Application of biomarker in evaluation of cervical cancer immunotherapy curative effect and / or prognosis

Abstract

The invention relates to the technical field of tumor immunotherapy evaluation, in particular to application of a biomarker in evaluation of cervical cancer immunotherapy curative effect and / or prognosis. It is found for the first time that the nPD-L1 + TAMs expression level of cervical cancer tumor hypoxia marginal (HM) is closely related to immunotherapy response and patient prognosis, and the highly expressed nPD-L1 + TAMs is not only significantly related to lymph node metastasis, advanced FIGO staging and other malignant clinical characteristics, but also can be used as a predictive index of immunotherapy resistance and poor prognosis. In addition, through combined detection of the expression levels of nPD-L1 + TAMs and CD163 + TAMs in HM, the accuracy of immune curative effect prediction can be remarkably improved. The invention provides a new biomarker and a new method for cervical cancer immunotherapy curative effect evaluation and prognosis judgment, has the advantages of real-time performance, specificity and high accuracy, and has important significance for optimizing cervical cancer individualized treatment strategies.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor immunotherapy evaluation, and particularly relates to application of a biomarker in evaluation of curative effect and / or prognosis of cervical cancer immunotherapy. BACKGROUND

[0002] Cervical cancer is the fourth most common malignant tumor in women worldwide, with more than 600,000 new cases and about 340,000 deaths each year. The 5-year survival rate of patients with advanced and recurrent metastatic cervical cancer is less than 20%. In recent years, the immune checkpoint inhibitor represented by PD-1 / PD-L1 has brought a breakthrough in the treatment of advanced cervical cancer. Drugs such as Pembrolizumab and Atezolizumab have been proven to prolong the survival of some patients. However, clinical data show that only about 15%-20% of patients can benefit from such treatment, and primary or acquired drug resistance is still the main bottleneck restricting the curative effect. How to accurately screen potential benefit population, predict treatment response and prognosis has become a key problem to be solved.

[0003] PD-L1, as a ligand of PD-1, inhibits anti-tumor immune response by binding to PD-1 on the surface of T cells, and its expression level is considered an important predictor of immunotherapy response. However, existing studies have shown that the expression of PD-L1 has significant spatial heterogeneity and subcellular heterogeneity: in tumor tissues, PD-L1 is not only expressed in tumor cells, but also widely exists in immune cells (such as macrophages, dendritic cells) in the tumor microenvironment (TME); and in subcellular localization, in addition to the classic cell membrane expression, PD-L1 can also be localized in the cytoplasm and nucleus (i.e. nPD-L1), and PD-L1 with different localization may have completely different biological functions. For example, nuclear PD-L1 has been confirmed to promote tumor progression through non-immune mechanisms (such as regulating cell cycle, DNA damage repair), but its role in immune cells and clinical significance have not been clearly defined.

[0004] Tumor-associated macrophages (TAMs) are the most abundant immune cell population in TME, which have high plasticity and can be polarized into pro-tumor phenotype (such as CD163 + M2 type) under the regulation of microenvironment signals such as hypoxia and cytokines, and inhibit T cell function by secreting IL-10, TGF-β and other cytokines, promote angiogenesis and tumor metastasis. Studies have shown that the infiltration density of TAMs is closely related to the malignant degree and poor prognosis of cervical cancer, but the correlation between TAMs and PD-L1 expression, especially the clinical value of nuclear PD-L1 in TAMs, has not been systematically elucidated.

[0005] Hypoxia is one of the core features of solid tumors, caused by the contradiction between rapid tumor proliferation and insufficient blood oxygen supply. In cervical cancer, the hypoxic region can be further divided into hypoxic center (HC) and hypoxic margin (HM): HC is the area with extremely low oxygen partial pressure (<1%) inside the tumor, accompanied by severe metabolic stress and necrosis; HM is located at the junction of hypoxic tumor and normoxic tissue, with relatively high oxygen partial pressure (1%-5%), and is rich in blood vessels and immune cells, which is the 'front line position' of tumor invasion and metastasis. Previous studies have shown that the immune microenvironment status (such as immune cell infiltration and cytokine distribution) of HM has a significantly greater impact on tumor progression and treatment response than HC, but the research on the expression of PD-L1 in immune cells in HM is still blank.

[0006] The existing PD-L1 detection methods (such as immunohistochemistry) are mostly based on the overall expression level of tumor cells, without considering the distribution and subcellular localization differences in TME immune cells, and ignoring the spatial heterogeneity of hypoxic regions, which limits the prediction accuracy. Therefore, it is necessary to explore nPD-L1 + The expression characteristics of TAMs and their association with immunotherapy response and patient prognosis are expected to provide new biomarkers and theoretical basis for the precise evaluation of cervical cancer immunotherapy. SUMMARY

[0007] The purpose of the present application is to overcome the deficiencies in the prior art and provide a method for detecting nPD-L1 + TAMs, or a combination of CD163 + The application of the substance for detecting TAMs density in the preparation of a product for evaluating the efficacy and / or prognosis of cervical cancer immunotherapy.

[0008] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the present application provides an application of a biomarker in the preparation of a product for evaluating the efficacy and / or prognosis of cervical cancer immunotherapy, wherein the biomarker is a nuclear PD-L1 positive tumor-associated macrophage in the hypoxic margin of cervical cancer tissue, i.e. nPD-L1 + TAMs.

[0009] The existing technology focuses on the expression of PD-L1 on the surface of tumor cells, ignoring the subcellular localization heterogeneity (such as nuclear localization nPD-L1) and spatial distribution heterogeneity (such as differences in different hypoxic regions) of PD-L1. The present application first found that the nPD-L1 + TAMs expression level in the hypoxic margin (HM) of cervical cancer tumor is closely related to the immune therapy response and patient prognosis, and the high expression of nPD-L1 +Tumor hypoxic margin markers (TAMs) are not only significantly associated with malignant clinical features such as lymph node metastasis and advanced FIGO stage, but can also serve as predictive indicators of immunotherapy resistance and poor prognosis. This invention detects nPD-L1 at the tumor hypoxic margin. + The expression level of TAMs provides a new biomarker and method for evaluating the efficacy and prognosis of cervical cancer immunotherapy, offering advantages such as real-time detection, specificity, and high accuracy. This is of great significance for optimizing individualized treatment strategies for cervical cancer. This invention detects nPD-L1 in HM (hypermocytes and stromal cells). + The density of TAMs enables precise assessment of immunotherapy response and patient survival.

[0010] Preferably, the hypoxic edge is defined by staining with the hypoxia marker CAIX, which is a hypoxic area around the tumor with high CAIX expression.

[0011] Current PD-L1 detection methods are mostly based on whole tumor tissue and do not distinguish functional differences in hypoxic regions. This invention uses CAIX staining to define the hypoxic center (HC) and hypoxic margin (HM). This study found that the HM, as a key region for tumor invasion and immune interaction, contains significant nPD-L1 levels. + The expression of tumor-associated antigens (TAAs) better reflects the core mechanisms of immunotherapy resistance, and the spatial specificity of detection is significantly improved. This strategy can guide precise sampling for pathological testing (such as prioritizing the analysis of hepatic muscular masses), reduce detection errors caused by tumor heterogeneity, and improve the reliability of results.

[0012] Preferably, the biomarker further includes CD163 at the hypoxic margin of cervical cancer tissue. + TAMs.

[0013] During the investigation, this invention discovered that joint detection of nPD-L1 in HM... + TAMs and CD163 + The predictive accuracy of TAMs (AUC = 0.9551) was significantly higher than that of single biomarkers, which greatly improved the predictive accuracy of immunotherapy response rate. It can effectively screen out patients who are most likely to benefit from PD-1 / PD-L1 inhibitor treatment, reducing ineffective treatment and waste of medical resources.

[0014] Preferably, by detecting the hypoxic margin of cervical cancer tissue nPD-L1 + TAMs and / or CD163 + Density assessment of TAMs.

[0015] Preferably, the detection technique used includes immunohistochemistry or multiplex immunofluorescence.

[0016] The detection methods used in this invention (immunohistochemistry and multiplex immunofluorescence) are all routine clinical pathological techniques that do not require special equipment or complex operations. They can be directly carried out by hospital pathology departments and have a technical basis for widespread application.

[0017] Preferably, the nPD-L1 + TAMs density ≥ critical value, and CD163 + If the density of TAMs is greater than or equal to a critical value, the patient is considered to have a low response rate to immunotherapy; conversely, if the density is less than or equal to a critical value, the patient is considered to have a high response rate. The critical value is determined using ROC curves. (nPD-L1) + The critical value for TAMs is 51 per mm. 2 CD163 + The critical value for TAMs is 10⁸ per mm².

[0018] This invention is based on a quantization standard using the "hotspot method" (counting per mm). 2 The positive cell count method is simple and easy to implement, and the consistency of results can be ensured through double-blind slide reading, making it suitable for large-scale clinical applications.

[0019] Preferably, the nPD-L1 + TAMs and / or CD163 + The higher the density of TAMs, the lower the overall survival rate of cervical cancer patients.

[0020] Survival analysis showed that nPD-L1 in HM + TAMs density is an independent prognostic factor affecting overall survival (OS) of patients. Its predictive value is superior to traditional clinicopathological indicators (such as tumor size and stage), which helps to identify high-risk patients earlier and develop individualized follow-up strategies.

[0021] Secondly, this invention provides a product for evaluating the efficacy and / or prognosis of immunotherapy for cervical cancer, including the detection of nPD-L1 at the hypoxic margin of cervical cancer tissue. + Reagents for TAM density, CAIX test reagents for defining the hypoxic margin of tumors, and specific antibodies.

[0022] Preferably, the product further includes CD163 for detecting hypoxic margins in cervical cancer tissue. + Reagents for TAMs density.

[0023] Preferably, the product includes detection reagents, detection kits, chips, membrane strips, or detection platforms.

[0024] The beneficial effects of this invention are as follows: 1. This invention overcomes the limitations of traditional PD-L1 detection and reveals the value of new biomarkers: This invention is the first to clearly define nPD-L1 in the hypoxic margin of tumors (HM).+ As independent biomarkers, TAMs are directly associated with the malignant phenotype of cervical cancer, resistance to immunotherapy, and poor prognosis, making up for the shortcomings of traditional biomarkers in reflecting the complexity of the tumor microenvironment.

[0025] This invention, for the first time, demonstrates the clinical significance of intranuclear PD-L1 in TAMs, a core cell population in the immune microenvironment, breaking through the limitations of previous research that only focused on PD-L1 in tumor cells and providing a new perspective for understanding immune escape mechanisms.

[0026] 2. This invention enables precise assessment of the efficacy and prognosis of immunotherapy: This invention discovers nPD-L1 in HM + High expression of tumor markers (TAMs) is significantly associated with clinical features such as lymph node metastasis, advanced FIGO stage, and poor differentiation, directly reflecting the malignant potential of tumors and providing quantitative indicators for clinical risk stratification. Survival analysis showed that nPD-L1 in HMs... + TAMs density is an independent prognostic factor affecting overall survival (OS) of patients. Its predictive value is superior to traditional clinicopathological indicators (such as tumor size and stage), which helps to identify high-risk patients earlier and develop individualized follow-up strategies.

[0027] Joint detection of nPD-L1 in HM + TAMs and CD163 + The predictive accuracy of TAMs (AUC = 0.9551) was significantly higher than that of single biomarkers, which greatly improved the predictive accuracy of immunotherapy response rate. It can effectively screen out patients who are most likely to benefit from PD-1 / PD-L1 inhibitor treatment, reducing ineffective treatment and waste of medical resources.

[0028] 3. This invention combines tumor microenvironment characteristics to optimize the spatial specificity of the detection strategy: This invention uses CAIX staining to define the hypoxia center (HC) and hypoxia margin (HM). This study found that the HM, as a key region for tumor invasion and immune interaction, contains nPD-L1... + The expression of tumor-associated antigens (TAAs) better reflects the core mechanisms of immunotherapy resistance, and the spatial specificity of detection is significantly improved. This strategy can guide precise sampling for pathological testing (such as prioritizing the analysis of hepatic muscular masses), reduce detection errors caused by tumor heterogeneity, and improve the reliability of results.

[0029] 4. This invention has high technical feasibility and is easy to translate into clinical applications: The detection methods used in this invention (immunohistochemistry and multiplex immunofluorescence) are all routine clinical pathological techniques that do not require special equipment or complex operations. They can be directly carried out by hospital pathology departments and have a technical basis for widespread application.

[0030] 5. This invention provides a theoretical basis for the development of new therapeutic targets: This invention discloses nPD-L1 in HM + The key role of TAMs in immunosuppression suggests that targeting and eliminating these cells or inhibiting their nuclear PD-L1 function may reverse immune resistance, providing a new direction for designing combination therapy strategies (such as TAM depletion agents combined with PD-1 inhibitors). Attached Figure Description

[0031] Figure 1 To define hypoxic regions (hypoxic center HC / hypoxic margin HM) in cervical cancer tissue and CD163 + Distribution and quantification results of TAMs, PD-L1, and nuclear PD-L1 (nPD-L1) (immunohistochemical staining); Figure A shows the representative immunohistochemical staining results of the hypoxia marker CAIX in cervical cancer tissue, dividing the hypoxic region into the tumor hypoxic center (HC) and the tumor hypoxic margin (HM) based on CAIX expression; Figure B shows the representative immunohistochemical staining results of the tumor-associated macrophage marker CD163 in cervical cancer tissue; Figure C shows the representative immunohistochemical staining results of PD-L1 in cervical cancer tissue; Figure D shows the CD163 in HC and HM of 103 cervical cancer patients. + Quantitative analysis results of the number of TAMs; Figure E shows the quantitative analysis results of PD-L1 expression levels in HC and HM in 103 cervical cancer patients; Figure F shows the quantitative analysis results of nPD-L1 levels in HC and HM in 103 cervical cancer patients.

[0032] Figure 2 This image shows the fluorescence colocalization of CAIX, CD163, and PD-L1 in hypoxic regions of cervical cancer tissue and the comparison results of immunotherapy-sensitive / resistant samples (multiplex immunofluorescence staining). Image A shows representative immunofluorescence staining results of CAIX (hypoxia marker, red), CD163 (TAMs marker, purple), PD-L1 (green), and DAPI (nuclear, blue) in cervical cancer tissue, comparing immunotherapy-sensitive (top) and immunotherapy-resistant (bottom) samples. Image B shows the quantitative analysis results of the expression levels of CAIX, CD163, PD-L1, and nuclear PD-L1 (nPD-L1) in immunotherapy-sensitive and immunotherapy-resistant tissues. Data are expressed as mean ± standard error (mean ± SEM), *P < 0.05 (using Student's...). (t-test); Figure C is a high-power fluorescent staining image of the hypoxic subregion (hypoxic center HC, hypoxic edge HM), showing the distribution of CAIX (red), CD163 (purple), PD-L1 (green) and DAPI (blue), with a scale bar of 50 μm.

[0033] Figure 3 PD-L1 in the hypoxia center (HC) and hypoxia margin (HM) of cervical cancer patients + TAMs, PD-L1 + The correlation analysis diagram between TAMs density and various clinicopathological indicators; among them, the AE diagram shows the nuclear PD-L1 in the hypoxia center (HC) and hypoxia margin (HM) after stratification according to different clinicopathological variables. + Quantitative analysis results of TAM density: Figure A is stratified by age (<45 years vs. ≥45 years), Figure B is stratified by lymph node metastasis (negative vs. positive), Figure C is stratified by FIGO staging (stages I-IIA vs. IIB-IV), Figure D is stratified by histological grade (high / moderate differentiation vs. low differentiation), and Figure E is stratified by tumor size (≤4cm vs. >4cm); Figure FI shows the PD-L1 concentration in the hypoxic center (HC) and hypoxic margin (HM) after stratification according to different clinicopathological variables. + Quantitative analysis results of TAM density: Figure F was stratified by age (<45 years vs. ≥45 years), Figure G was stratified by lymph node metastasis (negative vs. positive), Figure H was stratified by FIGO staging (stages I-IIA vs. IIB-IV), and Figure I was stratified by histological grade (high / moderate differentiation vs. low differentiation); all data are expressed as mean ± standard error (mean ± SEM), *P<0.05, **P<0.001 (Student's t-test was used).

[0034] Figure 4 To determine the nuclear PD-L1 in hypoxic regions + TAMs, PD-L1 + Kaplan-Meier survival analysis of overall survival (OS) in cervical cancer patients stratified by TAM density (high and low); Figure A shows the overall survival based on PD-L1 in the hypoxic center (HC). + Kaplan-Meier curves of overall survival (OS) for patients stratified by TAM density (low L expression vs. high H expression); Figure B shows the overall survival (OS) based on PD-L1 in hypoxia centers (HC). + Kaplan-Meier curves of overall survival (OS) for patients stratified by TAM density (low L expression vs. high H expression); Figure C shows the PD-L1 nucleus in the hypoxia margin (HM). + Kaplan-Meier curves of overall survival (OS) for patients stratified by TAM density (low L expression vs. high H expression); Figure D shows the overall survival (OS) of patients based on PD-L1 in the hypoxia margin (HM). +Kaplan-Meier curves of overall survival (OS) for patients stratified by TAM density (low L expression vs. high H expression); log-rank test was used to determine statistical significance, and the corresponding P-values ​​are marked in each figure.

[0035] Figure 5 The hypoxic subregion contains nuclear PD-L1 and CD163. + Receiver operating characteristic (ROC) analysis of TAMs alone and in combination for predicting immunotherapy response in cervical cancer patients; where plots A and C show the ROC curves of each indicator in the hypoxia center (HC) for predicting immunotherapy response: plot A shows the ROC curve of nuclear PD-L1 alone, and plot B shows the ROC curve of CD163. + ROC curves for TAMs detected individually; Figure C shows the nuclear PD-L1 and CD163. + ROC curves for combined TAMs detection; DF plot shows ROC curves for each indicator predicting immunotherapy response in the hypoxia margin (HM); D plot shows ROC curves for nuclear PD-L1 detection alone; E plot shows ROC curves for CD163. + ROC curves for TAMs detected individually; Figure F shows the nuclear PD-L1 and CD163. + ROC curves for combined TAMs detection; the corresponding area under the curve (AUC) value and statistical significance (P value) are marked in each figure. Detailed Implementation

[0036] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. The specific embodiments described herein provide a more detailed explanation of the purpose, technical solution, and beneficial effects of this invention. It should be understood that the above descriptions are merely specific embodiments of this invention and are not intended to limit the scope of protection of this invention. In particular, it should be noted that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention for those skilled in the art.

[0037] Note: nPD-L1 + TAMs: Nuclear PD-L1 positive tumor-associated macrophages.

[0038] Example 1: Spatial distribution of hypoxic regions in cervical cancer tissue and nPD-L1 + Locational characteristics of TAMs: 1. Clinical specimen collection Tissue specimens (formalin-fixed and paraffin-embedded (FFPE)) from 103 cervical cancer patients preserved at Guangdong Provincial People's Hospital were collected. All patients underwent radical hysterectomy. Clinical data included age, tumor size, FIGO stage, and lymph node metastasis status (see Table 1 for details).

[0039] 2. Delineation of hypoxic areas IHC was used to detect the hypoxia marker CAIX (antibody: ab184006, Abcam), and the cells were stratified according to staining intensity. Hypoxia center (HC): A tumor interior region that is strongly positive for CAIX and uniformly distributed; Hypoxic margin (HM): A tumor peripheral region with heterogeneous CAIX expression and adjacent to normal tissue.

[0040] 3. Immunostaining and Quantification IHC assay: Anti-CD163 (ab156769, Abcam) and anti-PD-L1 (ab205921, Abcam) antibodies were used, DAB staining was performed, and hematoxylin was used for counterstaining. Multiplex immunofluorescence: The Opal 4-Color Kit was used, with CAIX, CD163, and PD-L1 fluorescently labeled, and DAPI staining of the nuclei. Quantification method: Two pathologists performed a double-blind count, using the "hotspot method" to calculate nPD-L1 per mm² at ×400x magnification. + TAMs and CD163 + The number of TAMs was used to divide them into high / low expression groups based on the median value (see Table 1 for details).

[0041] 4. Main reagents for the experiment: Primary antibody working solutions: CAIX, model ab184006, brand Abcam, 1:1000; CD163, model ab156769, brand Abcam, 1:1000; PD-L1, model ab205921, brand Abcam, 1:1000; purchased from Guangzhou Xinjin Biotechnology Co., Ltd.

[0042] Secondary antibody working solution: Immunohistochemistry-specific secondary antibody, purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0043] DAB colorimetric reagent kit: purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0044] 5. Experimental steps: Immunohistochemistry was used to detect the protein expression levels of CAIX, CD163, and PD-L1 genes in serial sections of different clinical samples. The results are as follows: Figure 1 As shown.

[0045] The specific steps are as follows: (a) Routine dewaxing and dehydration: Paraffin sections of clinical samples were placed in a 65°C oven for 2 hours to dissolve the paraffin, then placed in xylene solutions (I, II, III) for 15 minutes each, and then placed in alcohol solutions of varying concentrations (100%, 95%, 95%, 90%, 80%, 70%) for 5 minutes each. Finally, they were washed with PBS for 5 minutes each time for 3 times. (b) Antigen retrieval: Place the slides treated in step (a) into an experimental pressure cooker, add antigen retrieval solution (1× sodium citrate buffer at pH=6.0) until the liquid covers the slides, pressurize the pressure cooker on high heat, then reduce to low heat and continue retrieval for 5 minutes, then allow to cool naturally at room temperature, wash with PBS, 5 minutes / time × 3 times. (c) Blocking endogenous peroxidase with H2O2: Immerse the slices treated in step (b) in 3% H2O2 working solution, incubate at 37°C for 15 minutes, wash with PBS, 5 minutes / time × 3 times; (d) Primary antibody incubation: Place the tissue sections processed in step (c) on a glass slide, circle the tissue specimen area on the slide with an immunohistochemistry pen, add the prepared primary antibody working solution to the tissue area of ​​each slide to cover the tissue block, incubate at 4°C overnight, warm up the next day, wash with PBS, 5 minutes / time × 3 times.

[0046] (e) Secondary antibody incubation: Add the corresponding secondary antibody working solution to each slide, incubate at 37°C for 1 hour, then discard the secondary antibody, wash with PBS, 5 minutes / time × 3 times; (f) DAB staining: According to the DAB staining kit, 20×DAB is prepared into 1×DAB. 1×DAB is added to the tissue area of ​​each section. The staining is observed under a microscope. After staining, the section is placed in 1×PBS to stop staining and avoid over-staining. (g) Nuclear staining: stain with hematoxylin for 10 minutes, rinse with tap water for 10 minutes after staining, differentiate with hydrochloric acid alcohol for 2-3 seconds, and rinse with tap water again for 15 minutes. (h) Dehydration, clearing and mounting: The slides were placed in alcohol of varying concentrations (70%, 80%, 90%, 95%, 95%, 100%) for 5 minutes each for dehydration, and then placed in xylene solutions (I, II, III) for 10 minutes each. After drying, the slides were mounted with neutral resin and observed and photographed under a microscope. (i) Evaluation of staining results: Immunohistochemical results were independently evaluated using a double-blind method. Five fields of view were randomly selected under an upright microscope. Based on the color intensity of the staining reaction, all cells in the same field of view were classified into four staining intensity levels: negative, weak, medium, and strong. The CAIX H score scoring system was used: H score = (percentage of cells with weak staining intensity level × 1) + (percentage of cells with medium staining intensity level × 2) + (percentage of cells with strong staining intensity level × 3). The median H score was selected to distinguish the expression levels of the proteins to be detected. CD163 and PD-L1 were statistically analyzed using a positive cell count method.

[0047] 6. Experimental Results: Experimental results showed that CAIX staining, a hypoxia marker, could clearly define two functional regions in cervical cancer tissue. Figure 1 A): Hypoxia centers (HC): CAIX are strongly positive and uniformly distributed, located inside the tumor, with extremely low oxygen partial pressure (<1%), accompanied by severe metabolic stress; Hypoxia margin (HM): CAIX staining shows significant heterogeneity. It is adjacent to normoxic tissues and blood vessels, with relatively high oxygen partial pressure (1%-5%), and is a key area for tumor invasion and immune cell interaction.

[0048] In cervical cancer tissue specimens, CD163 exhibits granular, brownish-red immunoreactivity and is clearly localized at the cell membrane. Figure 1 B); while PD-L1 staining presents as granular brown signals in cancer cells and macrophages (B); Figure 1 C). In both the hypoxia center (HC) and hypoxia margin (HM) regions, PD-L1 expression was correlated with CD163. + Significant spatial correlations exist among TAMs. Notably, CD163 is found near hypoxic regions. + Nuclear localization of PD-L1 exists in TAMs, and this nuclear PD-L1 (nPD-L1) phenotype is particularly pronounced in the hypoxia border (HM) region.

[0049] CD163 in hypoxia center (HC) and hypoxia margin (HM) regions + Quantitative analysis of TAMs, PD-L1, and nPD-L1 revealed significant region-specific differences in cell count and expression levels. Specifically, CD163... + The absolute counts of TAMs and the expression levels of PD-L1 and nPD-L1 were significantly higher in the hypoxia border (HM) region than in the hypoxia center (HC) region. Figure 1 DF; all P < 0.05, Student's t test).

[0050] Example 2: Co-localization characteristics of PD-L1 and CD163 in hypoxic regions of cervical cancer and differences in immunotherapy response. 1. Experimental subjects: Cervical cancer tissues from 12 patients treated with PD-1 inhibitors at Guangdong Provincial People's Hospital were selected. Among them, 8 cases showed treatment resistance (meaning that the tumor did not respond to PD-1 inhibitors or the response was short-lived, and the disease continued to progress), and 4 cases showed treatment sensitivity (meaning that the tumor responded significantly to PD-1 inhibitor treatment, manifested as tumor shrinkage, delayed disease progression, and prolonged survival).

[0051] 2. Main reagents for the experiment: Primary antibody working solutions: CAIX, model ab184006, brand Abcam, 1:1000; CD163, model ab156769, brand Abcam, 1:1000; PD-L1, model ab205921, brand Abcam, 1:1000; purchased from Guangzhou Xinjin Biotechnology Co., Ltd.

[0052] Secondary antibody working solution: Immunofluorescence-specific secondary antibody, purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0053] Multiplex immunofluorescence reagent kit: purchased from Guangzhou Weijia Biotechnology Co., Ltd.

[0054] 3. Experimental steps: Immunohistochemistry was used to detect the protein expression levels of CAIX, CD163, and PD-L1 genes in different clinical samples through serial sectioning.

[0055] The specific steps are as follows: (a) Baking: Routine dewaxing and dehydration: Paraffin sections of clinical samples were placed in a 65°C oven for 2 hours to dissolve the paraffin.

[0056] (b) Dewaxing: 15 min each of xylene I and II, 5 min of anhydrous ethanol, 5 min of 95% ethanol, 5 min of 70% ethanol, 30 s of sterile water rinsing of the slides, and 20 min of 10% neutral formalin immersion.

[0057] (c) Microwave antigen retrieval: Immerse the slide in 30 mL of AR6 (10×, diluted with 50× EDTA pH 8.0, and EDTA diluted with ddH2O) (the liquid level is above the tissue). Place the container (must be plastic, glass will break) in a microwave oven, turn to medium-high power, heat to boiling, then turn to low power and heat for 15 minutes (if the liquid evaporates severely, the time can be reduced appropriately).

[0058] (d) Allow to cool naturally at room temperature (about 30 minutes) or overnight. Rinse with sterile water for 30 seconds. Soak the tablets in 1×TBST (20×TBS diluted with ddH2O + 0.1% Tween 20) for 2 minutes.

[0059] (e) Blocking: Wipe the fluid around the tissue dry, circle the tissue with an immunohistochemical pen, add 2 drops of blocking solution (DAKO), shake at room temperature (about 50 rpm), and incubate in a humidified chamber for 10 min.

[0060] (f) Incubation with primary antibody: Wipe the liquid around the tissue dry, add primary antibody, shake at room temperature (about 50 rpm), incubate in a humidified chamber for 1 h, soak the slide with 1×TBST, shake at room temperature (about 120 rpm) for 3 min × 2 times.

[0061] (g) Incubation with HRP secondary antibody: Wipe the liquid around the tissue dry, add secondary antibody (depending on the species of the primary antibody), shake at room temperature (about 50 rpm), incubate in a humidified chamber for 10 min, soak the slide with 1×TBST, shake at room temperature (about 120 rpm) for 3 min × 2 times.

[0062] (h) Incubation with TSA (signal amplification, fluorescence): Wipe the liquid around the tissue dry, add TSA (red, green, purple, 1:50 dilution), shake at room temperature (about 50 rpm), incubate in a humidified chamber for 10 min, soak the slide with 1×TBST, shake at room temperature (about 120 rpm) for 10 min.

[0063] (i) Microwave treatment: Remove primary and secondary antibodies, following the same steps as (c). The slide can then be observed under a microscope (to prevent drying). (J) Restaining: Repeat steps (d)-(g) (k) Incubation with fluorescent secondary antibody: Wipe the liquid around the tissue dry, add the secondary antibody (depending on the species of the primary antibody), shake at room temperature (about 50 rpm), incubate in a humidified chamber for 10 min, soak the slide with 1×TBST, shake at room temperature (about 120 rpm) for 3 min × 2 times.

[0064] (l) DAPI staining: Wipe the liquid around the tissue dry, add DAPI (1:500 dilution), shake at room temperature (about 50 rpm), incubate in a humidified chamber for 10 min, soak the slide in 1×TBST, shake at room temperature (about 120 rpm) for 10 min.

[0065] (m) Mounting: Wipe the fluid around the tissue dry, add 30 μL of anti-fluorescence attenuation agent (DAKO), add 4 drops of nail polish around the tissue for fixation, and cover with a coverslip (first apply a small amount of nail polish to the four corners, let one side soak in the liquid, and slowly cover it). (n) Take a picture under an upright fluorescence microscope.

[0066] 4. Experimental Results: To further clarify the role of hypoxia and CD163 in the response to immunotherapy. + Spatial interactions between TAMs and PD-L1 expression were investigated in this study using multiplex immunofluorescence staining of CAIX, CD163, and PD-L1 in cervical cancer tissues from immunotherapy-resistant and immunotherapy-sensitive patients. Results showed that in immunotherapy-resistant cervical cancer tissues, strong co-expression of CD163 and PD-L1 was detected in hypoxic regions (marked by abundant CAIX expression); conversely, in immunotherapy-sensitive tissues, the expression levels of CD163 and PD-L1 were extremely low, and CAIX expression was minimal, suggesting a normoxic environment. Notably, CD163 was observed in the stromal microenvironment surrounding hypoxic cancer cell nests. + TAMs and PD-L1 exhibit spatial co-location phenomenon. Figure 2 A).

[0067] Quantitative analysis confirmed that, compared with immunotherapy-sensitive tissues, immunotherapy-resistant tissues showed significantly higher expression levels of CAIX, CD163, PD-L1, and nuclear PD-L1 (nPD-L1). Figure 2 B; all P < 0.05).

[0068] High-powered observation of the hypoxia center (HC) and hypoxia margin (HM) revealed that CD163 + TAMs and nPD-L1 primarily infiltrate the stroma surrounding the cancer nests in HM, while their distribution is less pronounced within HC (near the tumor necrosis core). Figure 2 C) suggests that the HM is a key region for the aggregation of immunosuppressive cells and the mediation of treatment resistance.

[0069] Example 3: Sample set: Clinical sample data from 103 patients collected in Example 1, to evaluate nPD-L1 in HC and HM. + TAMs and PD-L1 + The relationship between TAMs and clinicopathological features.

[0070] The results are as follows Figure 3 As shown in Table 1.

[0071] Table 1: Relationship between nPD-L1 and PD-L1 expression and clinicopathological parameters in hypoxic regions of HC and HM in cervical cancer tissues. Note: *P<0.05.

[0072] Quantitative analysis results showed that nPD-L1 in the hypoxia center (HC) region +TAMs density (trending) and hypoxia border (HM) region nPD-L1 + A statistically significant increase in TAM density is closely associated with aggressive clinicopathological features, including lymph node metastasis. Figure 3 B) Late FIGO stages (IIB-IV) Figure 3 C) Low degree of histological differentiation Figure 3 D) and larger tumor size (>4cm) Figure 3 E). Notably, the correlation with these malignant phenotypes was most pronounced in the hypoxia border (HM) region. Conversely, nPD-L1 + TAMs density was not significantly associated with patient age (P>0.05) Figure 3 A). For PD-L1 + TAMs, in lymph node metastasis positive ( Figure 3 G) and late FIGO stages (IIB-IV) Figure 3 In patients with hypoxia (H), PD-L1 in the hypoxia center (HC) and hypoxia margin (HM) regions + TAM counts all increased. However, PD-L1 + No significant correlation was detected between TAM density and age, tumor size, or histological grade (all P>0.05). Figure 4 F, I). These results indicate that, compared with PD-L1 + Compared to TAMs, nPD-L1 in the hypoxia border (HM) region + TAMs density is more strongly associated with the malignant phenotype of cervical cancer.

[0073] Example 4: Survival Analysis To evaluate nPD-L1 + TAMs and PD-L1 + The prognostic significance of TAMs: This study used the median as the cutoff value to divide 32 cervical cancer patients with follow-up data (follow-up time of 600 days) who received PD-1 inhibitor treatment at our hospital into a "low-density" group and a "high-density" group (i.e., density below the median was defined as "low-density," and density above the median was defined as "high-density"). Retrospective survival analysis showed that nPD-L1 in hypoxic marginal zone (HM) regions... + The overall survival (OS) of patients in the high-density TAM group was significantly shorter than that of patients in the low-density group. Figure 4 C; P = 0.0019), and this subgroup of patients had a higher frequency of tumor recurrence. A similar trend was also observed in the hypoxic center (HC) region: nPD-L1 + Patients in the high-density TAMs group had poorer overall survival (OS). Figure 4 A; P = 0.0217). For PD-L1+ TAMs, particularly high-density groups in the hypoxia border (HM) region, were also associated with shortened overall survival (OS). Figure 4 D; P = 0.0298), but its impact is not as great as that of nPD-L1. + TAMs were significant. Conversely, PD-L1 was significantly higher in hypoxia centers (HC). + TAMs density was not significantly correlated with overall survival (OS). Figure 5 B; P = 0.3398). These results indicate that nPD-L1, especially in the hypoxia margin (HM) region, + TAMs can serve as reliable prognostic biomarkers for predicting tumor recurrence and survival outcomes in cervical cancer patients.

[0074] Example 5: Given nPD-L1 + TAMs are closely associated with poor prognosis. This study further explored their potential to predict immunotherapy response by constructing a combined diagnostic model using logistic regression. The model uses immunotherapy response as the dependent variable and nPD-L1 and CD163 as the predictors. + With the density of TAMs as the independent variable, the regression equation is as follows: Hypoxia center (HC) region: Logit(P) = -13.621 + 0.061 × nPD-L1 + 0.064 × CD163 + TAMs; Hypoxia borderline (HM) region: Logit(P) = -53.624 + 0.274 × nPD-L1 + 0.225 × CD163 + TAMs.

[0075] Likelihood ratio tests confirmed that both models were statistically significant (hypoxia center HC: χ2 = 10.754, P = 0.005; hypoxia border HM: χ2 = 39.951, P < 0.001).

[0076] To compare predictive accuracy, this study analyzed receiver operating characteristic (ROC) curves and calculated the area under the curve (AUC). In the hypoxia center (HC) region, nPD-L1 and CD163... + The AUCs of TAMs and their combined detection were 0.7276 (P = 0.0110), 0.7041 (P = 0.0226), and 0.8419 (P = 0.0001), respectively. Figure 5 AC). Notably, in the hypoxia-border region (HM), the AUCs of all indicators were significantly higher: nPD-L1 had an AUC of 0.8974 (P<0.0001), CD163... +The AUC of TAMs was 0.8365 (P = 0.0002), and the AUC of the combined detection of the two was 0.9551 (P < 0.0001). ​ DF).

[0077] These results indicate that the combined detection of nPD-L1 and CD163 in the hypoxia margin (HM) region is effective. + TAMs achieve the highest AUC values ​​and are more accurate in predicting immunotherapy response than detecting any single biomarker.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The use of a biomarker in the preparation of products for evaluating the efficacy and / or prognosis of cervical cancer immunotherapy, characterized in that, The biomarker is nuclear PD-L1 positive tumor-associated macrophages at the hypoxic margin of cervical cancer tissue, i.e., nPD-L1. + TAMs.

2. The application as described in claim 1, characterized in that, The hypoxic margin is defined by CAIX staining, a hypoxic marker, and is a hypoxic area in the periphery of the tumor with CAIX staining heterogeneity.

3. The application as described in claim 1, characterized in that, The biomarkers also include CD163 at the hypoxic margin of cervical cancer tissue. + TAMs.

4. The application as described in claim 3, characterized in that, By detecting nPD-L1 at the hypoxic margin of cervical cancer tissue + TAMs and / or CD163 + Density assessment of TAMs.

5. The application as described in claim 4, characterized in that, The techniques used for the detection include immunohistochemistry or multiplex immunofluorescence.

6. The application as described in claim 4, characterized in that, The nPD-L1 + TAMs density ≥ critical value, and CD163 + If the density of TAMs is greater than or equal to a critical value, the patient is considered to have a low response rate to immunotherapy; conversely, if the density is less than or equal to a critical value, the patient is considered to have a high response rate. The critical value is determined using ROC curves. (nPD-L1) + The critical value for TAMs is 51 per mm. 2 CD163 + The critical value for TAMs is 10⁸ / mm. 2 .

7. The application as described in claim 4, characterized in that, The nPD-L1 + TAMs and / or CD163 + The higher the density of TAMs, the lower the overall survival rate of cervical cancer patients.

8. A product for evaluating the efficacy and / or prognosis of immunotherapy for cervical cancer, characterized in that, Including the detection of hypoxic margins in cervical cancer tissue using nPD-L1 + Reagents for TAM density, CAIX test reagents for defining the hypoxic margin of tumors, and specific antibodies.

9. The product for evaluating the efficacy and / or prognosis of cervical cancer immunotherapy as described in claim 8, characterized in that, The product also includes CD163 for detecting hypoxic margins in cervical cancer tissue. + Reagents for TAMs density.

10. The product for evaluating the efficacy and / or prognosis of cervical cancer immunotherapy as described in claim 8, characterized in that, The products include testing reagents, testing kits, chips, membrane strips, or testing platforms.

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