Application of Xu5P in evaluating immunotherapy responsiveness and prognosis of kidney cancer

Abstract

The invention provides application of Xu5P in evaluation of kidney cancer immunotherapy reactivity and kidney cancer prognosis, and finds that the kidney cancer metastasis incidence rate of patients with high Xu5P level is remarkably reduced, the average survival time is remarkably prolonged and the index of immune depletion in CD8 + T cells is remarkably reduced compared with those of patients with low Xu5P level. By evaluating the content of Xu5P in blood, kidney cancer tissue and metastatic kidney cancer tissue of a patient, the metastatic risk, survival time and immunotherapy reactivity of the patient are predicted, and a basis is provided for curative effect evaluation and prognosis of kidney cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to use of Xu5P in evaluating the responsiveness to immunotherapy of renal cancer and the prognosis of renal cancer. Background Art

[0002] Xylulose 5-phosphate (D-Xylulose 5-phosphate) is an intermediate metabolite in the pentose phosphate pathway, derived from the ketose ribulose 5-phosphate.

[0003] Kidney cancer, also known as renal cell carcinoma, is a malignant tumor that originates in the kidney and is a common malignant tumor of the urinary system. Due to the lack of obvious clinical symptoms in the early stages, there are currently no clear and effective early diagnostic markers. 25% of patients have already developed metastasis by the time of diagnosis, and approximately 40% of patients experience local recurrence. Distant metastasis is the main cause of death in patients with advanced kidney cancer. The identification of predictive markers that can predict kidney cancer metastasis and prognosis is crucial for monitoring kidney cancer.

[0004] Kidney cancer is insensitive to chemotherapy and radiotherapy, and surgery remains the mainstay of treatment. The use of immune checkpoint inhibitors has improved the treatment of kidney cancer patients to some extent, but overall response rates are low, and nearly all patients eventually develop resistance, resulting in a poor prognosis. Identifying biomarkers that can predict immune responses is crucial for better selecting patients who may benefit from immunotherapy. Summary of the Invention

[0005] In order to solve the above shortcomings, the present invention provides the use of Xu5P in evaluating the responsiveness to immunotherapy of renal cancer and the prognosis of renal cancer.

[0006] The present invention found that the Xu5P level in the blood samples of patients with renal cancer metastasis was significantly lower than that of patients without metastasis, and high Xu5P level was associated with long survival time. In addition, Xu5P level was also associated with low expression of PD-1, TIM3, and Lag3 on the surface of tumor-infiltrating immune CD8+ T cells and high expression of CD137.

[0007] Based on the above findings, the first aspect of the present invention provides the use of xylulose 5-phosphate Xu5P as a marker in evaluating the responsiveness to immunotherapy of renal cancer and / or the prognosis of renal cancer.

[0008] The immunotherapy responsiveness includes a response to an anti-PD-1 / PD-L1 antibody, or a response to an anti-TIM3 antibody, or a response to an anti-Lag3 antibody.

[0009] The prognosis can be the prediction of tumor metastasis or the prediction of survival time.

[0010] The renal cancer includes clear cell renal carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma, or other types of renal cancer.

[0011] The second aspect of the present invention provides the use of Xu5P and / or a substance for detecting Xu5P in preparing a product for detecting renal cancer immunotherapy responsiveness and / or renal cancer prognosis.

[0012] The substance for detecting Xu5P can quantitatively detect the level of Xu5P in blood or tissue samples.

[0013] The substance for detecting Xu5P is selected from any substance that can detect the content of Xu5P, for example, it can be a chemical detection reagent for detecting Xu5P, it can be a liquid chromatography detection reagent for detecting Xu5P, or it can be a mass spectrometry detection reagent for detecting Xu5P. In a specific embodiment of the present application, the substance for detecting Xu5P is a liquid chromatography detection reagent for detecting Xu5P and a mass spectrometry detection reagent for detecting Xu5P.

[0014] The Xu5P can be used as a standard in detection products to accurately quantify Xu5P in samples.

[0015] The immunotherapy responsiveness includes a response to an anti-PD-1 / PD-L1 antibody, or a response to an anti-TIM3 antibody, or a response to an anti-Lag3 antibody.

[0016] The prognosis can be the prediction of tumor metastasis or the prediction of survival time.

[0017] The renal cancer includes clear cell renal carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma, or other types of renal cancer.

[0018] The test sample of the test product is selected from blood, tissue, serum and plasma.

[0019] The third aspect of the present invention provides a product for detecting renal cancer immunotherapy responsiveness and / or renal cancer prognosis, comprising the Xu5P and / or a substance for detecting Xu5P described in the second aspect.

[0020] The substance for detecting Xu5P can quantitatively detect the level of Xu5P in blood or tissue samples.

[0021] The immunotherapy responsiveness includes a response to an anti-PD-1 / PD-L1 antibody, or a response to an anti-TIM3 antibody, or a response to an anti-Lag3 antibody.

[0022] The prognosis can be the prediction of tumor metastasis or the prediction of patient survival time.

[0023] The renal cancer includes clear cell renal carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma, or other types of renal cancer.

[0024] The test sample of the test product is selected from blood, tissue, serum and plasma.

[0025] A fourth aspect of the present invention provides a device for evaluating the responsiveness to immunotherapy of renal cancer and / or the prognosis of renal cancer, the device comprising:

[0026] Data module, used to obtain Xu5P data of samples;

[0027] An analysis module for evaluating renal cancer immunotherapy responsiveness and / or renal cancer prognosis based on Xu5P data of samples.

[0028] The immunotherapy responsiveness includes the response to anti-PD-1 / PD-L1 antibodies, or the response to anti-TIM3 antibodies, or the response to anti-Lag3 antibodies; the prognosis includes the prediction of tumor metastasis and / or the prediction of survival time.

[0029] The renal cancer includes clear cell renal carcinoma, papillary renal cell carcinoma, or chromophobe renal cell carcinoma.

[0030] The device uses any one or more of the following methods to assess renal cancer immunotherapy responsiveness and / or renal cancer prognosis:

[0031] 1) When the detection module detects that the level of Xu5P is higher than the threshold, the analysis module determines that the tumor immunotherapy is effective; when the detection module detects that the level of Xu5P is lower than the threshold, the analysis module determines that the tumor immunotherapy is ineffective;

[0032] 2) When the Xu5P level detected by the detection module is lower than a threshold, the renal cancer patient is judged to have a risk of tumor metastasis; when the Xu5P level detected by the detection module is higher than the threshold, the renal cancer patient is judged to have no risk of tumor metastasis;

[0033] 3) When the detection module detects that the level of Xu5P is higher than the threshold, it is judged that the renal cancer patient has the possibility of long-term survival; when the detection module detects that the level of Xu5P is lower than the threshold, it is judged that the renal cancer patient has a short survival time.

[0034] A fifth aspect of the present invention provides a computer-readable storage medium comprising a computer program, wherein when the computer program is executed by a processor, a method comprising the following steps is performed:

[0035] Xu5P data of the sample is obtained, and renal cancer immunotherapy responsiveness and / or renal cancer prognosis is evaluated based on the Xu5P data of the sample.

[0036] The immunotherapy responsiveness includes the response to anti-PD-1 / PD-L1 antibodies, or the response to anti-TIM3 antibodies, or the response to anti-Lag3 antibodies; the prognosis includes the prediction of tumor metastasis and / or the prediction of survival time.

[0037] The renal cancer includes clear cell renal carcinoma, papillary renal cell carcinoma, or chromophobe renal cell carcinoma.

[0038] The computer-readable storage medium uses any one or more of the following methods to assess renal cancer immunotherapy responsiveness and / or renal cancer prognosis:

[0039] 1) When the detection module detects that the level of Xu5P is higher than the threshold, the analysis module determines that the tumor immunotherapy is effective; when the detection module detects that the level of Xu5P is lower than the threshold, the analysis module determines that the tumor immunotherapy is ineffective;

[0040] 2) When the Xu5P level detected by the detection module is lower than a threshold, the renal cancer patient is judged to have a risk of tumor metastasis; when the Xu5P level detected by the detection module is higher than the threshold, the renal cancer patient is judged to have no risk of tumor metastasis;

[0041] 3) When the detection module detects that the level of Xu5P is higher than the threshold, it is judged that the renal cancer patient has the possibility of long-term survival; when the detection module detects that the level of Xu5P is lower than the threshold, it is judged that the renal cancer patient has a short survival time.

[0042] A sixth aspect of the present invention provides a terminal comprising a memory and a processor, wherein the memory comprises a computer program, wherein when the computer program is executed by the processor, a method comprising the following steps is performed:

[0043] Xu5P data of the sample is obtained, and renal cancer immunotherapy responsiveness and / or renal cancer prognosis is evaluated based on the Xu5P data of the sample.

[0044] The immunotherapy responsiveness includes the response to anti-PD-1 / PD-L1 antibodies, or the response to anti-TIM3 antibodies, or the response to anti-Lag3 antibodies; the prognosis includes the prediction of tumor metastasis and / or the prediction of survival time;

[0045] The terminal uses any one or more of the following methods to evaluate the responsiveness to renal cancer immunotherapy and / or the prognosis of renal cancer:

[0046] 1) When the detection module detects that the level of Xu5P is higher than the threshold, the analysis module determines that the tumor immunotherapy is effective; when the detection module detects that the level of Xu5P is lower than the threshold, the analysis module determines that the tumor immunotherapy is ineffective;

[0047] 2) When the Xu5P level detected by the detection module is lower than a threshold, the renal cancer patient is judged to have a risk of tumor metastasis; when the Xu5P level detected by the detection module is higher than the threshold, the renal cancer patient is judged to have no risk of tumor metastasis;

[0048] 3) When the detection module detects that the level of Xu5P is higher than the threshold, it is judged that the renal cancer patient has the possibility of long-term survival; when the detection module detects that the level of Xu5P is lower than the threshold, it is judged that the renal cancer patient has a short survival time.

[0049] The beneficial effects of the present invention are as follows: the present invention provides a new marker that can evaluate the responsiveness of renal cancer immunotherapy and the prognosis of renal cancer. By evaluating the content of Xu5P in the patient's blood, renal cancer tissue, and metastatic renal cancer tissue, the patient's metastasis risk, survival time, and immunotherapy responsiveness can be predicted, providing a basis for predicting the cancer cell metastasis risk and survival time of renal cancer patients, and providing a basis for judging the effectiveness of immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Shown is the relationship between Xu5P levels and renal cancer metastasis in Example 1.

[0051] Figure 2 Shown is the ROC curve of Xu5p level and renal cancer metastasis in Example 1.

[0052] Figure 3 Shown is the ROC curve of Xu5p level and immunotherapy responsiveness in Example 2.

[0053] Figure 4 Shown is the relationship between Xu5p level and survival time of renal cancer patients in Example 3.

[0054] Figure 5 Shown is the ROC curve of Xu5p level and survival time of renal cancer patients in Example 3.

[0055] Figure 6 Shown is the ROC curve of Xu5p level and renal cancer metastasis in Example 4.

[0056] Figure 7 Shown is the ROC curve of Xu5p level and immunotherapy responsiveness in Example 5.

[0057] Figure 8 Shown is the relationship between Xu5p level and survival time of renal cancer patients in Example 6.

[0058] Figure 9 Shown is the ROC curve of Xu5p level and survival time of renal cancer patients in Example 6.

[0059] Figure 10Indicated as a device for assessing renal cancer immunotherapy responsiveness and / or renal cancer prognosis. DETAILED DESCRIPTION

[0060] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the contents disclosed in this specification. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0061] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0062] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0063] Xu5P in this application, i.e. xylulose-5-phosphate, is an intermediate metabolite in the pentose phosphate pathway, derived from the ketose ribulose-5-phosphate, and has the structural formula shown in the following formula I:

[0064]

[0065] Specifically, the renal cancer in the present invention includes but is not limited to clear cell renal carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma, multilocular cystic renal cell carcinoma of low malignant potential, hereditary leiomyomatosis and renal cell carcinoma, related renal cell carcinoma, collecting duct carcinoma, renal medullary carcinoma, MiT family translocation renal cell carcinoma, succinate dehydrogenase deficiency-related renal cell carcinoma, mucinous tubular and spindle renal cell carcinoma, tubular cystic renal cell carcinoma, acquired cystic renal cell carcinoma-related renal cell carcinoma, clear cell papillary renal cell carcinoma, unclassified renal cell carcinoma, etc.

[0066] Generally, those skilled in the art will know that immunotherapy is to improve the body's non-specific immune response to treat cancer, including vaccines, checkpoint inhibitors, and adoptive cell therapy. The checkpoint inhibitors can be common checkpoint inhibitors such as PD-1 / PD-L1 inhibitors, TIM3 inhibitors, Lag3 inhibitors, and CD137 activators. Immunotherapy responsiveness is the change in indicators of immune exhaustion in CD8+T cells after immunotherapy is used on patients. In this application, the PD-1 / PD-L1 inhibitor is an anti-PD-1 / PD-L1 antibody, the TIM3 inhibitor is an anti-TIM3 antibody, and the Lag3 inhibitor is an anti-Lag3 antibody.

[0067] Furthermore, T cell exhaustion broadly refers to any state of functional loss of antigen-specific CD8+ T lymphocytes, whereby CD8+ T lymphocytes persist but are unable to eliminate pathogenic threats. The onset of exhaustion is accompanied by the surface expression of co-inhibitory receptors that control CD8+ T cell function, such as PD-1, TIM3, and Lag3, which inhibit T cell activation.

[0068] In a specific embodiment of the present invention, the detection object of the detection product provided by the present invention can be a biological sample of a kidney cancer patient, such as blood, tissue, serum, plasma and other samples.

[0069] In a specific embodiment of the present invention, the substance for detecting Xu5P can be a chemical detection reagent for detecting Xu5P, a liquid chromatography detection reagent for detecting Xu5P, or a mass spectrometry detection reagent for detecting Xu5P. According to the prior art, the chemical detection reagent for detecting Xu5P can be a reaction mixture prepared by mixing 54mM imidazole at pH 7.6, 10mM NAD, 6mM MgCI, 5mM sodium arsenate, 0.166mM D-erythrose-4-phosphate, 0.01wt% thiamine pyrophosphate, and 32 units of glyceraldehyde-3-phosphate dehydrogenase (EC1.2.1.12), and the content of Xu5P can be detected by spectrophotometry. The liquid chromatography detection reagent for detecting Xu5P can be a conventional reagent required for liquid chromatography detection. The mass spectrometry detection reagent for detecting Xu5P can be a conventional reagent required for mass spectrometry detection. Conventional reagents such as reagents, standards, and reference substances required for extracting Xu5P can also be included. Those skilled in the art will appreciate that, according to the prior art, the above reagents can be prepared using conventional methods according to actual conditions.

[0070] The term "level" in this application refers to the amount or quantity of a substance in a sample to be tested. Specifically, in this application, it refers to the amount of Xu5P in a sample to be tested, such as mass, content, concentration, etc. The level can be expressed as a percentage (%), a measure of weight (e.g., mg, μg, ng, etc.), a measure of concentration (e.g., mg / mL, μg / mL, ng / mL, etc.), a measure of volume (e.g., mL, μL, nL, etc.), a % change, etc.

[0071] The term "metastasis" in this application refers to the process by which cancer cells leave the site where they first appeared (primary lesion) and spread to other parts of the body through the blood system, lymphatic system or other pathways, and grow new tumors in appropriate locations. Metastasis can include local metastasis and distant metastasis. Local metastasis means that cancer cells have spread to nearby organs or tissues, such as lymph nodes, but have not yet spread to other organs or tissues in the body; distant metastasis means that cancer cells have spread to other organs or tissues in the body, and can spread to one organ or multiple organs.

[0072] The term "responsiveness" in this application refers to a favorable response of the patient's body to treatment. In patients, a beneficial response can be represented by many clinical parameters, including disappearance of detectable tumors (complete response, CR), reduction in tumor size and / or number of cancer cells (partial response, PR), stagnation of tumor growth (stable disease, SD), enhanced anti-tumor immune response, which may lead to tumor regression or rejection; one or more symptoms associated with the tumor are alleviated to some extent; survival time after treatment is increased; and / or mortality rate at a given time point after treatment is reduced.

[0073] The term "prognosis" in this application refers to the possible course of disease and outcome of a disease. It includes judging the specific consequences of the disease, predicting future survival time, and providing time clues for the corresponding disease, such as predicting the probability of a certain outcome occurring within a certain period of time in the future. Prognosis includes positive prognosis or negative prognosis, and the negative prognosis includes disease progression such as recurrence, tumor growth, metastasis and drug-resistant mortality (mortality), and positive prognosis includes disease remission such as no disease state, and disease improvement such as tumor regression or stabilization (stabilization). In a specific embodiment of the present invention, the prognosis is for predicting tumor metastasis situation and patient survival time, and survival time is divided into long-term survival and short-term survival, and the long-term survival is that survival time is greater than 12 months, and short-term survival is that survival time is less than 12 months.

[0074] The term "threshold" in this application can be replaced by cut-off value, critical value, limit value, etc., which refers to the critical point between two different states of a thing, the boundary at which the state of a thing changes, that is, the lowest or highest value that an effect can produce. It can be a numerical value, a numerical range, or a formula for calculating a numerical value or numerical range. In the ROC curve, the threshold is the coordinate point corresponding to the point closest to the upper left corner (0,1.0) on the ROC curve.

[0075] In this application, the threshold value can be obtained based on the correct index, which is also called the Youden index. The correct index refers to the total ability of the test method to detect positives and negatives. Correct index = sensitivity + specificity - 1. Different values are used as the basis for judging negatives and positives, and the negative and positive values of the sample are judged. Based on the actual negative and positive situations in the sample, the sensitivity and specificity of each value are calculated, and the correct index of each value is obtained according to the above formula. The value with the largest correct index is the threshold value of the detection method.

[0076] In a specific embodiment of the present application, according to the above method, a threshold value for determining the responsiveness of tumor immunotherapy is 150.1 uM. When the Xu5P level is higher than the threshold value of 150.1 uM, the analysis module determines that the tumor immunotherapy is effective; when the Xu5P level detected by the detection module is lower than the threshold value of 150.1 uM, the analysis module determines that the tumor immunotherapy is ineffective. A threshold value for determining the risk of tumor metastasis is 174.1 uM. When the Xu5P level detected by the detection module is lower than 174.1 uM, the renal cancer patient is determined to have a high risk of tumor metastasis. When the Xu5P level detected by the detection module is higher than 174.1 uM, the renal cancer patient is determined to have no risk of tumor metastasis. A threshold value for determining survival time is 452.2 uM. When the Xu5P level detected by the detection module is higher than 452.2 uM, the renal cancer patient is determined to have a high probability of long-term survival, with a survival time of more than 12 months. When the Xu5P level detected by the detection module is higher than 452.2 uM, the patient is determined to have a short survival time, with a survival time of less than 12 months.

[0077] In this application, "patient" is used interchangeably with "subject" and refers to a mammal suffering from kidney cancer and in need of treatment. Mammals can include, but are not limited to, primates (e.g., humans and non-human primates, such as monkeys), domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), and laboratory animals (e.g., mice, rats, guinea pigs, etc.). In preferred embodiments, the patient is a human.

[0078] It should be noted that, in the specific embodiments of the present invention, the division of the various modules of the device is merely a division of logical functions. In actual implementation, they can be fully or partially integrated into one physical entity, or they can be physically separated. These modules can all be implemented in the form of software called by a processing element; they can also all be implemented in the form of hardware; some modules can also be implemented in the form of software called by a processing element, and some modules can be implemented in the form of hardware. For example, the detection module can be a separately established processing element, or it can be integrated into a certain chip. In addition, it can also be stored in a memory in the form of program code, called by a certain processing element and execute the functions of the above database acquisition module. The implementation of other modules is similar. In addition, these modules can all or partly be integrated together, or they can be implemented independently. The processing element described here can be an integrated circuit with signal processing capabilities. In the implementation process, each step of the above method or each of the above modules can be completed by the hardware integrated logic circuit in the processor element or by instructions in the form of software.

[0079] For example, these modules in the embodiments of the present invention may be one or more integrated circuits configured to implement the above methods, such as one or more application specific integrated circuits (ASICs), or one or more digital singnal processors (DSPs), or one or more field programmable gate arrays (FPGAs) or graphics processing units (GPUs). For another example, when a module is implemented by scheduling program code through a processing element, the processing element may be a general-purpose processor, such as a central processing unit (CPU) or other processor that can call program code. For another example, these modules may be integrated together and implemented in the form of a system-on-a-chip (SOC).

[0080] In a certain embodiment of the present invention, the computer-readable storage medium may include a read-only memory, a random access memory, an EEPROM, a CD-ROM or other optical disk storage device, a magnetic disk storage device or other magnetic storage device, a flash memory, a USB flash drive, a mobile hard disk, or any other medium that can be used to store desired program code in the form of instructions or data structures and can be accessed by a computer. In addition, any connection can be appropriately referred to as a computer-readable storage medium. For example, if the instruction is sent from a website, server or other remote source using a coaxial cable, a fiber optic cable, a twisted pair, a digital subscriber line (DSL) or wireless technologies such as infrared, radio and microwaves, the coaxial cable, fiber optic cable, twisted pair, DSL or wireless technologies such as infrared, radio and microwaves are included in the definition of the medium. However, it should be understood that a computer-readable storage medium does not include carrier waves, signals or other temporary media, but is intended to be directed to non-temporary, tangible storage media.

[0081] In one embodiment of the present invention, a terminal may be a conventional device such as a mobile phone, a computer, a tablet device, a personal digital assistant, or factory backend processing equipment. The terminal may include at least one processor, a memory, at least one network interface, and a user interface. The various components within the device are coupled together via a bus system. It will be appreciated that the bus system is used to enable connectivity and communication between these components. In addition to a data bus, the bus system also includes a power bus, a control bus, and a status signal bus. Furthermore, the user interface may include a display, a keyboard, a mouse, a trackball, a click gun, keys, buttons, a touchpad, or a touch screen.

[0082] The present application is further illustrated by the following examples, but the scope of this application is not limited thereby. Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in this invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques are well described in existing literature.

[0083] The patient blood samples used in the examples of this application were all obtained from the Urology Department of Shanghai First People's Hospital, and all samples were collected with the patients' signed informed consent.

[0084] Example 1 Relationship between Xu5P levels in the blood of renal cancer patients and renal cancer metastasis

[0085] Blood samples were collected from 10 patients with and 25 patients without renal cell carcinoma. Plasma Xu5P levels were measured using mass spectrometry. Changes in Xu5P levels between the two groups were compared. The specific experimental steps are as follows:

[0086] 1. Sampling: After an overnight fast, collect blood samples between 6:00 and 8:00 AM into EDTA-anticoagulant serum collection tubes to minimize dietary effects. The tubes are then incubated at 4°C for 4 hours and centrifuged at 3000 g for 15 minutes. Serum samples are collected from the supernatant and stored at -80°C until use.

[0087] 2. Sample Pretreatment: Serum samples were thawed on ice before use. 80 μL of serum was mixed with 320 μL of 100% acetonitrile (ACN) and proteins were vortexed to precipitate. The samples were then centrifuged at 20,000 g for 15 min, and 300 μL of the supernatant was transferred and lyophilized using a SpeedVac vacuum concentrator. The samples were then derivatized and analyzed by LCMS.

[0088] 3. Derivatization: Dissolve a known amount of dried sample or Xu5P standard in 20 μL of methoxyamine and incubate at 60°C for 30 minutes. After incubation at room temperature overnight, add 6 μL of 1-methylimidazole and 12 μL of propionic anhydride and heat at 37°C for 30 minutes. Then, evaporate the mixture to dryness using nitrogen.

[0089] 4. LC-MS Analysis: Derivatized samples were dissolved in 100 μL of 0.1% formic acid (FA) for LC-MS analysis. Standards were separated and diluted to various concentrations to construct a standard curve for absolute quantification. Xu5P content in the samples was quantified using an Agilent 6495 UHPLC equipped with an Acquity BEH 1.7 μm, 2.1 × 100 mm column (Watts) in multiple reaction monitoring (MRM) mode.

[0090] Solvent A was 2% formic acid in water, and solvent B was 2% formic acid in 100% methanol. A linear gradient was set at 0.5 mL / min over 18 min as follows: 0% B for 1 min; 0.1-30% B for 2 min; 30-40% B for 3 min; 40% B for 4 min; 40-70% B for 2.5 min; 70% B for 2.5 min; 70-99% B for 2.5 min; and 99% B for 0.5 min.

[0091] Conditions: The column chamber was set at 40°C, and 1 μL of sample was injected for analysis. The mass spectrometer was operated in ESI negative mode with a gas temperature of 230°C, a gas flow of 12 L / min, a nebulizer pressure of 20 psi, a sheath gas temperature of 400°C, a sheath gas flow of 12 L / min, a capillary voltage of -4000 V, a nozzle voltage of 500 V, an iFunnel high-voltage RF of 150 V, and an iFunnel low-voltage RF of 60 V. A fragmentor voltage of 380 V and a cell acceleration voltage of 5 V were used. The MRM transitions used for Xu5P quantification were 429 (precursor ion) to 79 (product ion), with a CE of 49. Data were processed using MassHunter Qualitative Analysis Software.

[0092] 5. Calculate the absolute concentration of Xu5P in the sample using the standard curve.

[0093] The results are as follows Figure 1 As shown in the results, the concentration of Xu5P in the blood of patients with metastatic lesions was significantly lower than that of patients without metastasis (p < 0.001), indicating that the level of Xu5P in human blood is related to the metastasis of renal cancer. When the level of Xu5P is low, the probability of renal cancer patients with metastasis is high. ROC curve analysis was performed using GraphPad Prism 9.0 software ( Figure 2 ), its AUC (Areaunder the ROC curve) value was 0.839, p<0.05, and the cut-off value was 174.1uM. At this time, the detection sensitivity was 0.917 and the specificity was 0.727, indicating that Xu5P can predict the patient's metastasis and prognosis.

[0094] Example 2 Relationship between Xu5P Levels in the Blood of Renal Cancer Patients and Immunotherapy Responsiveness

[0095] In Example 1, 16 of the 35 renal cancer patients received immunotherapy with PD-1 inhibitors, of which 10 patients showed significant clinical remission after treatment, but the other 6 patients did not show significant remission or even progressed. The Xu5P level in the blood of the patients was detected according to the method of Example 1. The patients were divided into groups according to their clinical remission status, with one group showing clinical symptom remission and the other group showing no clinical symptom remission. The data were analyzed by ROC curve ( Figure 3 ), with an AUC of 0.816, p<0.05, and a cut-off value of 150.1 μM. At this point, the detection sensitivity was 0.800 and the specificity was 0.667. This suggests that Xu5P can assess a patient's responsiveness to tumor immunotherapy. When the patient's Xu5P level is higher than 150.1 μM, the patient is more responsive to PD-1 inhibitor immunotherapy.

[0096] Example 3 Relationship between Xu5P Levels in the Blood and Survival Time in Renal Cancer Patients

[0097] The 35 patients with renal cancer in Example 1 were divided into two groups, high-risk and low-risk, according to their median survival time. ROC curve analysis was performed on the two groups ( Figure 4 ), the results showed an AUC value of 0.747, p = 0.012, and a cut-off value of 452.2 uM. The detection sensitivity was 0.833 and the specificity was 0.588. The Xu5P levels in the patients' blood were detected according to the method of Example 1. The patients were then divided into two groups based on the Xu5P levels in the blood: a high Xu5P level group (22 patients) and a low Xu5P level group (13 patients). Based on the cut-off value, patients with a level above 452.2 uM were classified as the high-level group, and those with a level below 452.2 uM were classified as the low-level group. The survival of the 35 patients was analyzed and survival curves were plotted (Logrank test p < 0.001).

[0098] The results are as follows Figure 5 As shown in the results, it was found that the average survival time and the longest survival time of patients in the Xu5P high level group were significantly better than those in the Xu5P low level group, indicating that the Xu5P level can predict the patient's survival time and prognosis.

[0099] Example 4: Determining the Patient's Tumor Metastasis by Using the Technical Solution of This Example

[0100] This example verifies the accuracy of the method of Example 1 in judging the tumor metastasis of patients. Blood samples from another 25 patients with renal cancer were collected to verify the evaluation effect of the judgment method of Example 1. In the case of no grouping and unknown whether the tumor has metastasized, the Xu5P level in the patient's blood was detected according to the method of Example 1. Among them, 8 patients whose Xu5P levels were lower than the Cutoff value of 174.1uM were judged to have a risk of tumor metastasis, and 17 patients whose Xu5P levels were higher than the Cutoff value of 174.1uM were judged to have no risk of tumor metastasis. Then, whether the above judgment was correct was determined based on the patient's tumor metastasis situation. ROC curve analysis was performed using GraphPad Prism 9.0 software ( Figure 6 ), its AUC value was 0.787, p<0.05, and 174.1uM in Example 1 was used as the cut-off value. At this time, the detection sensitivity was 0.823 and the specificity was 0.750, indicating that Xu5P was able to predict the metastasis of patients with good sensitivity, specificity and accuracy.

[0101] Example 5: Determining a patient's immunotherapy responsiveness using the technical solution of this example

[0102] This example verifies the accuracy of the method of Example 2 in judging the immunotherapy responsiveness of patients. Among the 25 renal cancer patients in Example 4, 15 of them received immunotherapy with PD-1 inhibitors. After the patients received treatment, the Xu5P level in the patient's blood was detected according to the method of Example 1. In the absence of grouping and unknown treatment effects, the threshold value of Example 2 was used for judgment. Among the 25 patients receiving immunotherapy, the Xu5p levels of 8 patients were higher than the threshold value and were judged to have good immunotherapy responsiveness. The Xu5p levels of 7 patients were lower than the threshold value and were judged to have poor immunotherapy responsiveness. Then, whether the above judgment was correct was determined based on whether the patient had relief of clinical symptoms. ROC curve analysis was performed on the above data ( Figure 7 ), AUC value was 0.821, p<0.05. Taking 150.1uM of Example 2 as the cut-off value, the detection sensitivity was 0.812 and the specificity was 0.714, which verified the analysis results of Example 2. It shows that Xu5P can be used to evaluate the patient's responsiveness to tumor immunotherapy with good sensitivity, specificity and accuracy.

[0103] Example 6: Determining the patient's survival time using the technical solution of this embodiment

[0104] This example verifies the accuracy of the method of Example 3 in determining patient survival time. For the 25 renal cancer patients in Example 4, the Xu5p levels in the patients' blood were detected according to the method of Example 1. In the absence of grouping and unknown survival time, according to the threshold value in Example 3, the 13 patients below the threshold value were divided into the Xu5p low-level group, and the 12 patients above the threshold value were divided into the Xu5p high-level group. The Xu5p low-level group was judged to have the possibility of long-term survival, and the Xu5p high-level group was judged to have the possibility of short-term survival. By monitoring the survival time of the patients, the survival status of the 25 patients was analyzed and the survival curve was drawn (Logrank test p < 0.01). The results are as follows: Figure 8 As shown in Figure 2, the mean survival time and the longest survival time of patients in the Xu5P high level group were significantly better than those in the Xu5P low level group. The above judgment was correct based on the actual survival time of the patients. ROC curve analysis was performed on the two groups ( Figure 9 ), the results showed that the AUC value was 0.801, p = 0.011, and 452.2uM in Example 3 was used as the cut-off value. At this time, the detection sensitivity was 0.731 and the specificity was 0.750, indicating that it has been verified that the Xu5P level can predict the patient's survival time and prognosis, with good sensitivity, specificity and accuracy.

[0105] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

Claims

1. Use of xylulose 5-phosphate (Xu5P) as a biomarker in assessing renal cell carcinoma immunotherapy responsiveness and / or prognosis.

2. The use according to claim 1, characterized in that The immunotherapy response includes anti-PD-1 / PD-L1 The prognosis includes predicting tumor metastasis and / or predicting survival time; And / or, the renal cancer includes clear cell renal carcinoma, papillary renal cell carcinoma, or chromophobe renal cell carcinoma.

3. Use of Xu5p and / or substances for detecting Xu5P in the preparation of products for detecting renal cancer immunotherapy responsiveness and / or renal cancer prognosis.

4. The use according to claim 3, characterized in that The substance for detecting Xu5P is selected from a chemical detection reagent for detecting Xu5P, a liquid chromatography detection reagent for detecting Xu5P, and a mass spectrometry detection reagent for detecting Xu5P.

5. The use according to claim 3, characterized in that The immunotherapy response includes anti-PD-1 / PD-L1 The prognosis includes predicting tumor metastasis and / or predicting survival time; and / or, the renal cancer comprises clear cell renal carcinoma, or papillary renal cell carcinoma, or chromophobe renal cell carcinoma; And / or, the test sample of the test product is selected from any one of the following: blood, tissue, serum, plasma.

6. A product for detecting renal cancer immunotherapy responsiveness and / or renal cancer prognosis, comprising Xu5P according to any one of claims 3 to 5 and / or a substance for detecting Xu5P.

7. The detection product according to claim 6, characterized in that: The immunotherapy responsiveness includes the response to anti-PD-1 / PD-L1 antibodies, or the response to anti-TIM3 antibodies, or the response to anti-Lag3 antibodies; the prognosis includes the prediction of tumor metastasis and / or the prediction of survival time; and / or, the renal cancer comprises clear cell renal carcinoma, or papillary renal cell carcinoma, or chromophobe renal cell carcinoma; And / or, the test sample of the test product is selected from any one of the following: blood, tissue, serum, plasma.

8. A device for evaluating renal cancer immunotherapy responsiveness and / or renal cancer prognosis, the device comprising: Data module, used to obtain Xu5P data of samples; An analysis module for evaluating renal cancer immunotherapy responsiveness and / or renal cancer prognosis based on Xu5P data of samples.

9. A computer-readable storage medium comprising a computer program, characterized in that: When the computer program is executed by a processor, a method comprising the following steps is performed: Xu5P data of the sample is obtained, and renal cancer immunotherapy responsiveness and / or renal cancer prognosis is evaluated based on the Xu5P data of the sample.

10. A terminal comprising a memory and a processor, wherein the memory comprises a computer program, characterized in that: The computer program is executed by a processor to perform a method comprising the following steps: Xu5P data of the sample is obtained, and renal cancer immunotherapy responsiveness and / or renal cancer prognosis is evaluated based on the Xu5P data of the sample.

11. The apparatus according to claim 8, the computer-readable storage medium according to claim 9 or the terminal according to claim 10, wherein: The immunotherapy responsiveness includes the response to anti-PD-1 / PD-L1 antibodies, or the response to anti-TIM3 antibodies, or the response to anti-Lag3 antibodies; the prognosis includes the prediction of tumor metastasis and / or the prediction of survival time; And / or, the renal cancer includes clear cell renal carcinoma, papillary renal cell carcinoma, or chromophobe renal cell carcinoma.

12. The apparatus, computer-readable storage medium, or terminal according to claim 11, wherein: Any one or more of the following methods are used to assess renal cell carcinoma immunotherapy response and / or renal cell carcinoma prognosis: 1) When the detection module detects that the level of Xu5P is higher than the threshold, the analysis module determines that the tumor immunotherapy is effective; when the detection module detects that the level of Xu5P is lower than the threshold, the analysis module determines that the tumor immunotherapy is ineffective; 2) When the detection module detects that the level of Xu5P is lower than the threshold, the renal cancer patient is judged to have a risk of tumor metastasis; 3) When the detection module detects that the level of Xu5P is higher than the threshold, it is determined that the renal cancer patient has the possibility of long-term survival.

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