Methods for predicting the treatment effect of immunotherapy combined with chemotherapy in a subject and uses thereof

Abstract

The application provides a method for predicting the treatment effect of immunotherapy combined with chemotherapy on a subject and application thereof. The method for predicting the treatment effect of immunotherapy combined with chemotherapy on the subject comprises detecting the expression level of a biomarker in a biological sample of the subject before the subject is administered with the immunotherapy combined with chemotherapy, and the biomarker comprises STAT1 protein. The method can solve the problem of low accuracy in predicting whether a patient can receive immunotherapy, and is suitable for the technical field of biological medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a method for predicting the efficacy of immunotherapy combined with chemotherapy in subjects and its application. Background Technology

[0002] Gastric cancer (GC) is a malignant tumor with leading incidence and mortality rates worldwide. Its high invasiveness, late diagnosis, and poor prognosis make it a major challenge for global public health. Locally advanced gastric cancer (LAGC) accounts for 60%-70% of all gastric cancer cases. Due to its high preoperative tumor burden, low surgical resection rate, and high risk of postoperative recurrence, its treatment outcomes in clinical practice are poor.

[0003] Early clinical trials extending immunotherapy combined with chemotherapy to neoadjuvant therapy have shown promising results. For example, in neoadjuvant therapy for LAGC (lazy gastric cancer), neoadjuvant chemotherapy combined with immunotherapy, such as pembrolizumab + chemotherapy or durvalumab + FLOT (fluorouracil, leucovorin, oxaliplatin, and docetaxel), can significantly improve pathological regression in LAGC patients by enhancing tumor immunogenicity and promoting tumor cell killing. However, in clinical practice, the pathological response rate of immunotherapy combined with chemotherapy in gastric cancer patients remains only 30%-50%. Ineffective treatment not only leads to an increased incidence of immune-related adverse reactions but also delays the optimal surgical time and wastes medical resources.

[0004] Existing methods for predicting whether a patient is suitable for immunotherapy mainly include the detection of combined biomarkers such as programmed death ligand 1 (PD-L1) expression levels, microsatellite instability (MSI) / deficient mismatch repair (dMMR), tumor mutational burden (TMB), or messenger RNA (mRNA), as disclosed in Chinese patent application CN 113891748 A. However, these prediction methods all suffer from drawbacks such as low population coverage, poor detection convenience, or inability to predict independently, which means that existing protocols cannot meet the clinical needs of neoadjuvant immunotherapy combined with chemotherapy for LAGC. Summary of the Invention

[0005] The main objective of this invention is to provide a method for predicting the efficacy of immunotherapy combined with chemotherapy in subjects and its application, in order to solve the problem of low accuracy in predicting whether patients will receive immunotherapy in the prior art.

[0006] To achieve the above objective, according to a first aspect of the present invention, a method for predicting the efficacy of immunotherapy combined with chemotherapy in a subject is provided, the method comprising: detecting the expression level of a biomarker, including STAT1 protein, in a biological sample of the subject before the subject is administered the immunotherapy combined with chemotherapy.

[0007] Further, the above-mentioned immunotherapy combined with chemotherapy includes administering an immunotherapy agent and a chemotherapy regimen to the subject simultaneously; preferably, the chemotherapy regimen includes one or more of SOX, XELOX, FOLFOX, or DOX; preferably, the chemotherapy regimen includes SOX or XELOX; preferably, the immunotherapy agent includes one or more of PD-1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, or A2AR inhibitors; preferably, the immunotherapy agent includes a PD-1 inhibitor.

[0008] Furthermore, the aforementioned biological samples include one or more of the following: blood samples, urine samples, or tumor tissue samples.

[0009] Furthermore, the expression level of the STAT1 protein mentioned above includes the content of the STAT1 protein mentioned above.

[0010] Furthermore, the methods for detecting the expression level of the STAT1 protein include one or more of immunohistochemistry, mass spectrometry, protein microarray, flow cytometry, quantitative PCR, or Western blot; preferably, the detection method includes immunohistochemistry.

[0011] Furthermore, the immunohistochemistry described above includes: grading and scoring the expression level of the STAT1 protein based on the staining intensity of the cytoplasm in the immunohistochemically stained sections.

[0012] Furthermore, the reagents used in the above-mentioned method for detecting the expression level of STAT1 include one or more of anti-STAT1 antibodies, primers, or probes; preferably, the above-mentioned reagents include anti-STAT1 antibodies.

[0013] Furthermore, the subjects mentioned above are cancer patients; preferably, the cancers include one or more of gastric cancer, non-small cell lung cancer, colorectal cancer, or triple-negative breast cancer.

[0014] To achieve the above objectives, according to a second aspect of the present invention, the use of a reagent for detecting STAT1 protein expression levels in the preparation of a kit for predicting the efficacy of immunotherapy combined with chemotherapy in subjects is provided.

[0015] Further, the above reagents include one or more of anti-STAT1 antibodies, primers, or probes; preferably, the above reagents include anti-STAT1 antibodies.

[0016] Further, the aforementioned immunotherapy combined with chemotherapy includes administering an immunotherapy agent and a chemotherapy regimen to the subject simultaneously; preferably, the chemotherapy regimen includes one or more of SOX, XELOX, FOLFOX, or DOX; preferably, the chemotherapy regimen includes SOX or XELOX; preferably, the immunotherapy agent includes one or more of PD-1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, or A2AR inhibitors; preferably, the immunotherapy agent includes a PD-1 inhibitor; preferably, the subject is a cancer patient; preferably, the cancer includes one or more of gastric cancer, non-small cell lung cancer, colorectal cancer, or triple-negative breast cancer.

[0017] To achieve the above objectives, according to a third aspect of the present invention, an electronic device for predicting the efficacy of immunotherapy combined with chemotherapy in a subject is provided. The electronic device includes a data collection module and a prediction module. The data collection module is used to collect the expression levels of biomarkers in a subject's biological sample and input the data collected by the data collection module into the prediction module. The biomarkers include STAT1 protein. The prediction module is configured to output a prediction result of the efficacy of immunotherapy combined with chemotherapy in the subject based on the expression levels of the biomarkers in the subject's biological sample.

[0018] Furthermore, the expression level of the aforementioned biomarker can be obtained from one or more of the following methods: immunohistochemistry, mass spectrometry, protein microarray, flow cytometry, quantitative PCR, or Western blot. Preferably, the expression level of the aforementioned biomarker is obtained based on the analysis of the aforementioned immunohistochemically induced sections. The aforementioned data collection module is used to analyze the staining of the cytoplasm of the aforementioned STAT1 protein in the aforementioned immunohistochemically induced sections. The aforementioned data collection module includes a staining intensity module and an optional calculation module. The aforementioned staining intensity module is used to determine the staining status and percentage of the aforementioned cytoplasm in the aforementioned immunohistochemically induced sections and obtain a staining intensity score. The aforementioned calculation module is used to calculate the immunohistochemical score of the aforementioned immunohistochemically induced sections based on the staining intensity score obtained by the aforementioned staining intensity module.

[0019] To achieve the above objectives, according to a fourth aspect of the present invention, a method for predicting the efficacy of immunotherapy combined with chemotherapy is provided. The steps of the prediction method are performed by a computer. The prediction method is used to predict the effect of immunotherapy combined with chemotherapy on cancer patients. The prediction method includes: generating a risk prediction result for immunotherapy combined with chemotherapy for cancer patients based on the expression level of biomarkers in the biological samples of the cancer patients; the biomarkers include STAT1 protein.

[0020] To achieve the above objectives, according to a fifth aspect of the present invention, a computer device is provided, including a memory, a processor, and a computer program stored in the memory, wherein the processor executes the computer program to implement the steps of the electronic device or the prediction method described above.

[0021] To achieve the above objectives, according to a sixth aspect of the present invention, a computer-readable storage medium is provided having a computer program stored thereon, characterized in that the computer program, when executed by a processor, implements the steps of the above-described electronic device or the above-described prediction method.

[0022] To achieve the above objectives, according to a seventh aspect of the present invention, a computer program product is provided, comprising a computer program that, when executed by a processor, implements the steps of the electronic device or the prediction method described above.

[0023] By applying the technical solution of this invention, the levels of biomarkers in the biological samples of the aforementioned subjects, including STAT1 (Signal Transducer and Activator of Transcription 1), are detected before the administration of combined immunotherapy and chemotherapy. This addresses the technical problem of low accuracy in predicting patient response to immunotherapy, enabling the prediction of the therapeutic effect of combined immunotherapy and chemotherapy in subjects. This method provides clinicians with a simple and accurate tool for selecting immunotherapy patients, optimizing immunotherapy strategies, improving the precision of immunotherapy, and transforming clinical immunotherapy practice. Attached Figure Description

[0024] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0025] Figure 1 The graph shows the differential expression results of STAT1 in the pathological tumor regression grade 0-3 groups according to Embodiment 1 of the present invention.

[0026] Figure 2The graph shows the differential expression results of STAT1 in the treatment response group and the non-treatment response group according to Embodiment 1 of the present invention.

[0027] Figure 3 The image shows the STAT1 immunohistochemical staining results according to Example 3 of the present invention.

[0028] Figure 4 The results of the color intensity of STAT1 in the treatment response group and the non-treatment response group according to Embodiment 3 of the present invention are shown.

[0029] Figure 5 A graph showing the statistical results of the objective mitigation rate of the mainstream markers according to Embodiment 4 of the present invention is presented.

[0030] Figure 6 The graph shows the differential expression results of COL18A1 in the pathological tumor regression grade 0-3 groups according to Comparative Example 1 of the present invention.

[0031] Figure 7 The graph shows the differential expression results of COL18A1 in the treatment response group and the non-treatment response group according to Comparative Example 1 of the present invention.

[0032] Figure 8 The immunohistochemical results of COL18A1 in the treatment response group and the non-treatment response group according to Comparative Example 1 of the present invention are shown.

[0033] Figure 9 The results of color intensity of COL18A1 in the treatment response group and the non-treatment response group according to Comparative Example 1 of the present invention are shown. Detailed Implementation

[0034] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0035] As mentioned in the background section, existing technologies for predicting whether a patient is suitable for immunotherapy combined with chemotherapy have shortcomings such as low population coverage, poor detection convenience, or inability to predict independently.

[0036] Currently, radical surgery combined with perioperative systemic therapy is an important treatment strategy for LAGC. Neoadjuvant therapy (preoperative treatment) is a key component due to its multiple clinical advantages: ① Tumor downstaging can increase the surgical R0 resection rate to over 90%; ② The tumor blood supply is intact before surgery, and the delivery efficiency of chemotherapy drugs is significantly better than after surgery (postoperative tumor blood supply disruption can easily lead to uneven drug distribution); ③ Potential micrometastases can be cleared in advance, reducing the risk of distant recurrence after surgery; ④ Patients are in good physical condition before surgery, and their tolerance to chemotherapy (such as bone marrow suppression and gastrointestinal reactions) is significantly lower than after surgery (postoperative complications may lead to delayed chemotherapy or dose reduction). According to the Chinese Society of Clinical Oncology (CSCO) Guidelines for the Diagnosis and Treatment of Gastric Cancer (2024 Edition), based on the long-term follow-up results of the RESOLVE trial (median follow-up of 61.3 months), the perioperative use of S-1 (tegafur) combined with oxaliplatin (SOX) regimen can increase the 5-year disease-free survival rate (DFS) of LAGC patients from 52.8% to 61.6% compared with traditional postoperative adjuvant chemotherapy. The perioperative use of SOX regimen can increase the 5-year overall survival rate (OS) of LAGC patients from 52.1% to 60.0% compared with traditional postoperative adjuvant chemotherapy. Therefore, perioperative use of SOX has become the preferred recommended regimen for neoadjuvant chemotherapy of LAGC in China.

[0037] With the clinical application of immune checkpoint inhibitors (ICIs), neoadjuvant immunotherapy combined with chemotherapy has brought breakthrough progress to the treatment of advanced gastric cancer (LAGC). In the field of advanced gastric cancer, the combination of programmed cell death protein 1 (PD-1) inhibitors with chemotherapy has been proven by multiple phase III clinical trials to significantly prolong patient survival. Examples include: the CheckMate-649 trial (nivolumab + chemotherapy) showed that in unselected patients, the median overall survival (OS) of combination therapy increased from 11.6 months to 13.8 months compared to chemotherapy alone, a significant difference (P < 0.001), with a hazard ratio (HR) of 0.79 for patients treated with combination therapy; and the KEYNOTE-859 trial (pembrolizumab + chemotherapy) showed that the median OS of combination therapy increased from 11.5 months to 12.9 months compared to chemotherapy alone, a significant difference (P < 0.001), with a hazard ratio of 0.78 for patients treated with combination therapy. The ORIENT-16 trial (sintilimab plus chemotherapy) showed that the median overall survival (OS) of combination therapy increased from 12.3 months to 15.2 months compared to chemotherapy alone, with a significant difference (P<0.001) and a hazard ratio of 0.70 for patients receiving combination therapy. The RATIONALE-305 trial (tislelizumab plus chemotherapy) showed that the median OS of combination therapy increased from 12.9 months to 15.0 months compared to chemotherapy alone, with a significant difference (P<0.001) and a hazard ratio of 0.78 for patients receiving combination therapy.

[0038] Early clinical trials extending the immunotherapy-chemotherapy strategy to neoadjuvant therapy for LAGC have shown promising results: the KEYNOTE-585 study (pembrolizumab + chemotherapy) showed a pathologic complete response (pCR) rate of 13.0% in the perioperative immunotherapy-chemotherapy group, significantly higher than the 2.4% in the chemotherapy-only group (P < 0.001); in the MATTERHORN phase II trial (durvalumab + FLOT chemotherapy), the pCR rate in the combination therapy group reached 19%, significantly higher than the 7% in the chemotherapy-only group (P = 0.012). These data indicate that neoadjuvant immunotherapy combined with chemotherapy can significantly improve the pathologic regression effect in LAGC patients by enhancing tumor immunogenicity and promoting tumor cell killing.

[0039] However, in clinical practice, the pathological complete response rate for gastric cancer patients undergoing immunotherapy combined with chemotherapy is still only 20%-30%, and the pathological major response rate (TRG0-1) is less than 50%. Ineffective treatment not only leads to an increased incidence of immune-related adverse reactions (grade 3 or higher rashes and colitis occur in 15%-20%), but also delays the optimal surgical timing (neoadjuvant therapy cycles are usually 2-4 cycles, and ineffective treatment will waste 4-12 weeks), while also wasting medical resources. Therefore, developing "precise, efficient, and convenient" efficacy prediction biomarkers has become the core clinical need to address the "precise screening of beneficiaries" in neoadjuvant immunotherapy combined with chemotherapy for LAGC, and is also the core research and development background of this invention.

[0040] Currently, the mainstream biomarkers used in clinical practice for predicting the efficacy of immunotherapy include PD-L1 expression level, MSI / dMMR status, and TMB. In addition, a few existing technologies mention STAT1 as one of the components of a combination of mRNA level biomarkers for immunotherapy prediction. The above approach is the closest implementation to this invention (STAT1 protein as an independent predictive biomarker for LAGC neoadjuvant immunotherapy combined with chemotherapy). Its specific content, technical features, and limitations are as follows:

[0041] Mainstream immunotherapy predictive biomarker regimens:

[0042] (1) Detection of PD-L1 expression level: PD-L1 is currently the most widely used predictive biomarker for immunotherapy in clinical practice. Its core mechanism is to detect the expression of PD-L1 protein on the surface of tumor cells (TC) or tumor-infiltrating immune cells (IC) through immunohistochemistry (IHC), which indirectly reflects the immune activity of the tumor microenvironment. High PD-L1 expression suggests that tumor cells inhibit T cell function through the PD-1 / PD-L1 pathway and have a higher probability of responding to PD-1 inhibitors. Currently, the commonly used PD-L1 detection antibody clone numbers include 28-8 (Dako), SP142 (Ventana), and SP163 (Ventana). The scoring system is mainly based on the Combined Positive Score (CPS) (CPS is defined as (number of PD-L1 positive cells / total number of tumor cells) × 100%, and positive cells include TC and IC).

[0043] This approach has three major limitations: ① High detection heterogeneity: The consistency of detection results among different antibody clones (such as 28-8, SP142, SP163) is only 60%-70%; ② Insufficient predictive accuracy: For patients with locally advanced gastric cancer diagnosed with PD-L1 CPS≥5, the objective response rate (ORR) of immunotherapy combined with chemotherapy is only 55%, and 45% of patients still do not respond; ③ Intratumoral heterogeneity: The difference in PD-L1 expression in different regions of the same tumor can reach 30%-50%, and biopsy samples are prone to "sampling bias".

[0044] (2) MSI / dMMR status detection: The core mechanism of MSI / dMMR is that dMMR tumors, due to the loss of function of mismatch repair proteins (MLH1, MSH2, MSH6 or PMS2), accumulate errors in the replication of microsatellite sequences (short tandem repeat sequences), generating a large number of tumor neoantigens, enhancing tumor immunogenicity, and having a significantly higher response rate to PD-1 inhibitors than proficient mismatch repair (pMMR) tumors. Currently, clinical detection methods include two types: ① IHC detection of the expression of four mismatch repair proteins (deficiency of ≥1 protein indicates dMMR); ② PCR detection of the instability of microsatellite sites such as BAT25, BAT26, NR21, NR24 or MONO27 (instability of ≥2 sites indicates MSI-H).

[0045] Clinical data show that the objective response rate (ORR) of immunotherapy in LAGC patients with MSI-H / dMMR is 75%. The core limitation of this regimen is its extremely low population coverage: the incidence of MSI-H / dMMR in LAGC is only 6% (significantly lower than the 15%-20% in colorectal cancer), and there is more than 50% overlap with TMB-H (i.e., some MSI-H patients are also TMB-H), which makes it impossible to expand the screening range of patients who benefit from immunotherapy.

[0046] (3) TMB detection: The core definition of TMB is the number of somatic nonsynonymous mutations per megabase (Mb) in the tumor genome. It is usually detected by next-generation sequencing (NGS). Among them, NGS detection often uses whole exome sequencing (WES) or large panel sequencing. Clinically, TMB-H is usually defined as ≥10 mutations / Mb. Its predictive mechanism is that a high mutation burden can generate more tumor neoantigens, activate the body's anti-tumor immune response, and thus improve the response rate to PD-1 / PD-L1 inhibitors.

[0047] Clinical data show that the objective response rate (ORR) of adjuvant immunotherapy combined with chemotherapy in LAGC patients with TMB-H reaches 71%. However, this approach has three key limitations: ① Low population coverage: The incidence of TMB-H in LAGC is only 13%, and it has a high overlap with MSI-H / dMMR, limiting the actual number of new patients to be screened; ② High testing costs and long processing time: A single WES test costs approximately 8,000-12,000 yuan, and large panel sequencing costs approximately 5,000-8,000 yuan, with a testing cycle of 2-3 weeks. Neoadjuvant therapy for LAGC needs to be initiated within 1-2 weeks after biopsy, which cannot meet the clinical timeliness requirements; ③ Lack of standardization: Different sequencing platforms (WES or large panel sequencing), different sequencing coverage (50% or 100% coverage), and bioinformatics analysis procedures (such as mutation filtering standards and synonymous mutation exclusion rules) can lead to differences of up to 20%-30% in TMB calculation results, resulting in poor comparability between results from different laboratories.

[0048] Existing technical solutions related to STAT1:

[0049] In the prior art, STAT1 is only used as a "combined biomarker of mRNA level" for predicting immunotherapy, without involving independent applications at the protein level. The most representative solutions are Chinese patent application CN 113891748 A and Chinese patent application CN114728070A.

[0050] Chinese patent application CN 113891748 A discloses "a method for treating tumors, which screens patients suitable for PD-1 monotherapy by detecting 'high inflammatory feature scores (including STAT1 mRNA) + TMB ≥ 10 mutations / Mb', and the tumor type includes gastric and esophageal cancer." Its technical features are: STAT1 is a member of the "inflammatory genome set (including CD274, CD8A, LAG3, or STAT1)," and its mRNA expression needs to be detected by reverse transcription polymerase chain reaction (RT-PCR), and it must be used in conjunction with TMB to achieve predictive function.

[0051] Chinese patent application CN 114728070 A discloses "a treatment method combining a PD-1 antagonist with an IDO1 inhibitor, which screens patients by detecting 'high IFNγ inflammatory signature score' (containing STAT1 mRNA + low TDO2 mRNA)". Its technical features are: STAT1 mRNA is one of the indicators of the IFNγ inflammatory pathway, and it needs to be detected in conjunction with TDO2 mRNA; the application scenario is "PD-1 + IDO1 inhibitor (immunotherapy combined with immunotherapy)," and it does not involve immunotherapy combined with chemotherapy.

[0052] The core limitations of the aforementioned existing STAT1-related technologies are as follows: ① The detection level is limited to mRNA. It is well known in the biological field that mRNA and protein expression are subject to post-transcriptional regulation (such as translational repression, protein degradation, and phosphorylation modification), and the correlation between the two is only 0.3-0.5. It is impossible to directly infer the protein expression level from the mRNA expression level, let alone assume that "mRNA markers can be replaced by protein markers"; ② It needs to be used in combination with other markers, none of which can be used as independent predictive markers, which increases the complexity and cost of detection and cannot meet the "fast and convenient" requirements of neoadjuvant therapy for LAGC; ③ The application scenarios are mismatched, only targeting PD-1 monotherapy or immunotherapy combined with immunotherapy, and not involving the neoadjuvant therapy scenario of "chemotherapy + PD-1 inhibitor". Chemotherapy will change the expression regulation mechanism of STAT1 by "inducing tumor cell immunogenic death and reshaping the immune microenvironment".

[0053] Summary of common shortcomings of existing solutions:

[0054] In summary, the existing technical solutions most similar to this invention (PD-L1, MSI / dMMR, TMB, and STAT1 mRNA combination biomarkers) all suffer from three core defects: ① Low population coverage: MSI-H / dMMR (6%) and TMB-H (13%) only cover a small number of LAGC patients, and although PD-L1 CPS≥5 covers about 30% of patients, its predictive accuracy is insufficient; ② Poor testing convenience: TMB relies on NGS, resulting in high costs and long processing times, and although PD-L1 / MSI / dMMR uses IHC, PD-L1 exhibits heterogeneity, and MSI / dMMR requires multi-protein detection; ③ Inability to predict independently: The STAT1 mRNA protocol must be combined with TMB / TDO2 and cannot be used alone. These defects directly result in the existing solutions failing to meet the clinical needs of LAGC neoadjuvant immunotherapy combined with chemotherapy for "high coverage, high accuracy, and rapid detection".

[0055] Therefore, in this application, the inventors attempted to develop a method for predicting the effect of immune-combined chemotherapy in subjects, and based on this, proposed a series of protection schemes for this application.

[0056] In a first typical embodiment of this application, a method for predicting the effect of immunotherapy combined with chemotherapy in a subject is provided. The method includes: before the subject receives the immunotherapy combined with chemotherapy, detecting the expression level of a biomarker in the subject's biological sample, wherein the biomarker includes STAT1 (signal transduction and activator of transcription 1) protein.

[0057] In this application, "immunotherapy combined with chemotherapy" refers to "neoadjuvant chemotherapy combined with immunotherapy." Immunotherapy combined with chemotherapy is a cancer treatment strategy that combines traditional chemotherapy with immunotherapy. Its purpose is to rapidly kill tumor cells and expose tumor antigens through chemotherapy, while simultaneously activating or enhancing the body's immune system's ability to recognize and eliminate tumors through immunotherapy, thereby improving the overall treatment effect and prolonging the patient's survival.

[0058] STAT1 protein is a core member of the JAK-STAT (Janus kinase-signal transducer and activator of transcription) signaling pathway, playing a crucial role in cytokine signaling and immune regulation. STAT is widely involved in immune responses. It is mainly activated by cytokines such as interferon (after phosphorylation, it enters the nucleus), thereby initiating the transcription of hundreds of downstream immune-related genes and participating in the regulation of key immune processes such as T cell (T lymphocyte) activation, NK cell (natural killer) killing, DC (dendritic cell) maturation, and macrophage polarization.

[0059] In this application, the inventors discovered that detecting biomarkers, including but not limited to STAT1, in biological samples from cancer patients can predict the therapeutic effect of combined immunotherapy and chemotherapy. This method provides clinicians with a simple and accurate tool for selecting immunotherapy patients, enabling the optimization of immunotherapy strategies and improving the precision of immunotherapy.

[0060] In a preferred embodiment, the above-mentioned immunotherapy combined with chemotherapy includes administering an immunotherapy agent and a chemotherapy regimen to the subject simultaneously; preferably, the chemotherapy regimen includes one or more of the following: SOX (composed of tegafur, oxaliplatin, and capecitabine), XELOX (composed of oxaliplatin and capecitabine), FOLFOX (composed of 5-fluorouracil, leucovorin, and oxaliplatin), or DOX (doxorubicin); preferably, the chemotherapy regimen includes SOX or XELOX; preferably, the above-mentioned immunotherapy agent includes one or more of the following: PD-1 (programmed death protein 1) inhibitor, CTLA-4 (cytotoxic T lymphocyte-associated protein 4) inhibitor, LAG-3 (lymphocyte activation gene 3) inhibitor, TIM-3 (T cell immunoglobulin mucin-3) inhibitor, TIGIT (T cell immunoglobulin and immune receptor tyrosine inhibitory motif domain protein) inhibitor, or A2AR (adenosine A2a receptor) inhibitor; preferably, the above-mentioned immunotherapy includes a PD-1 inhibitor.

[0061] The chemotherapy regimens mentioned above, including but not limited to SOX or XELOX, have shown significant efficacy against various types of gastric cancer. Moreover, SOX or XELOX chemotherapy regimens do not require hydration during application and have low nephrotoxicity to patients.

[0062] Among the aforementioned immunotherapies, patients treated with PD-1 inhibitors have a longer overall survival. PD-1 inhibitors have good safety and therapeutic efficacy. Therefore, in this application, the inventors preferably use PD-1 inhibitors as immunotherapeutic agents in the treatment process of patients.

[0063] In a preferred embodiment, the biological sample includes one or more of the following: blood sample, urine sample, or tumor tissue sample.

[0064] In the testing of the aforementioned biological samples from these patients, the sources of these biological samples typically include, but are not limited to, blood samples, urine samples, or tumor tissue samples. Among these, blood and urine samples are suitable for large-scale screening and liquid biopsies due to their non-invasiveness and convenience for patients. Tumor tissue samples can provide comprehensive information on tumor characteristics and microenvironment, making them more suitable for the discovery and validation of biomarkers.

[0065] In a preferred embodiment, the expression level of the STAT1 protein includes the content of the STAT1 protein.

[0066] Methods for detecting product content include, but are not limited to, protein content detection and mRNA expression level detection. Compared to mRNA expression level, protein content can more directly reflect the actual functional state of cells. Post-translational modifications such as phosphorylation, glycosylation, or splice variants, which determine the "functional switches" of protein activity, can only be captured at the protein level and cannot be detected at the mRNA expression level. Furthermore, protein detection methods (including but not limited to immunohistochemistry, enzyme-linked immunosorbent assay, or chemiluminescence) are simple and can yield results quickly, while mRNA detection usually requires reverse transcription and quantification, with many quality control points and a cumbersome process. Moreover, it is well known in the field of biology that mRNA and protein expression are subject to post-transcriptional regulation (such as translational repression, protein degradation, and phosphorylation modification), and their correlation is only 0.3-0.5. Therefore, protein expression level cannot be directly inferred from mRNA expression level, and it is incorrect to assume that "mRNA markers can be replaced by protein markers." For example, existing studies indicate that "the average protein-RNA correlation between samples is in the range of 0.4-0.6" (see *Functional Impact of Protein–RNA Variation in Clinical Cancer Analyses*, MCP, 2023). Furthermore, existing technologies also mention that "related studies have reported an average correlation between mRNA and protein of approximately 0.2-0.5" (see *Experimental reproducibility limits the correlation between mRNA and protein abundances in tumor proteomic profiles*, *Cell Reports Methods*, 2022). Numerous existing studies have also found that in samples such as tumor cells, the expression levels of mRNA and protein are not perfectly positively correlated; phenomena such as high mRNA expression levels with low corresponding protein expression levels, or low mRNA expression levels with high corresponding protein expression levels, exist. Therefore, in this application, the inventors preferably use protein content to detect STAT1 expression levels.

[0067] In a preferred embodiment, the method for detecting the expression level of the STAT1 protein includes one or more of immunohistochemistry, mass spectrometry, protein microarray, flow cytometry, quantitative PCR, or Western blot; preferably, the detection method includes immunohistochemistry.

[0068] Protein detection technologies include, but are not limited to, immunohistochemistry, mass spectrometry, protein chips, flow cytometry, quantitative PCR, or Western blot. Among these, immunohistochemistry is widely used in disease diagnosis, tumor research, and clinical applications due to its advantages such as mature technology, high visualization, and low sample requirements. Therefore, in this application, the inventors preferably use immunohistochemistry to detect the STAT1 biomarker protein in the biological samples of the subjects.

[0069] In a preferred embodiment, the color intensity of the cytoplasm in the immunohistochemically analyzed sections is used to grade and score the expression level of the STAT1 protein.

[0070] In this application, the inventors discovered that by using immunohistochemical sections of STAT1 protein and employing methods including but not limited to color intensity, it is possible to achieve the technical effect of classifying the expression level of STAT1 protein. In this invention, the inventors preferably use color intensity to classify the expression level of STAT1 protein. The different expression level grades of STAT1 after classification can be better used to predict the therapeutic effect of immunotherapy combined with chemotherapy in cancer patients.

[0071] In a preferred embodiment, the grading criteria for the color intensity include: negative, weakly positive, positive, or strongly positive; negative includes cells in the section showing no staining in the cytoplasm; weakly positive includes the percentage of cells in the section showing different degrees of cytoplasmic staining being >0% and ≤10%; positive includes the percentage of cells in the section showing strong brownish-red cytoplasmic staining being 11-30%, or the percentage of cells in the section showing weak or moderate cytoplasmic staining being 11-70%; strongly positive includes the percentage of cells in the section showing strong brownish-red cytoplasmic staining being >30%, or the percentage of cells in the section showing moderate cytoplasmic staining being >70%; wherein, negative is recorded as 0 points, weakly positive as 1 point (+), positive as 2 points (++), and strongly positive as 3 points (+++).

[0072] In this application, the inventors categorized the staining intensity of immunohistochemical sections of STAT1 protein into four levels: negative, weakly positive, positive, or strongly positive. For the classification of staining intensity, those skilled in the art can refer to the cytoplasmic type-related standards in the journal *Yang Jun, Kang Anjing, Su Baoshan, et al. Advances in the interpretation of immunohistochemical detection results [J]. Chinese Journal of Clinical Physicians (Electronic Edition)*.

[0073] The evaluation criterion for negative results is that there is no staining in the immunohistochemical section;

[0074] The evaluation criteria for weak positive are that the proportion of cells showing different degrees of cytoplasmic staining in the entire immunohistochemical section is, for example, >0% and ≤10% (including but not limited to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%).

[0075] The evaluation criteria for a positive result are: the proportion of cells with strong brown staining in the cytoplasm of the immunohistochemical section is, for example, >10% and ≤30% (including but not limited to 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%), or the proportion of cells with weak or moderate staining in the cytoplasm of the immunohistochemical section is, for example, 11-70% (including but not limited to 11%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%).

[0076] The criteria for strong positive results are that the proportion of cells with strong brown staining in the cytoplasm of the immunohistochemical section is, for example, >30% (including but not limited to 31%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%), or the proportion of cells with moderate staining in the cytoplasm of the immunohistochemical section is, for example, >70% (including but not limited to 71%, 75%, 80%, 85%, 90%, 95%, or 100%).

[0077] In this application, the calculation method for immunohistochemical scores includes, but is not limited to, staining intensity scoring. The inventors have found that the expression levels of STAT1 at different levels, classified according to the above-mentioned immunohistochemical staining intensity grading standard, can be effectively used to predict the therapeutic effect of immunotherapy combined with chemotherapy in cancer patients. For example, a staining intensity score of 0 and / or 1 is considered a negative result, and a score of 2 and / or 3 is considered a positive result.

[0078] Postoperative pathological specimens from the subjects were graded according to the pathological tumor regression grade (TRG) recommended by the National Comprehensive Cancer Network (NCCN): Grade 0 (complete pathological response, no residual tumor cells), Grade 1 (virtually no residual tumor, only a small number of cancer cells surviving), Grade 2 (significant tumor regression, but still a significant number of cancer cells remaining), and Grade 3 (minor tumor regression, most tumor tissue still present). Grade 0 was defined as pathologic complete response (pCR), and TRG 0-1 were defined as major pathological response (MPR). Based on postoperative pathological evaluation, patients were divided into the treatment response group (Response[R], TRG0-1 group) and the treatment non-response group (Non-Response[NR], TRG2-3 group) in this application.

[0079] In a preferred embodiment, the reagents used in the above-described method for detecting the expression level of STAT1 include one or more of anti-STAT1 antibodies, primers, or probes; preferably, the reagents include anti-STAT1 antibodies.

[0080] The detection of STAT1 expression levels includes, but is not limited to, anti-STAT1 antibodies, primers, or probes. Anti-STAT1 antibodies, due to their high specificity and sensitivity, can be directly used to detect proteins and their modified forms. In this application, the inventors preferably use anti-STAT1 antibodies to detect STAT1 expression.

[0081] In a preferred embodiment, the subject is a cancer patient; preferably, the cancer includes one or more of gastric cancer, non-small cell lung cancer, colorectal cancer, or triple-negative breast cancer.

[0082] In this application, the inventors discovered that the detection of STAT1 biomarkers can predict the efficacy of immunotherapy combined with chemotherapy in patients with cancers including, but not limited to, gastric cancer, non-small cell lung cancer, colorectal cancer, and triple-negative breast cancer.

[0083] Preferably, a specific method for predicting the efficacy of immunotherapy combined with chemotherapy in subjects, namely, a screening method for the population that benefits from STAT1-based immunotherapy, is as follows:

[0084] (1) Obtain tumor tissue biopsy samples from cancer patients and detect STAT1 expression using immunohistochemistry;

[0085] (2) Based on the STAT1 expression status, refer to the preset evaluation indicators to determine whether the patient has high or low STAT1 expression;

[0086] (3) Select gastric cancer patients with high STAT1 expression for immunotherapy combined with chemotherapy.

[0087] In a second typical embodiment of this application, the application of a reagent for detecting STAT1 protein expression levels is provided in the preparation of a kit for predicting the efficacy of immunotherapy combined with chemotherapy in subjects.

[0088] The application methods of the above-mentioned kit include, but are not limited to: using reagents capable of detecting STAT1 protein expression levels to detect the STAT1 protein expression level in the subject's biological samples. Further, after detection, the level of STAT1 protein expression is determined, and based on the result of a high STAT1 protein expression level, a conclusion is drawn that the patient is suitable for immunotherapy combined with chemotherapy.

[0089] Specifically, the usage methods of the above-mentioned reagent kits include, but are not limited to:

[0090] 1. Reagent preparation.

[0091] 2. Before administering the above-mentioned immunotherapy combined with chemotherapy, the expression levels of biomarkers in the biological samples of the subjects were detected. The biomarkers included, but were not limited to, STAT1 protein. The detection method for STAT1 protein included this kit.

[0092] 3. The biological samples tested by the kit include, but are not limited to, one or more of blood samples or tumor tissue samples.

[0093] 4. The expression level of the STAT1 protein mentioned above includes, but is not limited to, the content of the STAT1 protein or the expression level of the mRNA used to express the STAT1 protein.

[0094] 5. The reagents used in the above-mentioned method for detecting the expression level of STAT1 include, but are not limited to, one or more of anti-STAT1 antibodies, primers or probes; preferably, the above-mentioned reagents include anti-STAT1 antibodies.

[0095] 6. The subjects mentioned above are cancer patients, including but not limited to one or more of the following cancers: gastric cancer, non-small cell lung cancer, colorectal cancer, or triple-negative breast cancer.

[0096] In a preferred embodiment, the reagents include one or more of anti-STAT1 antibodies, primers, or probes; preferably, the reagents include anti-STAT1 antibodies.

[0097] In this application, the inventors discovered that the reagents for detecting STAT1 protein expression levels in a kit used to predict the efficacy of immunotherapy combined with chemotherapy in subjects include, but are not limited to, anti-STAT1 antibodies, primers, or probes, all of which can detect the expression level of STAT1. Anti-STAT1 antibodies, due to their high specificity and high sensitivity, can be directly used to detect proteins and their modified forms. In this application, the inventors preferably use anti-STAT1 antibodies to detect the expression of STAT1.

[0098] In a preferred embodiment, the above-mentioned immunotherapy combined with chemotherapy includes administering an immunotherapy agent and a chemotherapy regimen to the subject simultaneously; preferably, the chemotherapy regimen includes one or more of SOX, XELOX, FOLFOX, or DOX; preferably, the chemotherapy regimen includes SOX or XELOX; preferably, the immunotherapy agent includes one or more of PD-1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, or A2AR inhibitors; preferably, the immunotherapy agent includes a PD-1 inhibitor; preferably, the subject is a cancer patient; preferably, the cancer includes one or more of gastric cancer, non-small cell lung cancer, colorectal cancer, or triple-negative breast cancer.

[0099] In this application, the inventors discovered that the aforementioned kit can effectively predict the therapeutic effect of immunotherapy combined with chemotherapy in subjects. The chemotherapy regimens, including but not limited to SOX or XELOX, have significant efficacy against various types of gastric cancer. Furthermore, SOX or XELOX does not require hydration during use, resulting in lower nephrotoxicity for patients. The inventors also discovered that the aforementioned kit can predict the therapeutic effect of immunotherapy combined with chemotherapy in patients with, but not limited to, gastric cancer, non-small cell lung cancer, colorectal cancer, and triple-negative breast cancer.

[0100] In this application, the inventors detected the expression levels of biomarkers in the biological samples of the subjects before administering the aforementioned immunotherapy combined with chemotherapy. The biomarkers included STAT1 protein, and the detection methods for STAT1 protein included, but were not limited to, the aforementioned kit. The biological samples detected by the kit included, but were not limited to, one or more of blood samples or tumor tissue samples. The expression level of the STAT1 protein included, but was not limited to, the content of the STAT1 protein or the expression level of mRNA used to express the STAT1 protein. The reagents used in the method for detecting the expression level of STAT1 included, but were not limited to, one or more of anti-STAT1 antibodies, primers, or probes. Preferably, the reagents included, but were not limited to, anti-STAT1 antibodies. The subjects were cancer patients, and the cancer included, but was not limited to, one or more of gastric cancer, non-small cell lung cancer, colorectal cancer, or triple-negative breast cancer.

[0101] In a third typical embodiment of this application, an electronic device is provided for predicting the efficacy of immunotherapy combined with chemotherapy in a subject. The electronic device includes a data collection module and a prediction module. The data collection module is used to collect the expression levels of biomarkers in the subject's biological samples and input the data collected by the data collection module into the prediction module. The biomarkers include STAT1 protein. The prediction module is configured to output a prediction result of the efficacy of immunotherapy combined with chemotherapy in the subject based on the expression levels of the biomarkers in the subject's biological samples.

[0102] In this application, the inventors provide an electronic device whose core components include a data collection module and a prediction module. The data collection module automatically collects biomarker content data from a subject's biological sample, with particular focus on the key biomarker STAT1 protein. The electronic device may optionally include a physical device for detecting biomarker content, or may be configured solely as a device or system for collecting biomarker content. The data obtained by the data collection module is then transmitted to the prediction module, which, based on the biomarker content in the immunohistochemical sections of the subject's biological sample and based on built-in thresholds or judgment rules, outputs a diagnostic result or risk prediction result regarding the subject's response to immunotherapy combined with chemotherapy. The inventors have found that implementing the aforementioned electronic device can significantly improve the accuracy of predicting the suitability of cancer patients for immunotherapy combined with chemotherapy, effectively screen potentially benefiting individuals, avoid the waste of medical resources and potential adverse reactions caused by ineffective treatment, thereby optimizing clinical treatment strategies, improving treatment outcomes and patients' quality of life, and has significant guiding significance and practical application value for clinical practice.

[0103] In a preferred embodiment, the expression level of the biomarker is derived from one or more methods, including but not limited to immunohistochemistry, mass spectrometry, protein microarray, flow cytometry, quantitative PCR, or Western blot. Preferably, the expression level of the biomarker is obtained based on the analysis of the immunohistochemically analyzed sections. The data collection module is used to analyze the staining of the cytoplasm of the STAT1 protein in the immunohistochemically analyzed sections. The data collection module includes a staining intensity module and an optional calculation module. The staining intensity module is used to determine the staining status and percentage of the cytoplasm in the immunohistochemically analyzed sections and obtain a staining intensity score. The calculation module is used to calculate the immunohistochemical score of the immunohistochemically analyzed sections based on the staining intensity score obtained by the staining intensity module.

[0104] The staining intensity module is used to determine the staining status and percentage of the cytoplasm of the cells in the immunohistochemical sections, and to obtain a staining intensity score. In this application, the staining intensity of the STAT1 protein immunohistochemical sections can be divided into four levels: negative, weakly positive, positive, or strongly positive. The evaluation criterion for negative is that there is no staining in the immunohistochemical section. The evaluation criterion for weakly positive is that the proportion of cells showing different degrees of cytoplasmic staining in the entire section is, for example, >0% and ≤10% (including but not limited to 0.1%, 0.2%, 0.5%). 3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%; a positive evaluation criterion is that the proportion of cells with strong brownish chroma staining in the cytoplasm of the immunohistochemical section is, for example, >10% and ≤30% (including but not limited to 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 2...). A strong positive result is defined as the percentage of cells exhibiting weak or moderate cytoplasm staining in the immunohistochemical section being, for example, 11-70% (including but not limited to 11%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%). A strong positive result is defined as the percentage of cells exhibiting strong brownish cytoplasm staining in the immunohistochemical section being, for example, >30% (including but not limited to 31%, 35%, 40%, 45%, 50%, 55%, or 60%). The percentage of cells with moderately stained cytoplasm in the immunohistochemical section (65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) is greater than 70% (including but not limited to 71%, 75%, 80%, 85%, 90%, 95%, or 100%). A negative result is scored as 0 points, a weak positive as 1 point, a positive as 2 points, and a strong positive as 3 points. This electronic device can autonomously identify the cytoplasmic staining of cells in the immunohistochemical section and calculate its percentage, classifying the scores accordingly.

[0105] The calculation module is used to calculate the immunohistochemical score of the immunohistochemically analyzed slides based on the color intensity score obtained by the color intensity module.

[0106] The prediction module, based on the levels of biomarkers in immunohistochemical sections of the subject's biological samples, uses built-in thresholds or judgment rules to output diagnostic results or risk predictions of the subject's response to immunotherapy combined with chemotherapy. The inventors have found that implementing the aforementioned electronic device can significantly improve the accuracy of predicting the suitability of cancer patients for immunotherapy combined with chemotherapy, effectively screen potentially benefiting individuals, avoid the waste of medical resources and potential adverse reactions caused by ineffective treatment, thereby optimizing clinical treatment strategies, improving treatment outcomes and patients' quality of life, and has significant guiding significance and practical application value for clinical practice.

[0107] In a fourth typical embodiment of this application, a method for predicting the efficacy of immunotherapy combined with chemotherapy is provided. All steps in the prediction method are performed by a computer. The prediction method is used to predict the effect of immunotherapy combined with chemotherapy on cancer patients. The prediction method includes: generating a risk prediction result of immunotherapy combined with chemotherapy for cancer patients based on the expression level of biomarkers in biological samples of cancer patients; the biomarkers include STAT1 protein.

[0108] The above prediction method only includes computer-based steps. If the expression level of the biomarker used in the prediction method is the STAT1 protein content, the prediction method directly obtains the relevant results of the STAT1 protein without including specific detection steps. If the expression level of the biomarker used in the prediction method is the STAT1 protein level in immunohistochemical sections, the prediction method may optionally include a visual analysis method for the section photographs. This analysis method includes executing the following algorithm:

[0109] The calculation method of the above algorithm includes, but is not limited to, a data collection module and a prediction module, wherein the data collection module includes a color intensity module and an optional calculation module.

[0110] The staining intensity module is used to determine the staining status and percentage of the cytoplasm of the cells in the immunohistochemically analyzed sections, and to obtain a staining intensity score. In this application, the staining intensity of the STAT1 protein immunohistochemical sections can be divided into four levels: negative, weakly positive, positive, or strongly positive. The evaluation criterion for negative is no staining in the immunohistochemical section. The evaluation criterion for weakly positive is that the percentage of cells showing different degrees of cytoplasmic staining in the entire section is, for example, >0% and ≤10% (including but not limited to 0.1%, 0.2%, 0.5%). 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%; a positive evaluation criterion is that the percentage of brownish staining of cell cytoplasm in the immunohistochemical section is, for example, >10% and ≤30% (including but not limited to 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%). A strong positive result is defined as the percentage of cells exhibiting weak or moderate chromatographic staining in the immunohistochemical section being, for example, 11-70% (including but not limited to 11%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%). A strong positive result is defined as the percentage of cells exhibiting strong brownish chromatographic staining in the immunohistochemical section being, for example, >30% (including but not limited to 31%, 35%, 40%, 45%, 50%, 55%, 60%, 60%). The percentage of cells with moderately stained cytoplasm in the immunohistochemical section (5%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) is greater than 70% (including but not limited to 71%, 75%, 80%, 85%, 90%, 95%, or 100%). A negative result is scored as 0 points, a weak positive as 1 point, a positive as 2 points, and a strong positive as 3 points. This electronic device can autonomously identify the cytoplasmic staining of cells in the immunohistochemical section and calculate its percentage, classifying the scores accordingly.

[0111] The calculation module is used to calculate the immunohistochemistry score of the immunohistochemical slide based on the color intensity score obtained by the color intensity module. The calculation method includes, but is not limited to, calculating the color intensity score as the immunohistochemistry score.

[0112] The prediction module, based on the levels of biomarkers in immunohistochemical sections of the subject's biological samples, uses built-in thresholds or judgment rules to output diagnostic results or risk predictions of the subject's response to immunotherapy combined with chemotherapy. The inventors have found that implementing the aforementioned electronic device can significantly improve the accuracy of predicting the suitability of cancer patients for immunotherapy combined with chemotherapy, effectively screen potentially benefiting individuals, avoid the waste of medical resources and potential adverse reactions caused by ineffective treatment, thereby optimizing clinical treatment strategies, improving treatment outcomes and patients' quality of life, and has significant guiding significance and practical application value for clinical practice.

[0113] In a fifth typical embodiment of this application, a computer device is provided, including a memory, a processor, and a computer program stored in the memory, wherein the processor executes the computer program to implement the steps of the electronic device or the prediction method described above.

[0114] In a sixth typical embodiment of this application, a computer-readable storage medium is provided, on which a computer program is stored, which, when executed by a processor, implements the steps of the electronic device or the prediction method described above.

[0115] In a seventh typical embodiment of this application, a computer program product is provided, including a computer program that, when executed by a processor, implements the steps of the electronic device or the prediction method described above.

[0116] Those skilled in the art will understand that the modules or steps of the present invention described above can be implemented using general-purpose computing devices. They can be centralized on a single computing device or distributed across a network of multiple computing devices. They can be implemented using computer-executable program code, and thus can be stored in a storage device for execution by a computing device. In some cases, the steps shown or described can be performed in a different order than those presented herein, or they can be fabricated as separate integrated circuit modules, or multiple modules or steps can be fabricated as a single integrated circuit module. Thus, the present invention is not limited to any particular combination of hardware and software.

[0117] In an eighth typical embodiment of this application, a reagent for treating cancer in a subject is provided, the reagent comprising a reagent for detecting STAT1 protein content and an immunotherapy combined with chemotherapy drugs.

[0118] In this application, the inventors discovered the application of reagents for detecting STAT1 protein levels in the preparation of kits for predicting the efficacy of immunotherapy combined with chemotherapy in subjects. This kit enables a more convenient and accurate detection of STAT1 protein.

[0119] In a ninth typical embodiment of this application, a method for treating a subject suffering from cancer is provided, the method comprising: ① detecting the expression level of a biomarker, including STAT1 protein, in a biological sample of the subject prior to the treatment; and ② administering treatment to the subject who passes the detection. The type of treatment includes immunotherapy combined with chemotherapy for the subject.

[0120] In a tenth typical embodiment of this application, a method for assessing the prognostic effect of a subject suffering from cancer is provided. The method includes: ① detecting the expression level of a biomarker, including STAT1 protein, in a biological sample of the subject after the aforementioned treatment; ② assessing the prognostic effect of the subject suffering from cancer based on the expression level of the STAT1 protein. The type of treatment includes immunotherapy combined with chemotherapy for the subject.

[0121] In an eleventh typical embodiment of this application, a method for monitoring the treatment effect on a subject suffering from cancer is provided. The method includes: ① detecting the expression level of a biomarker, including STAT1 protein, in a biological sample of the subject during the treatment process; ② monitoring the treatment effect on the subject suffering from cancer by measuring the expression level of the STAT1 protein. The type of treatment includes immunotherapy combined with chemotherapy for the subject.

[0122] In this application, the inventors discovered that detecting the STAT1 biomarker in the biological samples of subjects can be applied in clinical trials treating subjects with cancer. Furthermore, the ORR (Objective Response Rate) after immunotherapy can effectively infer the prognosis of patients. For example, prior art mentions that "in the analysis of patient response levels, patients who meet the corresponding criteria have longer survival times compared to non-responding patients" (see Overall Response Rate, Progression-Free Survival, and Overall Survival With Targeted and Standard Therapies in Advanced Non-Small-Cell Lung Cancer: US Food and Drug Administration Trial-Level and Patient-Level Analyses, JCO, 2015). In addition, prior art also proposes predicting late-stage survival based on early efficacy ORR (see Predict progression-free survival and overall survival using objective response rate for anti-PD1 / PDL1 therapy development, BMC Cancer, 2024). Therefore, in this application, immunotherapy subjects with high ORR can be screened based on STAT1 expression levels, thereby further predicting the prognosis of patients.

[0123] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0124] Example 1

[0125] Discovery of differentially expressed proteins:

[0126] Pre-treatment biopsy samples and post-operative surgical resection samples were obtained from gastric cancer patients who received neoadjuvant chemotherapy combined with immunotherapy. These patients were divided into a treatment response group (R group) and a non-response group (NR group). The classification of the R and NR groups was based on the pathological tumor regression grade (TRG) recommended by the National Comprehensive Cancer Network (NCCN), including: Grade 0 (complete pathological response, no residual tumor cells), Grade 1 (virtually no residual tumor, only a small number of cancer cells surviving), Grade 2 (significant tumor regression, but still a significant number of cancer cells remaining), and Grade 3 (minor tumor regression, most tumor tissue still present). Grade 0 was defined as pathologic complete response (pCR), and TRG0–1 were defined as major pathological response (MPR). Based on post-operative pathological evaluation, patients were further divided into the treatment response group (TRG0–1 group) and the non-response group (TRG2–3 group).

[0127] (1) For tumor tissue samples from the R group and non-R group mentioned above, the protein expression profile of the enrolled samples was obtained by mass spectrometry-based proteomics method (data-dependent mode acquisition [DDA]), and the quality control conditions met the 1% false discovery rate (FDR) of peptide and protein levels.

[0128] (2) Bioinformatics methods were used to perform differential expression analysis on the data from group R and non-group R, and differentially expressed proteins (fold change > 1.2, P-value < 0.05) were screened. The screening results are as follows: Figure 1 and Figure 2 As shown; where, Figure 1 To detect STAT1 expression in the TRG0-3 group using a mass spectrometry-based proteomics method, the results showed that STAT1 expression was highest in the TRG0 group. Figure 2 To detect the differential expression of STAT1 in the R and NR groups using a mass spectrometry-based proteomics method, the results showed that the expression of STAT1 in the R group was higher than that in the NR group.

[0129] (3) Based on the molecular biological functions of differentially expressed proteins, candidate biomarker STAT1 was identified for subsequent validation.

[0130] Example 2

[0131] Sample collection:

[0132] (1) The research samples were obtained from pre-treatment biopsy samples and post-operative surgical resection samples of gastric cancer patients who received neoadjuvant chemotherapy combined with immunotherapy. It should be noted that the research samples used in this embodiment are completely different from those in Example 1, and there is no overlap or intersection.

[0133] (2) Pretreatment biopsy samples were subjected to mass spectrometry-based proteomics detection and immunohistochemical verification.

[0134] (3) Postoperative samples were used to evaluate the efficacy of neoadjuvant therapy, and the evaluation method was the same as the TRG method in Example 1.

[0135] Example 3

[0136] Immunohistochemical verification:

[0137] (1) Antibody selection:

[0138] Immunohistochemistry was performed using research antibodies.

[0139] Step 1: Preparation of white slides (preparation of paraffin sections):

[0140] Sectioning: Gastric cancer tissue is sectioned continuously from paraffin blocks using a pathological microtome, with a thickness typically of 4-5 μm;

[0141] Spreading and scooping: Spread the cut tissue slices flat in warm water (about 40-45°C), and then scoop them up with a glass slide to prevent them from falling off;

[0142] Baking: Place the slides on a slide warmer at 60-65°C and bake for at least 2 hours, or overnight at 37°C. The purpose is to ensure that the tissue adheres tightly to the slide and prevent it from falling off in subsequent steps.

[0143] Step 2: Dewaxing and Hydration

[0144] Xylene I: Soak for 10-15 minutes; Xylene II: Soak for 10-15 minutes; 100% Ethanol I: Soak for 5 minutes; 100% Ethanol II: Soak for 5 minutes; 95% Ethanol: Soak for 5 minutes; 85% Ethanol: Soak for 5 minutes; 75% Ethanol: Soak for 5 minutes; Rinse with distilled water: 5 minutes (Purpose: To thoroughly remove paraffin from the tissue and rehydrate the tissue to prepare for the aqueous reaction system).

[0145] Step 3: Antigen Repair

[0146] Place the slides in a container filled with EDTA antigen retrieval solution (pH=8.0 or 9.0), microwave on high until boiling, then reduce to medium-low heat and maintain a gentle boil for 15-20 minutes; allow to cool naturally to room temperature (about 30-40 minutes), do not cool rapidly to avoid damaging the tissue morphology; rinse the slides three times with phosphate-buffered saline (PBS) (pH=7.4), 5 minutes each time.

[0147] Step 4: Block endogenous peroxidase:

[0148] Place the slices in a 3% solution of freshly prepared methanol. Incubate in the solution at room temperature in the dark for 15-25 minutes, then rinse three times with PBS for 5 minutes each time.

[0149] Step 5: Seal off:

[0150] Draw a hydrophobic circle around the tissue using an immunohistochemistry pen to prevent fluid from spreading; add a sufficient amount of normal goat serum (or serum homologous to the source of the secondary antibody) blocking solution to completely cover the tissue; incubate at room temperature for 20-30 minutes. Discard the serum blocking solution; do not rinse with PBS or running water.

[0151] Step 6: Primary Antibody Incubation

[0152] Add the prepared STAT1 primary antibody working solution (Bio-Asia Biotech, STAT1 Recombinant Rabbit mAb, bsm-63166R) (initial concentration 1:200) to the tissue; place the slide flat in a humidified chamber and incubate overnight at 4°C (approximately 16-18 hours); remove the humidified chamber from the refrigerator the next day and allow it to equilibrate at room temperature for 30 minutes; wash three times with PBS for 5 minutes each time to thoroughly remove any unbound primary antibody.

[0153] Step 7: Secondary Antibody Incubation

[0154] Add horseradish peroxidase (HRP)-labeled rabbit secondary antibody polymer (Zhongshan Jinqiao, PV-6001) to completely cover the tissue; incubate at room temperature for 30-50 minutes. Wash three times with PBS for 5 minutes each time to thoroughly remove unbound secondary antibody.

[0155] Step 8: Color development with 3,3'-Diaminobenzidine (DAB):

[0156] Prepare the DAB working solution fresh for each use, following the instructions for the DAB kit (Beijing Solarbio Science & Technology Co., Ltd., DA1015). Add the DAB working solution to the tissue and observe the color development under a microscope in real time. A positive signal (brownish-yellow or brown granules) will typically appear gradually within seconds to minutes. The optimal termination point is when a strong positive signal appears in the positive control and the background becomes colorless. Once the ideal color depth is reached, immediately immerse the section in distilled water to stop the reaction.

[0157] Step 9: Counterstaining, dehydration, clearing, and mounting:

[0158] Counterstaining with hematoxylin: Counterstain cell nuclei with Harris hematoxylin staining solution (Zhongshan Jinqiao, ZLI-9610) for 1-2 minutes.

[0159] Differentiation: Place in 1% hydrochloric acid alcohol for a few seconds to differentiate, wash away excess hematoxylin, and make the cell nucleus turn pale blue;

[0160] Blueing: Place in ammonia or warm water to blue, making the cell nuclei appear a clear blue;

[0161] Dehydration: Pass through 75%, 85%, 95%, 100% I, and 100% II ethanol sequentially for 2-3 minutes each time;

[0162] To clarify: Place in xylene I and II for 5-10 minutes each to clarify.

[0163] Mounting: Remove the section from xylene, place a drop of neutral resin in the center of the tissue, cover with a coverslip, avoiding air bubbles. Allow to air dry at room temperature.

[0164] (2) Evaluation indicators:

[0165] STAT1 expression was classified into four categories based on staining intensity: 0, 1, 2, and 3 represent negative, weakly positive, positive, and strongly positive, respectively. The staining intensity of STAT1 was assessed and graded. The STAT1 expression level was also graded based on the staining of the cytoplasm in the immunohistochemical staining of the sections.

[0166] The specific evaluation method for color intensity is as follows: Under low magnification (4x or 10x), browse the entire section, observe the staining of the cell nuclei in the target cells and the distribution of positive cells in the nuclei, select the high expression area, and count the percentage of positive cells in the same type of cells in 5 high magnification (20x or 40x) fields as the interpretation result of the entire section.

[0167] The specific grading standards for staining intensity are as follows: 0 [Negative]: No staining; + [Weakly Positive]: >0% and ≤10% of cells show varying degrees of cytoplasmic staining; ++ (Positive): 11%-30% of cells show strong brownish cytoplasmic staining, or 11-70% of cells show weak or moderate cytoplasmic staining; +++ (Strongly Positive): >30% of cells show strong brownish cytoplasmic staining or >70% show moderate cytoplasmic staining. The staining intensity score is divided into 4 levels: 0 points for negative IHC cell staining, 1 point for weakly positive IHC cell staining, 2 points for positive IHC cell staining, and 3 points for strongly positive IHC cell staining.

[0168] In this experiment, we further calculated the immunohistochemistry score using a scoring method. The immunohistochemistry score is a staining intensity score, and an example of STAT1 immunohistochemical staining grading is shown below. Figure 3 As shown.

[0169] (3) Verification results:

[0170] Preoperative STAT1 immunohistochemical staining was performed on 96 patients with locally advanced gastric cancer who underwent radical gastrectomy after receiving neoadjuvant chemotherapy combined with immunotherapy (SOX or XELOX + PD-1 inhibitor). All patients had postoperative pathological results and were evaluated for TRG grading (NCCN criteria). The STAT1 staining intensity score and pathological TRG grade for each patient are shown in Table 1 below. The results showed that in patients with positive / strongly positive STAT1 expression (staining intensities 2 and 3) (n=63), the MPR rate (i.e., the proportion of patients in group R) was 58.7%; in patients with negative / weakly positive STAT1 expression (staining intensities 0 and 1) (n=33), the MPR rate (and the proportion of patients in group R) was 33.3%. According to the Pearson chi-square test, the difference in MPR rate between the two groups was statistically significant (χ2=5.587, P=0.018). These results suggest that high STAT1 expression (staining intensities 2 and 3) can effectively predict the pathological efficacy of neoadjuvant chemotherapy combined with immunotherapy in patients with locally advanced gastric cancer, and has potential clinical translational value for screening sensitive populations. The expression of STAT1 protein in groups R and NR is shown below. Figure 4 As shown; Figure 4 The immunohistochemical staining intensity of STAT1 protein in the R and NR groups was shown in Table 1. The immunohistochemical results showed that the immunohistochemical staining intensity of STAT1 in the R group was significantly higher than that in the NR group (P<0.01). The correlation between STAT1 expression before treatment and pathological efficacy in 96 patients undergoing neoadjuvant chemotherapy combined with immunotherapy is shown in Table 1.

[0171] Table 1

[0172]

[0173]

[0174]

[0175] Example 4

[0176] Comparison of STAT1 with mainstream biomarkers:

[0177] We statistically analyzed the positive detection rate (applicable proportion) and treatment efficacy (response rate, ORR: objective response rate) of mainstream immunotherapy biomarkers in gastric cancer. The trial results are shown in Table 2 and... Figure 5 As shown, the results indicated that when STAT1 protein expression showed a strong positive (3+) staining intensity on immunohistochemical detection, its applicable proportion in patients was 21%, and the response rate was 80%. The applicable proportion of MSI-H in patients was 5.5%, with MSI-H (Microsatellite Instability-High) determined by PCR detection of instability at microsatellite loci such as BAT25, BAT26, NR21, NR24, or MONO27 (≥2 loci instability are considered MSI-H), and a response rate of 62.5%. The applicable proportion of TMB-H (Tumor Mutational Burden-High) (>=10 mut / Mb) in patients was 13%, with a response rate of 71%. Here, mut represents mutation. TMB-H was determined by next-generation sequencing (NGS), with NGS often employing whole exome sequencing (WEE). Sequencing (WES) or large panel sequencing, clinically TMB-H is usually defined as ≥10 mutations / Mb; PD-L1 (Programmed DeathLigand 1) has an applicability rate of 19% and a response rate of 67% in patients with (CPS>=10%); PD-L1 has an applicability rate of 27% and a response rate of 55% in patients with (CPS>=5%). CPS stands for Combined Positive Score, which is defined as (number of PD-L1 positive cells / total number of tumor cells) × 100%. Positive cells include tumor cells or tumor-infiltrating immune cells.

[0178] Table 2

[0179]

[0180] Comparative Example 1

[0181] Discovery of differentially expressed proteins:

[0182] The differentially expressed protein COL18A1 was simultaneously screened using the method described in Example 1. The screening results are as follows: Figure 6 and Figure 7 As shown; where, Figure 6 To detect the expression of COL18A1 in the TRG0-3 group using a mass spectrometry-based proteomics method, the results showed that COL18A1 expression was highest in the TRG3 group. Figure 7 To detect the differential expression of COL18A1 in the R and NR groups using a mass spectrometry-based proteomics method, where the R group represents the treatment-responsive group and the NR group represents the treatment-non-responsive group, the results showed that the expression of COL18A1 in the NR group was higher than that in the R group.

[0183] COL18A1 protein immunohistochemical detection:

[0184] Taking STAT1 and COL18A1 proteins as examples, the protein detected in Example 3 was changed from STAT1 to COL18A1, while other operations remained the same as in Example 3. Immunohistochemical staining of COL18A1 showed no significant difference in expression between the treatment-responsive and non-responding groups. An example of COL18A1 protein immunohistochemical staining is shown in the figure below. Figure 8 As shown in the figure. The differential expression of COL18A1 protein staining intensity between the treatment-responsive group and the treatment-non-responsive group is shown in the figure. Figure 9 As shown, ns represents no significant difference, derived from... Figure 8 The results showed that the expression intensity of COL18A1 protein in the treatment response group was lower than that in the treatment non-response group, and there was no significant difference between the groups. This indicates that not all differentially expressed proteins obtained from omics screening can be used to predict the efficacy of immunotherapy combined with chemotherapy in gastric cancer patients.

[0185] Based on the staining intensity, COL18A1 immunohistochemical staining was performed on gastric cancer tissue samples from 18 patients before treatment. The COL18A1 immunohistochemical staining method was the same as that for STAT1 in Example 3, except that the primary antibody was replaced with the primary antibody corresponding to COL18A1 (bs-0547R, Bio-Sensing). The staining intensity of COL18A1 immunohistochemical staining and the pathological TRG grade are shown in Table 3. The results showed that there was no significant difference between the staining intensity of COL18A1 immunohistochemical staining and the response / non-response outcome (P>0.05). When color intensity 2 and 3 were defined as high COL18A1 expression, the MPR rate (i.e., the proportion of patients in group R) was 33.3% in patients with positive / strongly positive COL18A1 expression (n=9); and the MPR rate (and the proportion of patients in group R) was 44.4% in patients with weak COL18A1 expression (n=9). According to the Pearson chi-square test, the difference in MPR rate between the two groups was statistically significant (χ2=0.234, P=0.629). These results suggest that COL18A1 is not a reliable predictor of the sensitivity of patients to neoadjuvant chemotherapy combined with immunotherapy in locally advanced gastric cancer.

[0186] Table 3

[0187]

[0188] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0189] This invention provides a method for predicting the efficacy of immunotherapy combined with chemotherapy in subjects. The method achieves this prediction by detecting the STAT1 biomarker content in the subjects' biological samples before administration of immunotherapy combined with chemotherapy. This invention has the following advantages: ① Broad coverage: STAT1, as a predictive biomarker, can be applied to most gastric cancer patients, regardless of MSI-H / dMMR status. Even in MSI-S / MSS type gastric cancer patients, high STAT1 expression is associated with better immunotherapy response. ② Simple detection method: Immunohistochemistry is a commonly used pathological detection method in clinical practice. It is simple to operate, low in cost, and easy to promote and apply. This method can be performed during routine pathological examinations without the need for additional complex equipment and techniques. ③ Can be combined with other biomarkers: STAT1 can be used in combination with biomarkers such as PD-L1, MSI-H / dMMR, and TMB to improve predictive accuracy. Multi-biomarker combined models can more comprehensively assess the patient's immune status, providing a more reliable basis for clinical decision-making. ④ High clinical application value: This invention can guide clinicians in selecting the optimal treatment plan for gastric cancer patients, improving treatment efficacy and patient prognosis. By predicting the likelihood of a patient's response to immunotherapy, side effects and waste of medical resources caused by ineffective treatment can be avoided. ⑤ Broad application prospects: STAT1 is associated with immunotherapy response in various cancer types (such as non-small cell lung cancer, colorectal cancer, and triple-negative breast cancer), showing broad application prospects. ⑥ This application directly reads the immunohistochemical staining intensity score of STAT1 protein in patient tissues using a computer system, which is faster and more accurate than traditional manual discrimination methods.

[0190] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for predicting the efficacy of combined immunotherapy and chemotherapy in a subject, characterized in that, The method includes detecting the expression levels of biomarkers, including STAT1 protein, in the subject's biological samples prior to administration of the immunotherapy combined with chemotherapy.

2. The method according to claim 1, characterized in that, The combined immunotherapy and chemotherapy regimen includes administering immunotherapy agents and chemotherapy regimens to the subject simultaneously. Preferably, the chemotherapy regimen includes: One or more of SOX, XELOX, FOLFOX, or DOX; Preferably, the chemotherapy regimen includes SOX or XELOX; Preferably, the immunotherapy agent comprises one or more of the following: PD-1 inhibitor, CTLA-4 inhibitor, LAG-3 inhibitor, TIM-3 inhibitor, TIGIT inhibitor, or A2AR inhibitor; Preferably, the immunotherapy agent includes a PD-1 inhibitor.

3. The method according to claim 1, characterized in that, The biological samples include one or more of the following: blood samples, urine samples, or tumor tissue samples.

4. The method according to claim 1, characterized in that, The expression level of the STAT1 protein includes the content of the STAT1 protein.

5. The method according to claim 4, characterized in that, The methods for detecting the expression level of the STAT1 protein include one or more of the following: immunohistochemistry, mass spectrometry, protein microarray, flow cytometry, quantitative PCR, or Western blot. Preferably, the detection method includes immunohistochemistry.

6. The method according to claim 5, characterized in that, The immunohistochemistry includes: grading and scoring the expression level of the STAT1 protein based on the staining intensity of the cytoplasm in the immunohistochemically stained sections.

7. The method according to claim 1, characterized in that, The reagents used in the method for detecting the expression level of STAT1 include one or more of anti-STAT1 antibodies, primers, or probes; preferably, the reagents include anti-STAT1 antibodies.

8. The method according to claim 1, characterized in that, The subjects were cancer patients; Preferably, the cancer includes one or more of gastric cancer, non-small cell lung cancer, colorectal cancer, or triple-negative breast cancer.

9. Application of reagents for detecting STAT1 protein expression levels in the preparation of kits for predicting the efficacy of immunotherapy combined with chemotherapy in subjects.

10. The application according to claim 9, characterized in that, The reagent includes one or more of anti-STAT1 antibodies, primers, or probes; preferably, the reagent includes anti-STAT1 antibodies.

11. The application according to claim 9, characterized in that, The combined immunotherapy and chemotherapy regimen includes administering immunotherapy agents and chemotherapy regimens to the subject simultaneously. Preferably, the chemotherapy regimen includes: One or more of SOX, XELOX, FOLFOX, or DOX; Preferably, the chemotherapy regimen includes SOX or XELOX; Preferably, the immunotherapy agent comprises one or more of the following: PD-1 inhibitor, CTLA-4 inhibitor, LAG-3 inhibitor, TIM-3 inhibitor, TIGIT inhibitor, or A2AR inhibitor; Preferably, the immunotherapy agent comprises a PD-1 inhibitor; Preferably, the subject is a cancer patient; Preferably, the cancer includes one or more of gastric cancer, non-small cell lung cancer, colorectal cancer, or triple-negative breast cancer.

12. An electronic device for predicting the efficacy of combined immunotherapy and chemotherapy in a subject, characterized in that, The electronic device includes a data collection module and a prediction module. The data collection module is used to collect the expression levels of biomarkers in the subjects' biological samples and input the data collected by the data collection module into the prediction module; wherein the biomarkers include STAT1 protein; The prediction module is configured to output a prediction of the efficacy of the subject's combined immunotherapy and chemotherapy based on the expression levels of biomarkers in the subject's biological samples.

13. The electronic device according to claim 12, characterized in that, The expression levels of the biomarkers can be obtained from one or more of the following methods: immunohistochemistry, mass spectrometry, protein microarray, flow cytometry, quantitative PCR, or Western blot. Preferably, the expression level of the biomarker is obtained based on the analysis of the immunohistochemical sections; The data collection module is used to analyze the staining of the cytoplasm of the STAT1 protein in the immunohistochemical slices. The data collection module includes a staining intensity module and an optional calculation module. The color intensity module is used to determine the staining status and percentage of the cell cytoplasm in the immunohistochemically analyzed section, and to obtain a score for the color intensity. The calculation module is used to calculate the immunohistochemistry score of the immunohistochemical slice based on the score of the color development intensity obtained by the color development intensity module.

14. A method for predicting the efficacy of combined immunotherapy and chemotherapy, characterized in that, The steps of the prediction method are performed by a computer, and the prediction method is used to predict the effects of immunotherapy combined with chemotherapy on cancer patients. The prediction method includes: Based on the expression levels of biomarkers in the biological samples of the cancer patients, a risk prediction result for the combined immunotherapy and chemotherapy of the cancer patients is generated; the biomarkers include the STAT1 protein.

15. A computer device comprising a memory, a processor, and a computer program stored in the memory, characterized in that, The processor executes the computer program to implement the steps of the electronic device of any one of claims 12 to 13 or the prediction method of claim 14.

16. 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 steps of the electronic device according to any one of claims 12 to 13 or the prediction method according to claim 14.

17. A computer program product, comprising a computer program, characterized in that, When the computer program is executed by a processor, it implements the steps of the electronic device according to any one of claims 12 to 13 or the prediction method according to claim 14.

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