A method, system, and apparatus for cholangiocarcinoma treatment decision making based on idh1 / 2 genotype

Abstract

The application discloses a cholangiocarcinoma treatment decision method, system and equipment based on IDH1 / 2 genotype. Based on a genotype-confirmed mouse model and a clinical patient, it is found that IDH1 mutant tumors are only effective for immune checkpoint inhibitor (ICB) treatment, and IDH2 mutant and wild type tumors need to be combined with radiotherapy (RT) to enable patients to benefit from ICB treatment; FLASH-RT can inhibit tumors while reducing liver damage, reducing tumor fibrosis and enhancing CD8 + effector cell infiltration, thereby improving the efficiency of ICB treatment and overcoming immune tolerance. Based on this, the application provides a cholangiocarcinoma treatment decision method, system and equipment based on IDH1 / 2 genotype, which provides a new means for realizing personalized treatment of intrahepatic cholangiocarcinoma.

Description

Technical Field

[0001] This invention belongs to the field of intelligent medicine, specifically relating to a method, system, and device for decision-making in the treatment of cholangiocarcinoma based on the IDH1 / 2 genotype. Background Technology

[0002] Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive primary malignant liver tumor with a rising global incidence, but limited treatment options. The tumor microenvironment (TME) of ICC is typically characterized by dysfunctional CD8+ cells. + Characterized by T cells, abundant suppressive myeloid cell populations, and dense pro-fibrotic stroma, these factors may limit the effectiveness of immunotherapy.

[0003] Currently, there are no effective biomarkers in clinical practice that can predict which patients will respond to immunotherapy. This results in some patients not benefiting even after long-term treatment, wasting valuable treatment windows and creating a significant medical and economic burden. Therefore, screening for biomarkers that can predict the effectiveness of immunotherapy for cholangiocarcinoma is crucial in this field. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a method, system, and device for cholangiocarcinoma treatment decision-making based on the IDH1 / 2 genotype.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a method for decision-making in cholangiocarcinoma treatment based on IDH1 / 2 genotype, the method comprising: Data Acquisition: Obtain IDH1 / 2 genotype information from the patients to be tested; Data processing: Input the above IDH1 / 2 genotype information into the mutation determination model, wherein the IDH1 / 2 genotype includes IDH1 / 2 wild type, IDH1 mutant type, and IDH2 mutant type; Output results: The treatment plan for the patient to be tested is selected based on the IDH1 / 2 genotype information; if the genotype is IDH1 mutant, the immunotherapy plan is selected; if the genotype is IDH1 / 2 wild type, the combined immunotherapy and ultra-high dose rate radiotherapy plan is selected; if the genotype is IDH2 mutant, the combined immunotherapy and conventional radiotherapy plan is selected.

[0006] Furthermore, the immunotherapy is anti-PD-1 and CTLA-4 antibody therapy.

[0007] Furthermore, the ultra-high dose rate radiotherapy is selected from FLASH; the conventional radiotherapy is selected from SBRT or CONV-RT.

[0008] Furthermore, the radiation source of the FLASH is proton beam radiotherapy, and the radiation source of the SBRT or CONV-RT is photon radiotherapy.

[0009] Furthermore, the single dose of FLASH is 15 Gy, with a dose rate of 110-127 Gy / s; the dose of SBRT is 8 Gy × 3 times, with a dose rate of 3.52 Gy / min; and the single dose of CONV-RT is 15 Gy, with a dose rate of 3.52 Gy / min.

[0010] Furthermore, the cholangiocarcinoma mentioned is intrahepatic cholangiocarcinoma.

[0011] A second aspect of the present invention provides a cholangiocarcinoma treatment decision-making system based on the IDH1 / 2 genotype, the system comprising: Data acquisition module: Acquires IDH1 / 2 genotype information of the patients to be tested; Data processing module: Inputs the above IDH1 / 2 genotype information into the mutation determination model, wherein the IDH1 / 2 genotype includes IDH1 / 2 wild type, IDH1 mutant type, and IDH2 mutant type; Results output module: Selects treatment plan for the patient to be tested based on the IDH1 / 2 genotype information; if the genotype is IDH1 mutant, selects immunotherapy; if the genotype is IDH1 / 2 wild type, selects immunotherapy combined with ultra-high dose rate radiotherapy; if the genotype is IDH2 mutant, selects immunotherapy combined with conventional radiotherapy.

[0012] Furthermore, the immunotherapy is anti-PD-1 and CTLA-4 antibody therapy.

[0013] Furthermore, the ultra-high dose rate radiotherapy is selected from FLASH; the conventional radiotherapy is selected from SBRT or CONV-RT.

[0014] Furthermore, the radiation source of the FLASH is proton beam radiotherapy, and the radiation source of the SBRT or CONV-RT is photon radiotherapy.

[0015] Furthermore, the single dose of FLASH is 15 Gy, with a dose rate of 110-127 Gy / s; the dose of SBRT is 8 Gy × 3 times, with a dose rate of 3.52 Gy / min; and the single dose of CONV-RT is 15 Gy, with a dose rate of 3.52 Gy / min.

[0016] Furthermore, the cholangiocarcinoma mentioned is intrahepatic cholangiocarcinoma.

[0017] A third aspect of the present invention provides an apparatus comprising a memory and a processor, the memory being used to store program instructions; the processor being used to invoke the program instructions, which, when executed, implement the method described in the first aspect of the present invention.

[0018] A fourth aspect of the present invention provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the method described in the first aspect of the present invention.

[0019] A fifth aspect of the present invention provides a computer program product comprising a computer program that, when executed by a processor, implements the method described in the first aspect of the present invention.

[0020] A sixth aspect of the present invention provides the use of a reagent for detecting IDH1 / 2 genotypes in the preparation of products for selecting treatment regimens for cholangiocarcinoma; The IDH1 / 2 genotypes include IDH1 / 2 wild type, IDH1 mutant type, and IDH2 mutant type.

[0021] Advantages and beneficial effects of the present invention: Based on genotype-defined mouse models and clinical patients, this application found that IDH1 mutant tumors are only effective with immune checkpoint inhibitor (ICB) therapy, while IDH2 mutant and wild-type tumors require combined radiotherapy (RT) to allow patients to benefit from ICB treatment. FLASH-RT, while inhibiting tumors, can reduce liver damage, alleviate tumor fibrosis, and enhance CD8 activity. + Effector cell infiltration improves the efficacy of ICB therapy and overcomes immune tolerance. Based on this, this application provides a treatment decision-making method, system, and device for cholangiocarcinoma based on the IDH1 / 2 genotype, offering a new approach to achieving personalized treatment of intrahepatic cholangiocarcinoma. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the cholangiocarcinoma treatment decision-making method based on IDH1 / 2 genotype provided in this application; Figure 2 This diagram illustrates the genotype-dependent survival outcomes after RT and dual ICB treatment in an in situ ICC model. 2A shows the experimental workflow of the survival study, and 2B represents SS49-ICC (Idh2). R172K Kaplan-Meier survival curves of the mut mouse model after treatment with IgG, ICB, XRT+IgG, and XRT+ICB. 2C is the Kaplan-Meier survival curve of the 425-ICC (Tp53null) mouse model after treatment with IgG, ICB, XRT+IgG, and XRT+ICB. 2D is the Kaplan-Meier survival curve of the 2205 (Idh1) mouse model.R132C Kaplan-Meier survival curves of mut mouse models after treatment with IgG, ICB, XRT+IgG, and XRT+ICB; Figure 3 These are graphs showing disease progression and complication incidence in mouse ICC models after SBRT radiotherapy (XRT), immune checkpoint blockade (ICB), or a combination of both. 3A is an evaluation graph of mice treated with XRT (8 Gy × 3) and dual ICB (anti-PD-1 and anti-CTLA-4), and 3B is an evaluation graph of lung metastases in the SS49-ICC model. Figure 4 This is a graph showing the effects of radiotherapy (XRT) on the colony formation survival rate and gene expression of mouse ICC cells. Among them, 4A is a graph showing the colony formation survival rate of mouse ICC cell lines with different genotypes after XRT, 4B is a graph showing the gene set enrichment analysis of mouse ICC cells after radiotherapy, 4C is a volcano plot of differentially expressed genes in mouse ICC cells after radiotherapy, and 4D is a scatter plot of gene expression changes in mouse ICC cells and control mouse ICC cells. Figure 5 This is a diagram showing the transcriptome changes in mouse ICC cells after radiotherapy. In this diagram, 5A shows the relative expression levels of genes related to myeloid recruitment and depletion in mouse ICC cells after radiotherapy, 5B shows the results of KEGG pathway enrichment analysis, and 5C shows the gene set analysis related to the regulation of IL-2 production in radiotherapy-treated mouse ICC cells. Figure 6 It is Idh2 R172K Flow cytometry analysis of the frequency and phenotype of tumor-infiltrating and lymph node (LN) immune cells in a mouse ICC model, where 6A and 6B are CD8+ cells. + Representative flow cytometry and quantitative analysis of T cells; 6C and 6D represent Ki67. + IFN-γ + CD8 + Representative flow cytometry and quantitative analysis of T cells; 6E and 6F are IFN-γ. + CD4 + Representative flow cytometry and quantitative analysis of T cells; 6G and 6H represent TNF-α in lymph nodes. + CD8 + Representative flow cytometry and quantitative analysis of T cells; 6I and 6J represent TNF-α within the tumor. + CD8 + Representative flow cytometry and quantitative analysis of T cells; 6K and 6L are granzyme B in tumors. + CD8 + Representative flow cytometry and quantitative analysis of T cells; 6M and 6N are CD45+.+ CD11c + MHC-IIa + CD80 + CD86 + Flow cytometry and quantitative analysis of dendritic cells; 6O and 6P are CD206. low CD80 high Flow cytometry and quantitative analysis of M1 tumor-associated macrophages (TAMs), where 6Q and 6R are CD206. high Flow cytometry and quantitative analysis of M2 type TAM, 6S is the M1 / M2 TAM ratio, 6T is the intratumoral CD8. + / CD4 + T cell ratio plot; Figure 7 This is a graph showing the changes in tumor growth in an ICC mouse model after treatment with conventional radiotherapy (CONV-RT) or FLASH-RT with or without immune checkpoint blockade (ICB). Figure 8 This is a comparison chart of FLASH-RT / ICB and CONV-RT / ICB in a mouse ICC model, where 8A represents Idh1 / 2wt / Kras. G12D / p53null Experimental design diagrams for FLASH-RT and CONV-RT (with or without ICB) in a mouse ICC model; 8B is a Kaplan-Meier survival curve; 8C is an experimental setup diagram of the liver and spleen tissues adjacent to the tumor in radiotreated mice; 8D is a graph showing the changes in transaminase levels after radiotherapy; 8E is a graph showing the quantitative analysis of cleaved caspase-3 in the liver tissue adjacent to the proximal tumor after radiotherapy; 8F is a graph showing the quantitative analysis of cleaved caspase-3 in the liver tissue adjacent to the distal tumor after radiotherapy; 8G is a graph showing the quantitative analysis of cleaved caspase-3 in the spleen tissue adjacent to the tumor after radiotherapy; 8H is a histological analysis diagram of the liver tissue adjacent to the distal tumor. Figure 9 Is conventional radiotherapy (CONV-RT) or FLASH-RT suitable for patients with Idh1 / 2wt / Kras? G12D / p53null The effect of ICC on transaminase and circulating blood cells in mice, where 9A is the change of AST and ALT, and 9B is the change of white blood cells (WBC) and lymphocytes (LYM). Figure 10 These are representative images of cleavage caspase 3 staining in the liver and spleen tissues adjacent to the tumor. Among them, 10A is the image of the liver tissue adjacent to the proximal tumor, 10B is the image of the liver tissue adjacent to the distal tumor, and 10C is the image of the spleen tissue adjacent to the tumor. Figure 11Figure 8 shows time-matched analysis of tumor growth dynamics after conventional radiotherapy (CONV-RT) or FLASH-RT (15 Gy) and dual ICB (anti-PD-1 and anti-CTLA-4) therapy or radiotherapy combined with ICB therapy. Figure 12 This is a graph showing the impact of conventional radiotherapy (CONV-RT) or FLASH-RT with or without immune checkpoint blockade (ICB) on lung metastases. Figure 13 This is a graph showing the effect of conventional radiotherapy (CONV-RT) or FLASH-RT combined with or without immune checkpoint blockade (ICB) on mouse body weight; Figure 14 It is Idh1 / 2wt / Kras G12D / p53null Flow cytometry analysis of the frequency and phenotype of tumor-infiltrating immune cells after treatment in a mouse ICC model, where 14A represents CD45 + CD45 in cells + CD3 + Flow cytometry and quantitative analysis of T cells, 14B is CD3 + CD45 in T cells + CD3 + CD8 + Flow cytometry and quantitative analysis of T cells; 14C is CD8 expressing granzyme B. + Flow cytometry and quantitative analysis of T cells, 14D is Ki67 + Proliferative CD8 + Flow cytometry and quantitative analysis of T cells, 14E is GzmB + Ki67 + CD8 + Flow cytometry and quantitative analysis of T cells; 14F is defined as Tim3. + PD-1 + depletion CD8 + Flow cytometry and quantitative analysis of T cells; 14G represents MDSCs of monocytes (CD45). + CD11b + Ly6C + Flow cytometry and quantitative analysis of cells; Figure 15 It is Idh1 / 2wt / Kras G12D / p53null Flow cytometry analysis of FLASH and CONV-RT / ICB treatments in a mouse ICC model. 15A represents a representative flow cytometry image and activated dendritic cells (CD80). + CD86 +DCs) flow cytometry analysis and quantitative analysis graph, 15B is CD11b + F4 / 80 + MHC-II hi Flow cytometry analysis and quantitative analysis of M1-like tumor-associated macrophages (TAMs), 15C is CD11b + F4 / 80 + CD206 hi CD80 low M2-like TAMs flow cytometry analysis and quantitative analysis image, 15D is CD4 + T cell quantification map, 15E is CD8 + / CD4 + T cell ratio plot, 15F is MHC-II hi M1TAM and CD206 hi M2 TAM ratio chart; Figure 16 It is Idh1 / 2wtKras G12D / p53null Matrix effect diagram of FLASH-RT and CONV-RT with or without ICB treatment in a mouse ICC model, where 16A is CD31 + The tumor tissue area percentage map shows the percentage of positive areas adjacent to CD31 and α-smooth muscle actin (α-SMA), the percentage of positive areas of myofibroblasts (α-SMA) in ICC tissue, the percentage of type I collagen areas in ICC tissue, the percentage of positive areas of type I collagen in ICC tissue, the representative immunofluorescence map of CD31 (white), α-SMA (red) and cell nucleus (DAPI, blue), and the representative immunofluorescence map of type I collagen (red) and cell nucleus (DAPI, blue). Figure 17 This is a survival curve of mice in the IDH2 mutant ICC mouse model who received CONV-RT or FLASH radiotherapy with or without ICB treatment; Figure 18 These are prognostic charts for ICC patients after first-line treatment for different genotypes. Among them, 18A is the progression-free survival chart for IDH wild-type (wt) and IDH1 mutant (mut) patients; 18B is the overall survival chart for IDH wild-type (wt) and IDH1 mutant (mut) patients; 18C is the progression-free survival and overall survival chart for IDH1 / 2 wild-type ICC patients who received standard anti-PD(L)-1 / chemotherapy (with or without radiotherapy); 18D is the optimal tumor response chart for IDH1 / 2 mutant and wild-type patients; and 18E is the tumor regression spider chart for IDH1 mutant and IDH2 mutant ICC patients. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0024] In some of the processes described in the specification, claims, and accompanying drawings of this invention, multiple operations appearing in a specific order are included. However, it should be clearly understood that these operations may not be executed in the order they appear herein, or may be executed in parallel. The operation numbers, such as 101, 102, etc., are merely used to distinguish different operations and do not represent any execution order. Furthermore, these processes may include more or fewer operations, and these operations may be executed sequentially or in parallel. It should be noted that the descriptions such as "first," "second," etc., in this document are used to distinguish different messages, devices, modules, etc., and do not represent a sequential order, nor do they limit "first" and "second" to different types.

[0025] Figure 1 This is a schematic diagram of the cholangiocarcinoma treatment decision-making method based on the IDH1 / 2 genotype provided in this application, specifically including: 101 Data Acquisition: Obtain the IDH1 / 2 genotype information of the patients to be tested.

[0026] 102 Data Processing: Input the above IDH1 / 2 genotype information into the mutation determination model. The IDH1 / 2 genotype includes IDH1 / 2 wild type, IDH1 mutant type, and IDH2 mutant type.

[0027] 103 Output Results: Based on the IDH1 / 2 genotype information, select the treatment plan for the patient to be tested; if the genotype is IDH1 mutant, select the immunotherapy plan; if the genotype is IDH1 / 2 wild type, select the combined immunotherapy and ultra-high dose rate radiotherapy (FLASH) plan; if the genotype is IDH2 mutant, select the combined immunotherapy and conventional radiotherapy (SBRT or CONV-RT) plan.

[0028] This application provides a decision-making system for cholangiocarcinoma treatment based on the IDH1 / 2 genotype, specifically including: 201 Data Acquisition Module: Acquires IDH1 / 2 genotype information of the patients to be tested.

[0029] 202 Data Processing Module: Input the above IDH1 / 2 genotype information into the mutation determination model. The IDH1 / 2 genotype includes IDH1 / 2 wild type, IDH1 mutant type, and IDH2 mutant type.

[0030] 203 Result Output Module: Selects a treatment plan for the patient to be tested based on the IDH1 / 2 genotype information; if the genotype is IDH1 mutant, selects an immunotherapy plan; if the genotype is IDH1 / 2 wild type, selects a combined immunotherapy and ultra-high dose rate radiotherapy (FLASH) plan; if the genotype is IDH2 mutant, selects a combined immunotherapy and conventional radiotherapy (SBRT or CONV-RT) plan.

[0031] In some embodiments, the immunotherapy may also be referred to as immune checkpoint inhibitor therapy (ICB), specifically anti-PD-1 and CTLA-4 antibody therapy.

[0032] In some implementations, the conventional radiotherapy is CONV-RT (Conventional Radiotherapy, 15 Gy single dose). Alternatively, conventional radiotherapy can also be SBRT (Stereotactic Body Radiotherapy, 8 Gy × 3 fraction). The ultra-high dose rate proton radiotherapy is FLASH-RT (FLASH Radiotherapy, 15 Gy single dose), with a dose rate of 110-127 Gy / s.

[0033] In some embodiments, implementation of the methods and / or systems of this application may include performing or completing selected tasks manually, automatically, or in combination thereof. Furthermore, the actual instruments and equipment according to the embodiments of the methods and / or systems of this application may implement multiple selected tasks via hardware, software, firmware, or a combination thereof using an operating system.

[0034] For example, the hardware used to perform the selected task according to embodiments of this application may be implemented as a chip or circuit. As software, the selected task according to embodiments of this application may be implemented as multiple software instructions executed by a computer using any suitable operating system. In exemplary embodiments of this application, one or more tasks according to exemplary embodiments of the methods and / or systems described in this application may be performed by a data processor, such as a computing platform for executing multiple instructions. Optionally, the data processor includes volatile memory for storing instructions and / or data, and / or non-volatile memory for storing instructions and / or data, such as a magnetic hard disk and / or removable media. Optionally, a network connection is also provided. A display and / or user input device such as a keyboard or mouse may also be optionally provided.

[0035] This application provides a device, specifically including: a memory and a processor, wherein the memory is used to store program instructions; and the processor is used to invoke the program instructions, and when the program instructions are executed, implement the above method.

[0036] The present invention also provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the above-described method.

[0037] In some implementations, the computer-readable storage medium may be FRAM, ROM, PROM, EPROM, EEPROM, flash memory, magnetic surface memory, optical disc, or CD. ROM and other memory; or it can be any device that includes one or any combination of the above-mentioned memory.

[0038] In some implementations, executable instructions may take the form of programs, software, software modules, scripts, or code, written in any form of programming language (including compiled or interpreted languages, or declarative or procedural languages), and may be deployed in any form, including as stand-alone programs or as modules, components, subroutines, or other units suitable for use in a computing environment.

[0039] As an example, executable instructions may, but do not necessarily, correspond to files in a file system. They may be stored as part of a file that holds other programs or data, for example, in one or more scripts in a Hyper Text Markup Language (HTML) document, in a single file dedicated to the program in question, or in multiple collaborating files (e.g., a file that stores one or more modules, subroutines, or code sections).

[0040] As an example, executable instructions can be deployed to execute on a single computing device, or on multiple computing devices located in one location, or on multiple computing devices distributed across multiple locations and interconnected via a communication network.

[0041] In some embodiments, executable instructions can be implemented by a computer program instructing related hardware. The computer program can be stored in a non-volatile computer-readable storage medium. When executed, the computer program can include the processes described in the embodiments of the methods above. Any references to memory, storage, databases, or other media used in the embodiments provided in this application can include non-volatile and / or volatile memory. Non-volatile memory can include read-only memory (ROM), programmable ROM (PROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM), or flash memory. Volatile memory can include random access memory (RAM) or external cache memory. By way of illustration and not limitation, RAM is available in various forms, such as static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), dual data rate SDRAM (DDRSDRAM), enhanced SDRAM (ESDRAM), synchronous link DRAM (SLDRAM), Rambus direct RAM (RDRAM), direct memory bus dynamic RAM (DRDRAM), and memory bus dynamic RAM (RDRAM), etc.

[0042] This invention provides the application of reagents for detecting IDH1 / 2 genotypes in the preparation of products for selecting treatment regimens for cholangiocarcinoma; the IDH1 / 2 genotypes include IDH1 / 2 wild type, IDH1 mutant type, and IDH2 mutant type.

[0043] The reagent also includes a detectable marker.

[0044] In some embodiments, a detectable label refers to any composition that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. This includes, but is not limited to, UV-Vis labels, near-infrared labels, fluorescent groups, phosphorescent groups, magnetic spin resonance labels, photosensitizers, photolytically cleavable moieties, chelating centers, heavy atoms, radioactive isotopes, isotope-detectable spin resonance labels, paramagnetic moieties, chromophores, and luminescent organisms.

[0045] In some embodiments, the fluorescent groups include, but are not limited to, FAM (Carboxyfluorescein, green fluorescent), FITC (Fluorescein isothiocyanate), TET (Tetrachlorofluorescein), HEX (Hexachlorofluorescein), JOE (2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein), rhodamine dyes (such as R110, TAMRA, Texas Red, etc.), ROX, and AlexaFluor dyes (such as Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 500, Alexa 514, Alexa 532, Alexa 546, Alexa 555, Alexa 568, Alexa 594, Alexa 610, Alexa 633, Alexa 635, Alexa 647, Alexa 633, Alexa 635, Alexa 647, Alexa 633, Alexa 634, Alexa 645, Alexa 635, Alexa 636, Alexa 637, Alexa 638, Alexa 639 ... 660, Alexa 680, Alexa 700, Alexa 750, Alexa 790), ATTO dyes (such as ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rho11, ATTO Rho12, ATTO Thio12, ATTO Rho101, ATTO 590, ATTO 594, ATTO Rho13, ATTO 610, ATTO 620, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxa12, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), DyLight dyes, cyanine dyes (such as Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), FluoProbes dyes, SulfoCy dyes, Seta dyes, IRIS dyes, SeTau dyes, SRfluor dyes, Square dyes.

[0046] The products include reagent kits, chips, and test strips.

[0047] In some embodiments, the kit may also include a fluorescent dye, and a variety of known fluorescent dyes may be used. Examples include methods using an intercalator with a marking function, and methods using probes that bind fluorescent substances to nucleotides that specifically hybridize to the relatively amplified DNA sequence. Examples of intercalators include ethidium bromide and SYBR Green I as unsaturated fluorescent dyes, and Resolight and EvaGreen as saturated fluorescent dyes. The dosage should be as recommended by the manufacturer or distributor of the fluorescent dye used.

[0048] The kit also includes an instruction manual, which may include guidance on obtaining and processing samples.

[0049] In some embodiments, a kit refers to any delivery system used to deliver materials. The components of the kit may be packaged in an aqueous medium or in a lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container in which one component can be placed, and preferably, appropriately aliquoted. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in a single vial. The kit of this application will also typically include a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers in which the desired vials can be held.

[0050] The specific experimental methods and results of this application are as follows: I. Experimental Methods 1. Cell lines and culture Mouse ICC cell lines 425-ICC and 254-ICC (Idh) wt p53 – / – / Kras G12D ), 2205-ICC and 2275-ICC (Idh1) R132C / Kras G12D ) and SS49-ICC (Idh2 R172K / Kras G12DAll cells were derived from previously described genetically engineered mouse models. Specifically, 425-ICC and 254-ICC cells were derived from hybrid mice of C57 / BL6 and FVB mice; 2205-ICC and SS49-ICC cells were derived from hybrid mice of C57 / BL6 and 129S mice. Cells were cultured in DMEM or RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37°C in a 5% CO2 incubator. Cell identification and mycoplasma detection were performed before in vivo use.

[0051] 2. In vitro irradiation and RNA sequencing analysis ICC cells were irradiated using a 225 kV X-ray apparatus (XRad225, Precision X-ray). In clonogenic assays, cells received single irradiations of 2, 4, 6, or 8 Gy. Irradiated cells were seeded at extremely low dilutions (1:20–1:2000), and clones containing at least 50 cells were stained after 10–12 days to assess clonogenic viability. Viability fractions were calculated relative to the unirradiated control group.

[0052] For RNA sequencing analysis, all cells received a single 8 Gy irradiation. Twenty-four hours later, total RNA was extracted from ICC cells using a Qiagen kit. RNA sequencing was performed by Novogene. Raw RNA-seq reads were aligned to the mouse reference genome GRCm39 using STAR v2.7.9a software with default parameters. Gene-level read counts were quantified using featureCounts v2.0.1 and Ensembl annotation (GRCm39.107). Differentially expressed genes (DEGs) were identified using DESeq2 v1.48.2, with significance defined as p-value < 0.05 and |log2(FC)| > 1.5. Functional enrichment analysis of DEGs was performed using clusterProfiler v4.16.0, including gene ontology (GO) and KEGG pathway enrichment analysis, as well as gene set enrichment analysis (GSEA).

[0053] 3. In-situ ICC model All animal experiments were approved by the Massachusetts General Hospital Animal Care and Use Committee (IACUC) (Protocol No.: 2021N000137). We used 6- to 8-week-old immunocompetent F1 generation mice (Charles River Laboratory). 1 × 10 6One ICC cell was mixed with Matrigel at a 1:1 ratio and injected into the left lobe of the mouse liver to establish an orthotopic tumor model. The mice were anesthetized with isoflurane, and tumor growth was monitored every 3 days using high-frequency ultrasound. Researchers were unaware of the group assignments during tumor measurement and endpoint assessment.

[0054] 4. Internal radiation therapy When the tumor diameter reaches 4-5 mm, fractionated photon radiotherapy (SBRT) is used: mice are irradiated with 8 Gy × 3 for 3 consecutive days using a 320 kV X-RAD irradiator. A 15 mm collimated field is used to irradiate the tumor while the whole body is shielded. Conventional radiotherapy (CONV-RT) is performed under the same irradiation conditions, with a single 15 Gy photon beam at a dose rate of 3.52 Gy / min. FLASH radiotherapy (FLASH-RT) uses a 228 MeV dual-scatter beam to irradiate the tumor with a proton beam at an ultra-high dose rate (~110-127 Gy / s). The tumor receives a single 15 Gy dose.

[0055] The dosimetry results were validated using EBT3 film and an ionization chamber. A dose of 15 Gy was chosen based on the technical feasibility of FLASH-RT and to simulate high-dose irradiation in order to assess stromal and systemic effects under conditions compatible with FLASH-RT therapy.

[0056] 5. Immune checkpoint blockade (ICB) Anti-PD-1 (clone RMP1-14, BioXcell, catalog number: BE0146) and anti-CTLA-4 (clone 9D9, BioXcell, catalog number: BE0164) antibodies (BioXCell) were administered intraperitoneally at a dose of 10 mg / kg every 3 days. For the 8 Gy × 3 radiotherapy regimen, immune checkpoint blockade (ICB) was initiated on the day of the last radiotherapy and continued for 5 fractions. For single radiotherapy, 3 doses of antibodies were administered after radiotherapy. Control group mice received isotype-matched IgG (BioXCell).

[0057] 6. Flow cytometry Tumor tissue was collected on day 8 after the start of radiotherapy. Single-cell suspensions were prepared and stimulated with an activation mixture containing brefeldin A for 4 hours, followed by intracellular staining. Cell staining was used to detect CD45, CD3, CD4, CD8, Ki-67, granzyme B, PD-1, TIM-3, CD11b, Ly6C, Ly6G, F4 / 80, CD206, CD80, CD86, TNF-α, and IFN-γ.

[0058] Data were acquired using a flow cytometer and analyzed using FlowJo software.

[0059] 7. Immunofluorescence and HE analysis 5 μm thick paraffin-embedded sections were stained, and endothelial cells were detected using CD31 (DIA-310, clone SZ31), myofibroblasts using α-SMA (C6198, clone 1A4), and apoptotic cells using type I collagen (ab21286) and cleavage caspase-3 (CST 9664S, clone 5A1E). All secondary antibodies were purchased from Jackson ImmunoResearch. "Tumor adjacent to spleen" was defined as the area likely to be irradiated, immediately adjacent to the left lobe of the liver. "Proximal tumor adjacent to liver" included tissue within the three central hepatic lobule veins at the tumor margin, while "distal tumor adjacent to liver" was defined as the area 5–6 mm from the tumor margin, within the initial irradiation field. Quantitative analysis was performed on five random high-power fields of each tumor and its adjacent liver and spleen using QuPath software. Vascular area and pericyte coverage were expressed as a percentage of tissue area.

[0060] 8. Liver injury and hematological assessment Peripheral blood was collected before treatment and on day 4 (complete blood count) and day 8 (AST, ALT) after radiotherapy. Serum transaminase levels were measured using standard enzymatic methods.

[0061] 9. Transfer Assessment Lung metastases were counted during autopsy through gross examination. The presence or absence of pleural effusion, ascites, and lymph node metastasis were recorded as indicators of systemic progression.

[0062] 10. Cohort analysis of patients with biliary tract cancer This study analyzed a cohort of 103 patients with biliary tract cancer treated at Shandong Cancer Hospital in China. Genomic data were obtained using next-generation sequencing. Clinical outcomes were stratified according to IDH1 / 2 mutation status (Table 1). This study was approved by the institutional ethics committee (SDTHEC202601010), exempting the patient from informed consent requirements. Tumor response was assessed using RECIST 1.1 criteria.

[0063] Table 1 Clinical Patient Information

[0064] 11. Statistical Analysis The sample size was based on prior experience with the in situ ICC model and had sufficient statistical power to detect differences in tumor volume ≥40% (α=0.05). Data are expressed as mean ± standard error (SEM) unless otherwise stated. Survival analyses were performed using the Kaplan-Meier method with log-rank tests. Hazard ratios were calculated using the Cox proportional hazards model. Comparisons between two groups were performed using Student's t-test or Mann-Whitney U test, depending on the circumstances. Comparisons among multiple groups were performed using one-way ANOVA with Tukey correction. Due to the exploratory nature of the clinical analysis, no multiple comparison correction was performed. All tests were two-tailed. P < 0.05 was considered statistically significant.

[0065] II. Experimental Results 1. Genotype is associated with response to RT / ICB treatment in in situ ICC models. To investigate whether tumor genotype affects the efficacy of radiotherapy (RT) and dual immune checkpoint blockade (ICB), we first evaluated treatment responses in three homologous mouse ICC models representing different molecular subtypes: Idh1 / 2 wild-type (425), Idh2 mutant (SS49), and Idh1 mutant (2205). Figure 2 In the in situ Idh2 mutant model, ICB slightly delayed tumor growth, while ICB combined with hypofractionated radiotherapy (XRT, 8 Gy × 3) significantly inhibited tumor growth and improved survival, superior to either therapy alone. Figure 2 A). Idh1 / 2 wild-type ICC has intrinsic resistance to ICB treatment ( Figure 2 B). In this model, a combined radiotherapy regimen is required to control the tumor, while the addition of ICB significantly enhances the treatment effect. In Idh1 mutant ICC, only ICB significantly inhibited tumor growth, while RT and RT+ICB failed to inhibit tumor growth, but the combination of the two did not bring any additional benefit. Figure 2 C). Evaluation of distant metastasis and complications showed that RT / ICB reduced the incidence of distant metastasis and pleural effusion only in the Idh2 mutant ICC model, while in the Idh1 mutant ICC model, only ICB reduced the incidence of distant metastasis and pleural effusion. These findings suggest that treatment sensitivity is genotype-dependent, and the 425 tumors represent an ICB-resistant environment in which RT is required for ICB immunotherapy to be effective. Figure 3 ).

[0066] 2. Photon irradiation-induced genotype-dependent immunomodulatory transcriptional program To determine whether tumor genotype influences radiotherapy-induced immune signaling pathways, we performed RNA sequencing on Idh1 / 2 wild-type and Idh2 mutant ICC cells after radiotherapy (8 Gy). In Idh1 / 2 wild-type cells, radiotherapy induced enrichment of chemokines and cytoplasmic DNA sensing, consistent with activation of innate immune programs. RT also upregulated transcripts associated with compensatory immune regulatory signals, including Ccl2, Ccl7, Havcr2 (TIM-3), and Adora2a, suggesting the induction of inversely regulated myeloid recruitment and depletion pathways. Conversely, Idh2 mutant ICC cells showed enrichment of NF-κB, TNF-α, and TGF-β-related signaling pathways after photon irradiation, while IL-2 production-related transcripts were reduced compared to Idh1 / 2 wild-type ICC cells. These results suggest that tumor genotype determines RT-induced immune-matrix signaling, thereby influencing the dependence of ICC cells on RT-mediated immune initiation. Figure 4 , Figure 5 ).

[0067] 3. RT can reshape the immune tumor microenvironment of ICC and induce CD8. + T cell expansion and reduction of the spread of drug-resistant genotypes. To elucidate the cellular immune mechanism by which radiotherapy combined with immune checkpoint inhibitors (RT-ICBs) exerts therapeutic effects in intrahepatic cholangiocarcinoma (ICC), we analyzed the immune tumor microenvironment (TME) of Idh2-mutant tumors by flow cytometry 8 days after the start of treatment. Figure 6 Compared with immune checkpoint inhibitors (ICBs) alone, RT combined with ICBs can increase intratumoral CD8. + T cell infiltration and proliferation enhance dendritic cell (DC) activation (CD80). + CD86 + It also increased the proportion of M1 / M2 tumor-associated macrophages (TAMs); furthermore, compared with the control group, the combined use of PD-1 / CTLA-4 dual immune checkpoint inhibitors after RT increased intratumoral Ki67. + Proliferation and activation of CD8 + The number of T cells.

[0068] Importantly, in these drug-resistant models, RT / ICB treatment significantly reduced the lung metastasis burden at this stage, indicating that RT-induced immune reprogramming not only enhances local tumor control but also inhibits systemic spread. These findings suggest that RT can convert ICB-resistant tumors into an immune-allowed state, characterized by CD8+. + T cell proliferation and effector differentiation are enhanced, while metastasis and diffusion are reduced.

[0069] 4. FLASH-RT can reduce the toxicity caused by high-dose radiotherapy and restore the therapeutic effect of immune checkpoint inhibitors. Next, we tested whether ultra-high dose-rate proton FLASH-RT (15 Gy per dose) improved treatment efficacy compared to CONV-RT (15 Gy per dose) in an Idh1 / 2 wild-type orthotopic ICC model resistant to both ICB and RT. Both CONV-RT and proton FLASH-RT significantly delayed tumor growth, but due to distant metastasis progression, they did not improve overall survival compared to the IgG control group. Compared to RT alone, both CONV-RT / ICB and FLASH-RT / ICB combination therapy significantly enhanced tumor suppression. Figure 7 Importantly, no statistically significant difference was observed between CONV-RT / ICB and FLASH-RT / ICB in terms of primary tumor growth inhibition. These results suggest that the limitation of high-dose radiotherapy in this model is not due to insufficient tumor cytotoxicity, but rather to adverse effects on the tumor microenvironment and host tissues. However, FLASH-RT / ICB significantly improved overall survival compared to conventional radiotherapy / immune checkpoint blockade (CONV-RT / ICB). This indicates that the survival benefit mediated by FLASH-RT is not entirely attributable to tumor cell reduction. Importantly, FLASH-RT better preserved the integrity of normal tissues compared to CONV-RT, as evidenced by reduced serum transaminase levels and decreased caspase-3 staining in the tumor-adjacent liver and spleen. These results suggest that FLASH-RT / ICB improves the therapeutic index while maintaining or enhancing anti-tumor immunotherapy efficacy. Importantly, despite similar primary tumor control, differences in survival rates were observed, suggesting that factors influencing survival are systemic and microenvironmental mechanisms, rather than inherent tumor cytotoxicity. Figure 8-10 ).

[0070] 5. FLASH-RT combined with ICB can enhance CD8 + T cell proliferation and effector differentiation Next, we repeated the experiment and performed flow cytometry analysis 8 days after treatment to investigate the tumor-infiltrating immune cell population. In this time-matched experiment, both the FLASH-RT / ICB group and the CONV-RT / ICB group significantly inhibited tumor growth compared to the IgG group (p<0.001). Figure 11 ), and lung metastasis was significantly suppressed in the FLASH-RT / ICB group ( Figure 12 Furthermore, the FLASH-RT group experienced a smaller degree of weight loss than the CONV-RT group. Figure 13 Flow cytometry analysis of ICC tissue showed that RT / ICB increased intratumoral CD8. +T cell infiltration and proliferation.

[0071] However, in the cold tumor Idh1 / 2 wild-type ICC model, FLASH-RT / ICB treatment induced CD8+ oxidase activity. + T cell frequency, Ki67 + Proliferative CD8 + T cells, granzyme B + Effect CD8 + T cells and the CD8 / CD4 ratio are elevated, while PD-1 is elevated. + TIM-3 + Exhaustion CD8 + T cell reduction. Furthermore, FLASH-RT / ICB treatment also increased CD80. + CD86 + The frequency of activated dendritic cells (DCs) and the M1 / M2 tumor-associated macrophage (TAM) ratio were increased, and the number of monocyte-derived myeloid-derived suppressor cells (MDSCs) was reduced. These immune changes were more significant than with CONV-RT / ICB treatment. These data collectively suggest that, in the context of dual CTLA-4 / PD-1 immune checkpoint blockade therapy, FLASH-RT, compared with conventional radiotherapy, significantly enhances CD8+ immune function. + Effector cell differentiation and reduction of exhaustion markers are associated with ( Figure 14-15 ).

[0072] 6. FLASH-RT can reshape the pro-fibrotic ICC matrix and reduce pro-fibrotic signal transduction. Furthermore, we analyzed vascular and fibrotic changes in ICC tissue collected on day 8 post-radiotherapy using immunofluorescence (IF). Tumor vascular area slightly increased after CONV-RT / IgG treatment, but decreased after FLASH-RT / ICB treatment (p<0.05). However, the proportion of mature (pericylloid) vessels did not change significantly. Histological analysis also showed that CONV-RT increased fibroblast activation and collagen deposition in the liver adjacent to the tumor, consistent with radiation-induced wound healing responses. Conversely, FLASH-RT significantly reduced myofibroblast (α-SMA) activity. + The study results indicate that FLASH-RT can attenuate the radiation-induced stromal proliferative remodeling induced by CONV-RT, while maintaining a mature tumor vascular system and preserving antitumor immunity, thereby potentially improving the migration and effector function of immune cells within the immunotumor microenvironment (TME). These findings suggest that the therapeutic index of combined radiotherapy / immune checkpoint blockade in intrahepatic cholangiocarcinoma (ICC) may depend on the balance between immune activation and radiation-induced stromal and vascular remodeling. Figure 16In the IDH2 mutant ICC mouse model, regardless of the radiotherapy modality received, combined immunotherapy resulted in a better survival benefit. Figure 17 ).

[0073] 7. Retrospective clinical cohort studies support a genotype-dependent pattern. Radiation therapy and anti-CTLA-4 immune checkpoint blockade therapy are not standard treatments for cholangiocarcinoma. To better understand preclinical study results, we analyzed clinical response data from 103 patients with cholangiocarcinoma from the same institution.

[0074] Compared with IDH1 / 2 wild-type ICC patients (n=40), patients with IDH1 mutant tumors (n=6) had longer overall response rate (ORR) and progression-free survival (PFS), but no extended overall survival. In IDH1 / 2 wild-type patients receiving chemotherapy combined with immunotherapy, compared with chemotherapy or immunotherapy alone, combined radiotherapy significantly prolonged median progression-free survival (PFS) (10.5 months vs. 5.0 months; HR=0.39, 95% CI: 0.17–0.89, p=0.006), and showed a non-significant trend toward prolonged overall survival (OS) (29.0 months vs. 13.0 months; HR=0.41, 95% CI: 0.13–1.23, p=0.09) (n=15 per group). Figure 18 ).

[0075] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A treatment decision-making method for cholangiocarcinoma based on IDH1 / 2 genotype, characterized in that, The method includes: Data Acquisition: Obtain IDH1 / 2 genotype information from the patients to be tested; Data processing: Input the above IDH1 / 2 genotype information into the mutation determination model, wherein the IDH1 / 2 genotype includes IDH1 / 2 wild type, IDH1 mutant type, and IDH2 mutant type; Output results: The treatment plan for the patient to be tested is selected based on the IDH1 / 2 genotype information; if the genotype is IDH1 mutant, the immunotherapy plan is selected; if the genotype is IDH1 / 2 wild type, the combined immunotherapy and ultra-high dose rate radiotherapy plan is selected; if the genotype is IDH2 mutant, the combined immunotherapy and conventional radiotherapy plan is selected.

2. The method according to claim 1, characterized in that, The immunotherapy is anti-PD-1 and CTLA-4 antibody therapy; Preferably, the ultra-high dose rate radiotherapy is selected from FLASH; the conventional radiotherapy is selected from SBRT or CONV-RT. Preferably, the radiation source of the FLASH is proton beam radiotherapy, and the radiation source of the SBRT or CONV-RT is photon radiotherapy; Preferably, the single dose of FLASH is 15 Gy, and the dose rate is 110-127 Gy / s; the dose of SBRT is 8 Gy × 3 times, and the dose rate is 3.52 Gy / min; the single dose of CONV-RT is 15 Gy, and the dose rate is 3.52 Gy / min.

3. The method according to claim 1, characterized in that, The cholangiocarcinoma mentioned is intrahepatic cholangiocarcinoma.

4. A decision-making system for cholangiocarcinoma treatment based on IDH1 / 2 genotype, characterized in that, The system includes: Data acquisition module: Acquires IDH1 / 2 genotype information of the patients to be tested; Data processing module: Inputs the above IDH1 / 2 genotype information into the mutation determination model, wherein the IDH1 / 2 genotype includes IDH1 / 2 wild type, IDH1 mutant type, and IDH2 mutant type; Results output module: Selects treatment plan for the patient to be tested based on the IDH1 / 2 genotype information; if the genotype is IDH1 mutant, selects immunotherapy; if the genotype is IDH1 / 2 wild type, selects immunotherapy combined with ultra-high dose rate radiotherapy; if the genotype is IDH2 mutant, selects immunotherapy combined with conventional radiotherapy.

5. The system according to claim 4, characterized in that, The immunotherapy is anti-PD-1 and CTLA-4 antibody therapy; Preferably, the ultra-high dose rate radiotherapy is selected from FLASH; the conventional radiotherapy is selected from SBRT or CONV-RT. Preferably, the radiation source of the FLASH is proton beam radiotherapy, and the radiation source of the SBRT or CONV-RT is photon radiotherapy; Preferably, the single dose of FLASH is 15 Gy, and the dose rate is 110-127 Gy / s; the dose of SBRT is 8 Gy × 3 times, and the dose rate is 3.52 Gy / min; the single dose of CONV-RT is 15 Gy, and the dose rate is 3.52 Gy / min.

6. The system according to claim 4, characterized in that, The cholangiocarcinoma mentioned is intrahepatic cholangiocarcinoma.

7. A device, characterized in that, The device includes a memory and a processor, the memory being used to store program instructions; the processor being used to invoke the program instructions, and when the program instructions are executed, to implement the method according to any one of claims 1-3.

8. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by a processor, it implements the method described in any one of claims 1-3.

9. A computer program product, characterized in that, Includes a computer program that, when executed by a processor, implements the method described in any one of claims 1-3.

10. Application of reagents for detecting IDH1 / 2 genotypes in the preparation of products for selecting treatment regimens for cholangiocarcinoma; The IDH1 / 2 genotypes include IDH1 / 2 wild type, IDH1 mutant type, and IDH2 mutant type.

Images