A method for evaluating the relative biological effect of radiotherapy rays

Abstract

The present application discloses a method for evaluating the relative biological effect of radiotherapy rays. The method uses the ray to be evaluated and the reference ray to irradiate several samples to be irradiated at different irradiation doses, wherein the samples to be irradiated are three-dimensional cell cultures; the irradiated samples are stained to obtain stained images of all samples at different irradiation doses; the stained images are analyzed to obtain the positive cell ratio of the irradiated samples; based on the relationship between the positive cell ratio and the irradiation dose, a biological effect probability model is established; based on the biological effect probability model, the relative biological effect value of the ray to be evaluated is obtained, and a more accurate evaluation of the relative biological effect can be obtained.

Description

Technical Field

[0001] The present application relates to the technical field of radiotherapy, and in particular to a method for evaluating the relative biological effect of radiotherapy rays. Background Art

[0002] With the continuous development of modern radiotherapy technology, stereotactic radiotherapy, proton and heavy ion radiotherapy, boron neutron radiotherapy, ultra-high dose rate radiotherapy and other treatment technologies have emerged and gradually been promoted and applied. Compared with conventional X-ray-based fractionated radiotherapy, new radiotherapy technologies use different radiation sources, dose fractionation strategies or beam parameters. These emerging technologies can reduce the toxic side effects of patients while improving the treatment effect of tumors, providing patients with a better quality of life.

[0003] The quality of radiotherapy plans is closely related to the dose strategy used, which includes dose fractionation and biological dose. Biological dose refers to the difference in biological effects of different types of radiation by weighting after considering the physical dose. The difference in biological effects of different radiotherapy techniques can usually be evaluated using RBE (Relative Biological Effectiveness).

[0004] Before clinical radiotherapy, in order to effectively kill tumor cells while reducing damage to surrounding normal tissues, it is necessary to accurately evaluate the RBE values ​​of different types of radiation in order to develop a reasonable radiotherapy plan. At present, the evaluation method of RBE value generally uses the LEM model (Local Effect Model) or the MKM model (MicrodosimetricKinetic Model) to calculate the biological effect. However, these algorithms have some defects. For example, the LEM model has the problem of overestimating RBE in the low LET area (Linear Energy Transfer) and underestimating RBE in the high LET area. The MKM model has a large deviation when applied to large-fraction high-dose radiotherapy and tumor cells with small biological parameter α / β values, and cannot accurately reflect the real biological effect. Summary of the invention

[0005] To this end, an embodiment of the present application provides a method for evaluating the relative biological effects of radiotherapy rays, which can obtain a more accurate evaluation of the relative biological effects.

[0006] The present application provides a method for evaluating the relative biological effect of radiotherapy rays.

[0007] This application is achieved through the following technical solutions:

[0008] A method for evaluating the relative biological effect of radiotherapy rays, the method comprising:

[0009] Using the radiation to be evaluated and the reference radiation to irradiate a plurality of samples to be irradiated at different irradiation doses, wherein the samples to be irradiated are three-dimensional cell cultures;

[0010] The irradiated samples are stained to obtain staining images of all samples at different irradiation doses;

[0011] Analyzing the staining image to obtain the positive cell ratio of the sample after irradiation;

[0012] Based on the relationship between the positive cell ratio and the irradiation dose, a biological effect probability model was established;

[0013] The relative biological effect value of the ray to be evaluated is obtained through a biological effect probability model.

[0014] In a preferred example of the present application, it can be further configured that the three-dimensional cell culture is an organoid.

[0015] In a preferred example of the present application, it can be further configured that the step of using the radiation to be evaluated and the reference radiation to irradiate a plurality of samples to be irradiated with different radiation doses comprises:

[0016] A number of samples to be irradiated are divided into a first group and a second group; the first group is divided into n first subgroups, and the samples to be irradiated in the n first subgroups are irradiated with n different irradiation doses using the radiation to be evaluated; the second group is divided into n second subgroups, and the samples to be irradiated in the n second subgroups are irradiated with n different irradiation doses using reference radiation; wherein n>0.

[0017] In a preferred example of the present application, it can be further set that the biological effect probability model is:

[0018] Tumor control probability TCP model or normal tissue complication probability NTCP model.

[0019] In a preferred example of the present application, it can be further configured that the tumor control probability TCP model adopts any one of the TCP-Schultheiss logic model, the LQ-Poisson-TCP basic model, the LQ-Poisson-TCP two-parameter model or the Zaider-TCP model.

[0020] In a preferred example of the present application, it can be further configured that the normal tissue complication probability NTCP model adopts any one of the NTCP-Schultheiss logistic model, the NTCP-Lyman-Kutcher-Berman model or the NTCP-RSM model.

[0021] In a preferred example of the present application, it can be further configured that the steps of analyzing the staining image to obtain the positive cell ratio of the sample after irradiation are specifically as follows:

[0022] The stained image is placed under a microscope and manually analyzed under the microscope to obtain the number of positive cells, and the number of positive cells is divided by the number of all cells in the stained image to obtain the positive cell ratio of the stained image.

[0023] In a preferred example of the present application, it can be further configured that the staining image is analyzed to obtain the positive cell ratio of the sample after irradiation, specifically the steps of: importing the staining image into image analysis software, automatically counting the positive cells in the staining image to obtain the number of positive cells, dividing the number of positive cells by the number of all cells in the staining image, to obtain the positive cell ratio of the staining image.

[0024] In a preferred example of the present application, it can be further configured that the step of obtaining the relative biological effect value of the radiation to be evaluated through the biological effect probability model is: calculating the ratio of the dose of the reference radiation to the dose of the radiation to be evaluated when the sample reaches the same positive cell ratio according to the biological effect probability model.

[0025] In a preferred example of the present application, it can be further arranged that the reference ray and the ray to be evaluated are rays of different radiation types.

[0026] In summary, compared with the prior art, the technical solution provided in the embodiment of the present application has at least the following beneficial effects:

[0027] The irradiation of several samples to be irradiated with different irradiation doses is performed using the irradiation radiation to be evaluated and the reference radiation, wherein the samples to be irradiated are three-dimensional cell cultures; the irradiated samples are stained to obtain the stained images of all samples at different irradiation doses; the stained images are analyzed to obtain the positive cell ratio of the irradiated samples; based on the relationship between the positive cell ratio and the irradiation dose, a biological effect probability model is established; through the biological effect probability model, the relative biological effect value of the irradiation to be evaluated is obtained. Compared with the traditional in vitro cell scheme, the measurement and analysis using three-dimensional cell culture can more accurately simulate the structure and biological microenvironment of tissues or organs. By establishing a TCP model or NTCP model of dose and positive cell percentage under different radiation conditions, the ratio of the irradiation dose to be irradiated and the reference radiation dose when the same positive cell ratio is produced is calculated to obtain the RBE value, which has the advantages of high accuracy and strong repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A schematic flow chart of a method for evaluating the relative biological effect of radiotherapy rays provided by an exemplary embodiment of the present application;

[0029] Figure 2 A schematic diagram of an NTCP curve provided for an exemplary embodiment of the present application. DETAILED DESCRIPTION

[0030] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

[0031] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0032] In addition, the term "and / or" in this application is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this application, unless otherwise specified, generally indicates that the associated objects before and after are in an "or" relationship.

[0033] In this application, the terms "first", "second", etc. are used to distinguish identical or similar items with basically the same effects and functions. It should be understood that there is no logical or temporal dependency between "first", "second", and "nth", nor is there any limitation on quantity and execution order.

[0034] The embodiments of the present application are further described in detail below in conjunction with the drawings in the specification.

[0035] In one embodiment of the present application, a method for evaluating the relative biological effect of radiotherapy rays is provided, such as Figure 1 As shown, the evaluation method includes:

[0036] S10: using the radiation to be evaluated and the reference radiation to irradiate a plurality of samples to be irradiated at different irradiation doses, wherein the samples to be irradiated are three-dimensional cell cultures.

[0037] The reference ray and the ray to be evaluated are rays of different radiation types. The reference ray can be any ray, and in this embodiment, 250kV X-ray or 6MV X-ray is preferably used.

[0038] Three-dimensional cell cultures are organoids. Organoids are formed by three-dimensional (3D) culture of stem cells or pluripotent stem cells in vitro. They have a certain spatial structure and can simulate the structure and function of real organs. Organoids can more accurately simulate the structure and function of real organs. Compared with traditional cell or tissue models, organoids can form tissue analogs with spatial structures under three-dimensional culture in vitro, which enables them to better simulate the complex environment and cell-to-cell interactions of in vivo tissues. Therefore, in the assessment of irradiation dose, the use of organoids can more realistically reflect the impact of radiation on tissues and provide more accurate data support.

[0039] Specifically, a number of samples to be irradiated are divided into two groups, namely, a first group and a second group; the first group is divided into n first subgroups, and the samples to be irradiated in the n first subgroups are irradiated with the radiation to be evaluated at n different irradiation doses, that is, the n first subgroups are irradiated with a dose x 1 , irradiation dose x 2 , irradiation dose x 3 ...radiation dose x n irradiation; dividing the second group into n second subgroups, irradiating the samples to be irradiated in the n second subgroups with reference rays at n different irradiation doses, that is, irradiating the n second subgroups with irradiation doses x 1 , irradiation dose x 2 , irradiation dose x 3 ...radiation dose x nIrradiation is performed; wherein n>0. Dividing the samples to be irradiated into different groups and irradiating them under different conditions is helpful to improve the reliability and accuracy of the data.

[0040] S20: Staining the irradiated samples to obtain staining images of all samples at different irradiation doses.

[0041] Specifically, after the irradiated sample is fixed, embedded, sliced, etc., the sample is stained. Among them, the staining method can be any one of histopathology (HE) staining, immunohistochemistry staining or immunofluorescence staining. Histopathology (HE) staining is the hematoxylin-eosin staining method. The specific steps are to put the fixed tissue specimen into ethanol solutions of different concentrations for dehydration to remove moisture from the tissue; put the dehydrated tissue specimen into a transparent agent, such as xylene, to make the tissue transparent for subsequent processing; immerse the transparent tissue specimen in melted paraffin to fully penetrate it, and then embed it; cut the embedded wax block into thin slices and stick it on a slide; put the slice into hematoxylin staining solution for staining, and then put it into eosin staining solution after washing, differentiation, rinsing and blueing; the stained slices are dehydrated and transparent, and then sealed with neutral gum. The specific steps of immunohistochemical staining are as follows: slice the tissue specimen into thin slices and attach them to a glass slide; remove the paraffin on the slices with an organic solvent and hydrate them through a series of alcohols with decreasing concentrations; select an appropriate antigen retrieval method based on the nature of the target antigen to expose the antigen epitope; cover the slices with a blocking solution containing normal serum to reduce nonspecific binding; add specific antibodies (such as γH2AX, 53BP1, Ki67, BrdU, EdU, etc.) to the slices to bind to the corresponding antigens; wash to remove unbound primary antibodies, and then add secondary antibodies labeled with fluorescence or enzymes to bind to the primary antibodies; observe and record the results using a fluorescence microscope or enzyme substrate colorimetric method. The specific steps of immunofluorescence staining are slice preparation, dewaxing and hydration, antigen repair, blocking, etc., which are the same as immunohistochemical staining. Then, specific antibodies are added to the slices to bind to the corresponding antigens; unbound primary antibodies are washed to remove them, and then fluorescently labeled secondary antibodies are added to bind to the primary antibodies; fluorescent dyes such as DAPI are used to stain the cell nuclei; after the slices are sealed, the results are observed and recorded under a fluorescence microscope.

[0042] S30: Analyze the staining image to obtain the positive cell ratio of the sample after irradiation.

[0043] The analysis of the staining image to obtain the positive cell ratio can be automatically processed by image analysis software, or can be counted manually. Specifically, the staining image can be imported into the image analysis software, and the positive cells in the staining image can be automatically counted using the image analysis software to obtain the number of positive cells, and the number of positive cells is divided by the number of all cells in the staining image to obtain the positive cell ratio of the staining image. The image analysis software can be any one of HALO digital pathology image analysis software, ImageJ software, or a self-written analysis program.

[0044] The stained image can also be placed under a microscope and manually analyzed to identify cells in the stained image, determine whether the cells are negative cells or positive cells, count the number of positive cells, divide the number of positive cells by the number of all cells in the stained image, and obtain the positive cell ratio of the stained image.

[0045] S40: Based on the relationship between the positive cell ratio and the irradiation dose, a biological effect probability model is established.

[0046] The biological effect probability model is established according to the positive cell ratio (percentage) of the samples irradiated with different irradiation doses. The biological effect probability model can be a tumor control probability TCP model or a normal tissue complication probability NTCP model.

[0047] If the biological effect probability model adopts the tumor control probability TCP model (Tumor Control Probability, TCP), any one of the TCP-Schultheiss logistic model, LQ-Poisson-TCP basic model, LQ-Poisson-TCP two-parameter model or Zaider-TCP model can be further adopted. If the biological effect probability model adopts the normal tissue complication probability NTCP model (NTCP), any one of the NTCP-Schultheiss logistic model, NTCP-Lyman-Kutcher-Berman model or NTCP-RSM model can be adopted.

[0048] The Lyman-Kutcher-Berman algorithm is used to establish the NTCP model. The specific formula is:

[0049]

[0050]

[0051] in, D 50It refers to the irradiation dose when the probability of side effects is 50%, D refers to the irradiation dose of the sample, and m is the parameter that controls the slope of the NTCP dose-effect curve.

[0052] S50: Obtain the relative biological effect value of the radiation to be evaluated through the biological effect probability model.

[0053] The biological effect probability model can be used to calculate the reference radiation dose D when the same positive cell ratio is achieved. R The dose D of the radiation to be evaluated D , calculate the dose D of the reference ray R The dose D of the radiation to be evaluated D The relative biological effect value BRE of the radiation dose to be evaluated can be obtained by the ratio of

[0054] This application uses organoids for RBE measurement and analysis. Compared with traditional in vitro cell schemes, the scheme of this application can more accurately simulate the function, structure and biological microenvironment of tissues or organs, and obtain accurate RBE values ​​for different irradiation types.

[0055] In some preferred embodiments, all samples to be irradiated are divided into two groups; the first group is then divided into 6 first sub-groups, and the samples to be irradiated in the 6 first sub-groups are gradient irradiated with 6 different irradiation doses using the radiation to be evaluated; the second group is divided into 6 second sub-groups, and the samples to be irradiated in the 6 second sub-groups are gradient irradiated with 6 different irradiation doses using the reference radiation.

[0056] The present application is further described below in conjunction with specific embodiments. A 6MV X-ray with a dose rate of 0.1 Gy / s is used as a reference ray, a proton ray with a dose rate of 230 MeV is used as the ray to be evaluated, and an in vitro cultured mouse intestinal organoid is used as the sample to be irradiated. All organoids are divided into two groups. The first group is irradiated with 6MV X-rays. The first group is divided into 6 first subgroups. The first subgroup The radiation dose is 0Gy. The radiation dose is 4Gy. The radiation dose is 8Gy. The radiation dose is 12Gy. The radiation dose is 16Gy. The irradiation dose is 20Gy. The second group is irradiated with 230MeV proton beams, and the second group is divided into 6 second sub-groups at the same time The radiation dose is 0Gy. The radiation dose is 4Gy. The radiation dose is 8Gy. The radiation dose is 12Gy. The radiation dose is 16Gy. The irradiation dose is 20 Gy. The number of samples to be irradiated in each subgroup of the first group and the second group is 5, and the specific grouping is shown in Table 1 below.

[0057] Table 1

[0058]

[0059] After irradiation, the samples were placed for 3 hours, and γH2AX immunohistochemical staining was performed on the irradiated samples; the stained samples were scanned to obtain staining images. All staining images were imported into the HALO pathology analysis software to count the positive cells of the samples, and the positive cell ratio of each sample and the corresponding radiation type and irradiation dose of the sample were obtained. The results of analyzing the percentage of positive cells in the staining images of each sample by the HALO pathology analysis software are shown in Table 2.

[0060] Table 2

[0061]

[0062] According to the relationship between the positive cell ratio and the dose obtained by the above analysis, the Lyman-Kutcher-Berman algorithm was used to establish the NTCP model. In this embodiment, the NTCP-Lyman-Kutcher-Berman (LKB) model was used for calculation, and the specific formula is as follows:

[0063]

[0064]

[0065] in, D 50 It refers to the irradiation dose when the probability of side effects is 50%, D refers to the irradiation dose of the sample, and m is the parameter that controls the slope of the NTCP dose-effect curve. The experimental data obtained in the previous step (Table 2, the percentage of γH2AX-positive cells after irradiation of mouse intestinal organoids at different doses) were substituted into the NTCP model formula for fitting to obtain the NTCP curve. The results are as follows Figure 2 As shown in the figure, the blue squares are the experimental data of 6MV X-ray irradiation at a conventional dose rate, and the blue dotted line is the corresponding fitted NTCP curve, with the fitting parameter D 50=14.22Gy, m=0.704; the red dots are the experimental data of 230MeV proton irradiation at a conventional dose rate, and the red dotted line is the corresponding fitted NTCP curve, with the fitting parameter D 50 =12.72Gy, m=0.723. The reference radiation dose D when reaching the same positive cell count is calculated according to the NTCP model R The dose value D of the radiation to be measured D Ratio In this embodiment, the reference radiation dose D when the positive cell ratio is 50% R The dose value of the ray to be measured is 12.72Gy. D The RBE value of the conventional dose rate 230MeV proton beam can be obtained.

[0066] The present application also provides an evaluation system for the relative biological effect of radiotherapy rays, the evaluation system comprising:

[0067] An irradiation module, used to irradiate a plurality of samples to be irradiated with different irradiation doses using the irradiation to be evaluated and the reference irradiation, wherein the samples to be irradiated are three-dimensional cell cultures;

[0068] The analysis module is used to stain the irradiated samples to obtain staining images of all samples at different irradiation doses; and to analyze the staining images to obtain the positive cell ratio of the irradiated samples;

[0069] A model building module is used to establish a biological effect probability model based on the relationship between the positive cell ratio and the irradiation dose;

[0070] The evaluation module is used to obtain the relative biological effect value of the ray to be evaluated through a biological effect probability model.

[0071] The specific definition of the relative biological effect evaluation system of radiotherapy rays provided in this embodiment can be found in the embodiment of the relative biological effect evaluation method of radiotherapy rays mentioned above, and will not be repeated here. Each module in the above-mentioned relative biological effect evaluation system of radiotherapy rays can be implemented in whole or in part by software, hardware and a combination thereof. The above-mentioned modules can be embedded in or independent of the processor in the computer device in the form of hardware, or can be stored in the memory of the computer device in the form of software, so that the processor can call and execute the operations corresponding to the above-mentioned modules.

[0072] Those skilled in the art can clearly understand that for the convenience and simplicity of description, only the above-mentioned functional modules and module divisions are used as examples. In actual applications, the above-mentioned functions can be distributed and completed by different functional modules and modules as needed, that is, the internal structure of the system described in the present application is divided into different functional modules or modules to complete all or part of the functions described above.

Claims

1. A method for evaluating the relative biological effect of radiotherapy rays, characterized in that: The evaluation methods include: Using the radiation to be evaluated and the reference radiation to irradiate a plurality of samples to be irradiated at different radiation doses, respectively, wherein the samples to be irradiated are three-dimensional cell cultures, and the reference radiation and the radiation to be evaluated are radiations of different radiation types; The irradiated samples are stained to obtain staining images of all samples at different irradiation doses; Analyzing the staining image to obtain the positive cell ratio of the sample after irradiation; Based on the relationship between the positive cell ratio and the irradiation dose, a biological effect probability model was established; Calculate the ratio of the dose of the reference ray to the dose of the ray to be evaluated when the sample reaches the same positive cell ratio according to the biological effect probability model, and obtain the relative biological effect value of the ray to be evaluated; The steps of irradiating a plurality of samples to be irradiated with different irradiation doses using the irradiation to be evaluated and the reference irradiation include: A number of samples to be irradiated are divided into a first group and a second group; the first group is divided into n first subgroups, and the samples to be irradiated in the n first subgroups are irradiated with n different irradiation doses using the radiation to be evaluated; the second group is divided into n second subgroups, and the samples to be irradiated in the n second subgroups are irradiated with n different irradiation doses using reference radiation; wherein n>0.

2. The method for evaluating the relative biological effect of radiotherapy rays according to claim 1, characterized in that: The three-dimensional cell culture is an organoid.

3. The method for evaluating the relative biological effect of radiotherapy rays according to claim 1, characterized in that: The biological effect probability model is: Tumor control probability TCP model or normal tissue complication probability NTCP model.

4. The method for evaluating the relative biological effect of radiotherapy rays according to claim 3, characterized in that: The tumor control probability TCP model adopts any one of the TCP-Schultheiss logic model, the LQ-Poisson-TCP basic model, the LQ-Poisson-TCP two-parameter model or the Zaider-TCP model.

5. The method for evaluating the relative biological effect of radiotherapy rays according to claim 3, characterized in that: The normal tissue complication probability NTCP model adopts the NTCP-Schultheiss logistic model, Either the NTCP-Lyman-Kutcher-Berman model or the NTCP-RSM model.

6. The method for evaluating the relative biological effect of radiotherapy rays according to claim 1, characterized in that: The steps of analyzing the staining image to obtain the positive cell ratio of the sample after irradiation are specifically as follows: The staining image is imported into the image analysis software, the positive cells in the staining image are automatically counted to obtain the number of positive cells, and the number of positive cells is divided by the number of all cells in the staining image to obtain the positive cell ratio of the staining image.

7. The method for evaluating the relative biological effect of radiotherapy rays according to claim 1, characterized in that: The steps of analyzing the staining image to obtain the positive cell ratio of the sample after irradiation are specifically as follows: The stained image is placed under a microscope and manually analyzed under the microscope to obtain the number of positive cells, and the number of positive cells is divided by the number of all cells in the stained image to obtain the positive cell ratio of the stained image.

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