced CDBs by suture, which is the majority mode of their repair, is studied. The number of pH2AX foci per cell is determined at t4 and optionally also at t1, t2 and possibly also at t3; the determination at t3 makes it possible to consolidate the definition of the kinetic rate of t1 to t4. In an advantageous embodiment, after the time t4, the level of micronucleus is also determined to deduce the level of radioinduced micronuclei. This makes it possible to estimate radiosensitivity according to the importance of unrepaired CBD. b) In a second embodiment, the study of the cellular response to ionizing radiation is further investigated by measuring the functionality of the ATM-dependent kinase activity. It is known that in control radio-resistant cells, the phosphorylated forms of the ATM protein (pATM) are spontaneously cytoplasmic. The applicant has discovered that, in the irradiated state, they tend to become nuclear. Once passed into the nucleus, the pATM forms activate the suture repair mechanisms and inhibit the MRE11-dependent fault repair pathway. For example, if after irradiation (for example with an absorbed dose of 2 Gy) the pATM forms mount a cytoplasmic localization, it is concluded that the pATM forms do not pass or can not pass normally from the cytoplasm to the nucleus . This can be caused by a mutation of ATM or any other ATM protein partner that would help it pass into the nucleus after irradiation: in any case, this indicates a significant radiosensitivity.
    [0018] This optional determination of the location of the pATM protein is performed at least at t1 and t2, and optionally also at t3 and t4. c) in a third embodiment that can be combined with the above, the study of the cellular response to ionizing radiation through the MRE11-dependent pathway is studied in greater depth. In addition to the major suture repair pathway whose capacity is quantifiable by pH2AX immunofluorescence, the applicant has identified another alternative route of repair, which is an alternative to the suture and which is likely to replace it in case of impairment: it is the repair by recombination MRE11 dependent. Its capacity is quantifiable by the kinetic study of the immunofluorescence of MRE11 foci. This measurement is performed at least at t1, t2 and t3, and optionally also at t4. According to the findings of the applicant, in the radio-resistant control lines, MRE11 is cytoplasmic and the number of MRE11 foci is very low up to 4 hours after a 2 Gy dose (typically 7 ± 2 MRE11 foci); the labeling becomes cytoplasmic about 24 hours after irradiation. In a final step, the results are evaluated by calculating the scores in order to predict the radioinduced damage state and / or the radiosensitivity of the patient, and in particular the DTNPD that is specific to him.
    [0019] Description A. General definitions35 The terms "radioinduced damage", "radioinduced", "radiosensitivity", "radioresistance", "radiotoxicity", "radiotherapy" all refer to ionizing radiation, including particulate radiation, as constituted by particles of the alpha (a) or beta ([3) type, or else at high energy electromagnetic radiation, in particular of the gamma (y) or XB type Detailed description We describe here an embodiment with several variants which is suitable for a human patient. 1 Preparation of the test Before any cell collection and before any manipulation of the prelévées 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 so that 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. Upon receipt, the cell sample (typically the biopsy) is established as an amplifiable cell line without viral or chemical transformation agent following an ancillary procedure and well known to the culture laboratories. 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. 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). The irradiation can be carried out for example with a medical accelerator which delivers 6 MV photons with an absorbed dose rate of 3 Gy min-1. 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). 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 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.), Can be used: the cells were fixed in 3% paraformaldehyde and 2% sucrose for 15 minutes at room temperature and permeabilized in 20 mM solution HEPES buffer (4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid) pH 7.4, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose, 0.5% Triton X-100 (a nonionic surfactant) of formula t-Oct-C6H4- (OCH2CH2) 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 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 primary antibodies at 1: 100. Incubations with FITC anti-mouse or TRITC anti-rabbit secondary antibodies (1: 100, provided by Sigma Aldrich) were performed at 27 ° C in 2% BSA for 20 minutes. Glass slides were treated with VectashieldTM containing DAPI (4,6-Diamidino-2-phenylindole) to mark the core. Staining with DAPI also indirectly determines the number of cells in phase G1 (nuclei with homogeneous DAPI staining), in phase S (nuclei with many pH2AX foci), in phase G2 (nuclei with heterogeneous DAPI staining ) and metaphases (visible chromosomes).
    [0020] 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 GB / 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 a basis for 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 General and markers used The invention is based inter alia on the use of acquired data for the pH2AX marker 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 lapse of time determined as of the end of the irradiation, and their immunofluorescence is studied. It is possible to measure the complete kinetic curves, for example represented by 5 points advantageously located at t0, t1 (preferably 10 minutes), t2 (preferably 1h), t3 (preferably 4h) and t4 (preferably 24h), knowing that t0 corresponds to the state before irradiation (spontaneous state). It is advantageous to associate the acquired doonates with two other markers, namely pATM and MRE11. But the plaintiff has realized that some points (corresponding to certain times of repair) are more important than others, and that some points 30 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.
    [0021] The averages of each point and each dose with each marker are calculated with the standard deviation errors of the mean (SEM) since the sampling is n = 3 (no Gaussian standard deviation 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 applicant's finding, ATM passes from the cytoplasm to the nucleus after irradiation under normal conditions (radioresistant 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. (iii) MRE11 is an endonuclease that breaks DNA. According to the plaintiff's findings, MRE11 marks the badly repaired CBDs when the repair process is finalized. The marker MRE11 can be either cytoplasmic without foci, or cytoplasmic and nuclear without foci, or cytoplasmic and nuclear with foci.
    [0022] 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 for MRE11 and pATM under the influence of ionizing radiation), for quantify the micronuclei, apoptotic bodies and nuclei size that are complementary cell markers to the foci data. 2.2 Biological parameters One defines and determines as indicated: - NpH2Ax (t), NpATm (t), NMRE11 (t) the average numbers of nuclear foci obtained with the markers pH2AX, pATM, and MRE11 with the observation times tO (non irradiated ) or t1, t2, t3, t4 after irradiation (absorbed dose: 2 Gy), knowing that the determination of the parameter NpH2Ax (t) is mandatory within the framework of the method according to the invention, while that of the other parameters NpATm ( t) and NMRE11 (t) is optional but advantageous; - NMN (t) the number of micronuclei observed spontaneously (at t = t0, i.e. without irradiation) or at t = t4 after irradiation with an absorbed dose of 2 Gy per 100 cells (in%). 2.3 Predictive evaluation It aims to predict clinical or radiotherapeutic parameters based on measured biological data. It is a quantitative analysis directly derived from the mathematical value of the scores or mathematical formulas linking the scores; the analysis concerns the total dose not to be exceeded to avoid a potentially lethal reaction (called DTNPD criterion) applicable to a patient who is going to undergo or who is undergoing radiotherapy: the total dose not to be exceeded (DTNPD), expressed in Gray (Gy), is an important parameter for the radiation therapist, who can predict which maximum dose a given patient can absorb without experiencing a potentially lethal reaction; this parameter also makes it possible to exclude from radiation therapy patients who have a particularly strong radiosensitivity. According to the invention, the DTNPD can be determined according to the formula DTNPD = 60 / NpH2Ax (t4) if NpH2Ax (t0) 3, or according to the formula DTNPD = 60 / [NpH2Ax (t4) + NpH2Ax (t0)] if NpH2Ax ( In a variant of the method according to the invention, the mean number of micronuclei observed at time t per 100 cells [in%] is also determined on said cell sample (this average number being called NMN (t)), the time t being at least t0 (non-irradiated) and t4 after irradiation with an absorbed dose D, the parameter NNM (t4) is used in the determination of DTNPD. The DTNPD is then determined according to the formula DTNPD = 120 / [2 x NpH2Ax (t4) + NNM (t4)], and / or according to the formula DTNPD = 120 / [2 x NpH2Ax (t4) + NNM (t4)] if Nel2Ax (t0) 3, or according to the formula DTNPD = 120 / [2 x NpH2Ax (t4) + Nel2Ax (t0) + NNM (t4)] if Nel2Ax (t0)> 3. On the basis of this quantitative analysis a diagnosis more qualitative can then be done; it will be influenced by the quantitative analysis but will take into account any clinical elements brought to the knowledge of the practitioner.
    权利要求:
    Claims (3)
    [0001]
    REVENDICATIONS1. A method for predicting the cellular radiosensitivity of a cell sample to ionizing radiation, said cell sample having been obtained from cells taken from a patient in a non-irradiated or poorly irradiated area, in which method (i) said cells removed, these amplified cells constituting "the cell sample"; (Ii) determining on said cell sample the average number of nuclear foci obtained with the pH2AX marker at the observation times t (this average number being called NpH2Ax (t)), said observation times t being the time t = 0 min (called t0, non-irradiated state) and the observation time t4 (and preferably in addition to the times t1, t2 and t3) after irradiation with an absorbed dose D; (iii) determining the total dose not to exceed (DTNPD), expressed in Gray, using at least the parameter NpH2Ax (t4), and in which method - t4 is a fixed value which represents the time for which the rate DNA breaks reaches its residual value, 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 12 and 48 hours, and which is even more preferably about 24 hours; t3 is a fixed value which represents the time at which about 25% of double-strand breaks (DSBs) are repaired in control cells from radioresistant 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, 10
    [0002]
    2. 15
    [0003]
    3. 204. 5. 25 6. 30 7. 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. 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 about 10 minutes. The method according to claim 1, wherein the average number of micronuclei observed at times t per 100 cells [in%] is determined on said cell sample (this average number being called NMN (t)), the times t being at least t0 ( unirradiated) and t4 after irradiation with an absorbed dose D, and wherein the parameter NNM (t4) is used in the determination of DTNPD. Process according to claim 1 or 2, wherein the DTNPD is determined according to the formula DTNPD = 60 / NpH2Ax (t4) if Nel2Ax (t0) <3, or according to the formula DTNPD = 60 / [NpH2Ax (t4) + Nel2Ax (t0) )] if Nel2Ax (t0)> 3. A method according to claim 2 or 3, wherein the DTNPD is determined according to the formula DTNPD = 120 / [2 x NpH2Ax (t4) + NNM (t4)]. A process according to any one of claims 2 to 4, wherein DTNPD is determined according to the formula DTNPD = 120 / [2 x NpH2Ax (t4) + NNM (t4)] if Nel2Ax (t0) <3, or according to the formula DTNPD = 120 / [2 x (NpH2AX (t4) NpH2AX (t0)) NNM (t4)] if NpH2Ax (t0)> 3. A method according to any one of claims 1 to 5, wherein said cells taken are cells fibroblasts from a skin biopsy of a patient. Process according to any one of Claims 1 to 6, in which the absorbed dose D is between 0.5 Gy and 4 Gy, preferably between 1 Gyet 3 Gy, preferably between 1.7 and 2.3 Gy, and is even more preferably 2 Gy. 8. Process according to any one of claims 1 to 7, wherein t1 is between 8 and 12 minutes, t2 is between 50 and 70 minutes, t3 is between 3.5 and 4.5 hours, and t4 is between 22 and 26 hours. 9. A process according to claim 7 or 8, wherein t1 is 10 minutes, t2 is 60 minutes, t3 is 4 hours, t4 is 24 hours, and D is 2 Gy.A process according to any one of claims 1 to 9, wherein the determination of NpH2Ax (t) involves an immunofluorescence test. The method according to any one of claims 1 to 10, wherein said control cells from radioresistant patients have been selected as cells exhibiting an in vitro clonogenic survival rate of greater than 55% after irradiation with an absorbed dose of A method according to any one of claims 1 to 11, wherein said control cells from radioresistant patients have been selected as cells taken from patients who have not shown significant tissue reactions during or at the following a radiotherapeutic treatment. 13. Method according to any one of claims 3 to 12, wherein the average number of nuclear foci obtained with the pH2AX mark at the observation times t1, t2 and t3 is used to verify the shape of the kinetic curve of recognition of the sites. CBD. 14. The method as claimed in claim 1, in which the average number of nuclear foci obtained with at least one of the pATM and MRE11 markers is also determined at observation times t (these average numbers being respectively called NpATm). t) and NMRE11 (t)) and at least one observation time selected from t = t1, t2, t3 and t4 after irradiation with an absorbed dose D.35
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    同族专利:
    公开号 | 公开日
    FR3017625B1|2018-03-23|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2017029450A1|2015-08-19|2017-02-23|Neolys Diagnostics|Individual method predictive of the dna-breaking genotoxic effects of chemical or biochemical agents|
    WO2017029449A1|2015-08-19|2017-02-23|Neolys Diagnostics|Predictive method for characterizing the sensitivity of a tumour in response to a dna-breaking treatment|
    FR3067467A1|2017-06-13|2018-12-14|Neolys Diagnostics|RAPID PREDICTIVE METHOD FOR CHARACTERIZING THE RADIOSENSITIVITY AND / OR THE TISSUE REACTION OF AN INDIVIDUAL TO AN IRRADIATION|
    WO2018229439A1|2017-06-13|2018-12-20|Neolys Diagnostics|Rapid predictive method for characterising the radiosensitivity and/or the risk of tissue toxicity of an individual to irradiation|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1451216|2014-02-17|
    FR1451216A|FR3017625B1|2014-02-17|2014-02-17|PREDICTIVE METHOD FOR DETERMINING TISSUE RADIOSENSITIVITY|FR1451216A| FR3017625B1|2014-02-17|2014-02-17|PREDICTIVE METHOD FOR DETERMINING TISSUE RADIOSENSITIVITY|
    PCT/FR2015/050370| WO2015121597A1|2014-02-17|2015-02-16|Predictive method for determining tissual radiosensitivity|
    EP15709253.7A| EP3108252B1|2014-02-17|2015-02-16|Predictive method of characterizing the radiosensitivity and tissular reaction of a patient to therapeutic ionizing radiation|
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