Use of tertiary lymphoid structures in preparation of renal cancer subtyping diagnosis or prognosis assessment

Abstract

The application relates to the technical field of medical biological detection, and provides application of a three-level lymphoid structure in preparation of a kidney cancer typing diagnosis or prognosis evaluation, specifically application in preparation of a kidney cancer typing diagnosis or prognosis evaluation kit and a corresponding kit and evaluation system; and application of a reagent for promoting formation of the three-level lymphoid structure or promoting formation of secondary follicles in the three-level lymphoid structure in preparation of a kidney cancer treatment drug and a kidney cancer treatment drug composition. Research results of the application show that intratumoral TLS has sensitive and independent prognostic efficiency for kidney cancer patients, and intratumoral TLS can significantly distinguish high-risk long-term recurrence of tumors. With TLS position heterogeneity, a precise prognosis evaluation system for kidney cancer patients can be established; a mature TLS structure can improve a tumor immunosuppressive microenvironment by activating effector T cells and reducing Treg cell recruitment. Therefore, the application provides a new precise pathological monitoring index for clinical treatment and prognosis evaluation of kidney cancer.

Description

Technical Field

[0001] This invention relates to the field of medical biological detection technology, and to new applications of tertiary lymphoid structures, particularly in the application of tertiary lymphoid structures in the preparation of renal cell carcinoma subtyping diagnosis or prognostic assessment. Background Technology

[0002] Tertiary lymphoid structures (TLSs) are structures within the tumor microenvironment (TME) formed by the aggregation of tumor-infiltrating lymphocytes (TILs) driven by chemokines, possessing anti-tumor immune functions. Previous studies have shown that TLSs are associated with favorable prognoses in patients with various solid tumors and can enhance the efficacy of immune checkpoint inhibitors (ICIs) in cancer patients. However, the role of TLSs in renal cell carcinoma (RCC) remains controversial, and the molecular mechanisms are unclear.

[0003] In 2020, there were approximately 19.3 million new cancer cases worldwide. Renal cell carcinoma (RCC) is the most common clinicopathological type of the urinary system. While treatment methods for RCC have improved significantly in recent years, the overall resection rate remains low, with high postoperative recurrence and mortality rates, resulting in a 5-year survival rate of only about 15% for RCC patients. Therefore, improving the survival time of RCC patients has become one of the most challenging issues. With the development of targeted and immunotherapies and the application of precision medicine in clinical practice, not only has the resectable rate of RCC increased, but the median survival of patients with advanced RCC has also been significantly prolonged. However, the low objective response rate and high resistance rate of RCC patients to ICIs and targeted drugs severely hinder the clinical application of neoadjuvant therapy.

[0004] The tumor microenvironment (TME), composed of tumor cells, tumor endothelial cells (TILs), and stromal cells, exhibits significant heterogeneity and is a major culprit in promoting tumor growth and developing immune tolerance, leading to immune escape by tumor cells. Therefore, TILs within the TME are considered the ultimate target for immunotherapy in patients with recurrent tumor cell carcinoma (RCC). TILs are primarily composed of CD3+. + T cells, CD8 + T cells, CD20 + Composed of B cells, plasma cells, dendritic cells, and macrophages, etc. Figure 1Germinal centers (TLSs) enhance immune function, thereby influencing the prognosis of cancer patients. TLSs can be categorized by maturity into lymphocyte aggregations, round lymphocyte clusters without germinal centers (primary follicles), and follicles with germinal centers. Germinal centers are sites of B-cell expansion, immunoglobulin transformation, somatic hypermutation, and affinity maturation; their presence can improve the prognosis of HCC patients. Studies have shown that TLSs are associated with good prognosis in patients with gastric cancer, colorectal cancer, pancreatic cancer, lung cancer, bladder cancer, breast cancer, head and neck squamous cell carcinoma, and melanoma, and that cancer patients with TLSs have significantly higher objective response rates to targeted therapies and ICIs than those without TLSs. However, in RCC studies, the relationship between TLSs and patient prognosis remains controversial, and their mechanism of action is still unclear.

[0005] Lymphoid tissue synthases (TLSs) are newly formed immune lymphoid structures that initiate immune responses and exert anti-tumor effects, formed by the aggregation of lymphocytes driven by chemokines. They commonly occur in diseases such as chronic infections, immune diseases, and tumors. The formation of TLSs is mainly induced by lymphoformation somatic cells (LTo) and lymphoid tissue inducer cells (LTi cells), such as Th17 cells, B cells, and M1 macrophages, within the tumor microenvironment. LTi cells are innate lymphoid cells, while LTo cells are mesenchymal cells that later differentiate into follicular dendritic cells (FDCs) and fibroblastic reticular cells (FRCs). Figure 2 ).

[0006] The antitumor mechanism of lymphocytes (TLSs) may involve the combined action of numerous components or the action of individual components, including T cell-mediated immunity, B cell humoral immunity, and dendritic cell antigen presentation capabilities. The formation of TLSs is inseparable from the induction of lymphocytes by chemokines CC motif ligand (CCL)2, -3, -4, -5, -8, -18, -19, -21 and CXC motif chemokine ligand (CXCL)9, -10, -11, -13. These 12 chemokines are essential in the formation of TLSs, each playing a crucial role: CCL19 and CCL21 regulate T cell activation and drive through CCR7, while CXCL13 promotes the formation and maturation of TLSs by recruiting and activating B cells. The antitumor immune capacity of TLSs is mainly related to B cells and T cells. T cells have been shown to be associated with a good clinical prognosis in solid tumors, but the role of B cells in the tumor microenvironment (TME) remains unclear. On the one hand, B cells can act on tumor cells by producing antibodies, acting as antigen-presenting cells to induce T cell-mediated immunity, promoting NK cell function, and enhancing the phagocytic capacity of macrophages. On the other hand, B cells can also promote tumor development by producing autoantibodies and expressing immunosuppressive ligands or cytokines, thereby suppressing antitumor immune responses. The mechanism of action of TLSs in renal cell carcinoma, their prognostic significance for renal cell carcinoma patients, and their role in renal cell carcinoma immunotherapy remain unsolved mysteries.

[0007] The body's anti-tumor immunity mainly consists of two parts: acquired immunity and innate immunity. In the body's acquired immune response, the immune system can form a cancer immunity cycle (CIC) that kills tumor cells. During this process, once tumor cells are killed and related antigens are released, the cycle restarts, further strengthening the immune system's recognition and killing of tumor cells. If any link in the CIC is disrupted, this cycle will be interrupted, thereby weakening the body's anti-tumor immune response and allowing the tumor to escape immunely. An ideal anti-tumor immunotherapy drug should act on all the above steps, unblocking all links to maximize its anti-tumor effect. Existing research shows that the tertiary lymphoid structure itself promotes several steps in the CIC, such as antigen release, attracting T cells to infiltrate the tumor, and enhancing T cell recognition of tumor cells, but it cannot completely cover all these steps.

[0008] Therefore, to fully understand all aspects of acquired immunity in patients with solid tumors and activate innate immunity, thereby maximizing the anti-tumor immune response, an increasing number of detection methods are being applied to detect tertiary lymphoid structures in tumor and adjacent normal tissues. Representative methods include hematoxylin-eosin (HE) staining, immunofluorescence staining, immunohistochemical staining, and immunohistochemical staining with multiple antibodies. Utilizing multiplex fluorescence immunohistochemistry platforms, researchers can comprehensively analyze the infiltration of cytotoxic T cells, exhausted T cells, macrophages, B cells, tertiary lymphoid structures, and immune checkpoint receptors in the tumor immune microenvironment. This allows for a comprehensive assessment of patient prognosis and the efficacy of immunosuppressant therapy, and uncovers the relationship and clinical molecular mechanisms between the tumor immune microenvironment and tumor development, recurrence, metastasis, and drug resistance. However, these detection methods have not yet formed unified standards for pathological identification and clinical application, thus possessing a solid theoretical foundation and significant potential for patent transfer to clinical application.

[0009] Renal cell carcinoma is one of the most common malignant tumors of the genitourinary system, accounting for approximately 5% of all new cases in adult men and 3% in women. Statistics show that in 2019, there were approximately 73,820 new cases of renal cell carcinoma and 14,770 deaths in the United States. In my country, approximately 66,800 new cases of renal cell carcinoma are diagnosed annually, ranking second in incidence among urinary system tumors. Clear cell renal cell carcinoma (ccRCC) is the most common and highly malignant pathological type of renal cell carcinoma, accounting for approximately 70-85% of all renal cell carcinoma cases. About 25-30% of ccRCC patients have metastases at initial diagnosis, and the 5-year survival rate for metastatic ccRCC is only 32%. For clinically localized tumors, treatment remains primarily nephron-sparing surgery or radical nephrectomy. Postoperative cytokine therapy or individualized precision adjuvant therapy can reduce tumor recurrence and metastasis rates and improve long-term survival rates. Currently, first-line treatment for advanced renal cell carcinoma primarily relies on tyrosine kinase inhibitors targeting the vascular endothelial growth factor receptor, such as pazopanib, sunitinib, sorafenib, axitinib, and cabozantinib. Although anti-angiogenic drugs can inhibit tumor proliferation to some extent and significantly prolong survival in low-risk ccRCC patients, they have significant side effects, resulting in poor overall efficacy. The objective response rate is less than 30%, and the median overall survival extension is less than 12 months. Furthermore, even patients who initially respond to treatment may experience disease progression after a period of time, at which point most will lack effective subsequent treatment options.

[0010] In recent years, novel immunotherapies, represented by PD-1 / PD-L1 and CTLA4 inhibitors, have rapidly emerged in the treatment of renal cell carcinoma, demonstrating encouraging efficacy in advanced and refractory patients. Since the FDA approved nivolumab for patients with advanced renal cell carcinoma who had previously received anti-angiogenic therapy in 2015 based on the Checkmate 025 study, and in 2020, ASCOGU announced the 5-year follow-up results of the Checkmate 025 study, showing a 5-year survival rate of up to 26% with nivolumab as second-line treatment, highlighting the survival advantage of immunotherapy, immune checkpoint inhibitors have gradually moved from second-line to first-line therapy. Currently, PD-1 monoclonal antibodies combined with CTLA-4 monoclonal antibodies and PD-1 / PD-L1 monoclonal antibodies combined with anti-angiogenic drugs have been successively approved by the FDA for first-line treatment of advanced renal cell carcinoma, ushering in a new chapter in the treatment of advanced renal cell carcinoma.

[0011] The unique tumor microenvironment of renal cell carcinoma (RCC) is one of the key reasons why its malignancy far exceeds that of other tumors, and it is also a major cause of the poor prognosis and insensitivity to radiotherapy, chemotherapy, and immunotherapy in RCC patients. Immune checkpoint inhibitors combined with TKIs exert their effects from multiple angles, including inducing the normalization of anti-tumor immunity, inhibiting major signaling pathways in the development and progression of advanced RCC, and the tumor microenvironment. Their success relies on a deep understanding of the interaction between tumor cells and the tumor microenvironment. With further research, more evidence suggests that not only does the efficacy of immunotherapy depend on the activation of the tumor immune microenvironment, but the efficacy of traditional treatments such as targeted therapy also depends on the strength of the body's anti-tumor immune response. Cells and molecules in the tumor microenvironment are in a dynamic process, reflecting the evolutionary nature of cancer and jointly promoting tumor immune escape, growth, and metastasis. Exploring the potential mechanisms of tumor occurrence and development driven by the tumor microenvironment is of great significance for developing potential cancer treatment methods, improving the effectiveness of various existing treatments, and discovering new precise targets for RCC treatment. Therefore, regulating tumor exogenous regulatory factors in the microenvironment is crucial for understanding the malignant biological behavior of tumor cells and the game between tumor immune response and tumor immune escape, providing new directions for regulating tumor immunity.

[0012] Studies have found that only about 20% of renal cell carcinoma (RCC) patients have relatively high PD-L1 positive expression. However, clinical data indicate that PD-1 / PD-L1 inhibitor monotherapy is not very effective in RCC patients, possibly mainly due to the lack of T cell infiltration in the tumor microenvironment. Therefore, increasing the proportion of T cells in the RCC tumor microenvironment can improve the efficacy of ICIs (intracytoplasmic sperm injections) in RCC patients who have poor response or tolerance. Research on how to enhance the therapeutic effect of ICIs in RCC is one of the hot topics in RCC research.

[0013] Based on the characteristics of the renal cell carcinoma (RCC) tumor microenvironment, designing novel immunotherapy strategies to eliminate the immunosuppressive environment is of great significance for the treatment of RCC. By comprehensively analyzing the tumor immune microenvironment, both locally and surrounding, pathologists and clinicians can establish a prognostic assessment system for RCC patients based on the location and maturity heterogeneity of tertiary lymphoid structures. This system can comprehensively assess patients' recurrence risk and the efficacy of immunosuppressant therapy, and explore the relationship and clinical molecular mechanisms between the tumor immune microenvironment and tumor development, recurrence, metastasis, and drug resistance. Furthermore, tertiary lymphoid structures can stimulate the release of cytokines into tumor tissue, remodel the tumor immune microenvironment, chemotactically attract immune cells to infiltrate tumor tissue, and promote the transformation of "cold tumors" into "hot tumors." The ability of tertiary lymphoid structures to significantly alter the tumor microenvironment and attract immune cell infiltration can effectively predict the response rate of RCC patients to comprehensive treatment. Currently, there are no literature reports on the application of tertiary lymphoid structures in the preparation of prognostic assessment systems for RCC. Summary of the Invention

[0014] This invention is based on the above research and aims to provide the application of tertiary lymphoid structures in the preparation of renal cell carcinoma classification diagnosis or prognostic assessment, as well as the derived renal cell carcinoma classification diagnosis or prognostic assessment system, and also to provide its application in the treatment of renal cell carcinoma.

[0015] This invention first confirmed the presence of tertiary lymphoid structures in renal cell carcinoma tumor tissue, tumor stroma tissue, and adjacent normal tissue using hematoxylin and eosin (HE) staining. These structures stably exhibit highly infiltrated T lymphocytes and aggregated B cells, and their characteristic germinal center structures were also detected. These results suggest that tertiary lymphoid structures exist in the pathological state of renal cell carcinoma and possess the potential to improve the tumor immune microenvironment and enhance synergistic anti-tumor immune killing.

[0016] The second step involved assessing the maturity of TLS at different stages under pathological conditions, due to significant differences in the maturity of the three levels of lymphoid structures. Each HPF image was used to evaluate the maturity stage of TLS as follows: early TLS (E-TLS), characterized by dense lymphocyte aggregates lacking CD20 and CD21 signals; primary follicle-like TLS (PFL-TLS), characterized by dense lymphocyte aggregates with CD21 signals but lacking CD20 signals; and secondary follicle-like TLS (SFL-TLS), characterized by dense lymphocyte aggregates with CD21 signals and germinal centers (GCs) with dense B cell (CD20 signal) aggregation. The results showed that TLS at different maturity levels existed in renal cell carcinoma tumor tissue, tumor stroma tissue, and adjacent normal tissue. This indicates that, similar to other solid tumors such as liver and lung cancer, TLS is widely present in tumor tissue and peritumoral tissue and can initiate an invasive anti-tumor immune response.

[0017] Third, there is no clear relationship between the peritumoral or intratumoral location of TLS and clinical outcomes. To explore the relationship between TLS location heterogeneity and prognosis in patients with renal cell carcinoma, we included two real-world sets with available clinical follow-up and clinicopathological data (training set, n=290, from Fudan University Cancer Hospital [FUSCC]; validation set, n=105, from an external cohort), totaling 395 patients with ccRCC, for survival analysis.

[0018] In 290 Chinese patients with ccRCC from the FUSCC cohort, the presence of positive intratumoral TLS significantly predicted better long-term survival (OS: P < 0.001, HR = 0.456) and suppressed progression (PFS: P = 0.002, HR = 0.387). Conversely, positive peritumoral TLS was significantly associated with poor long-term survival (OS: P = 0.021, HR = 1.444) and progressive disease (PFS: P = 0.020, HR = 1.486) in ccRCC. Similarly, in the external validation set, the presence of intratumoral TLS was significantly associated with prolonged OS (P = 0.012, HR = 0.263) and suppressed PFS (P = 0.042, HR = 0.167). However, positive peritumoral TLS in 105 patients with ccRCC was also significantly associated with poor overall survival (OS) (P = 0.009, HR = 2.024) and progressive disease-free survival (P = 0.043, HR = 1.993). These results indicate that the local heterogeneity of TLS in renal cell carcinoma can effectively differentiate risk stratification and prognostic differences among renal cell carcinoma patients.

[0019] Fourthly, to further leverage the heterogeneity of TLS location to establish a precise prognostic assessment system for renal cell carcinoma (RCC) patients, we performed univariate and multivariate Cox regression analyses on 395 RCC patients from two different cohorts. In the univariate Cox regression analysis, traditional prognostic predictors, such as pathological TNM stage, ISUP nuclear grade, AJCC stage, and the presence or absence of intratumoral and peritumoral TLS, were significantly associated with overall survival (OS) and progression-free survival (PFS) in RCC patients. In the multivariate Cox regression analysis, pT stage, pM stage, ISUP grade, and the presence or absence of intratumoral TLS remained associated with both PFS and OS in the FUSCC cohort. Similarly, in the external validation set including 105 RCC patients, pT stage, pM stage, ISUP grade, and the presence of intratumoral TLS were also significantly associated with PFS and OS.

[0020] Next, we used the FUSCC set as the training cohort and constructed a nomogram based on multivariate Cox regression analysis, involving intratumoral TLS localization and maturity heterogeneity, to predict 2-year, 5-year, and 8-year overall survival (OS) and progression-free survival (PFS) in patients with renal cell carcinoma (ccRCC). Including these four variables, the intratumoral TLS-related risk score in 290 ccRCC patients was significantly associated with abnormally aggressive PFS. The AUC values ​​of the time-dependent ROC curves were higher than 0.80 (2-year AUC = 0.82, 5-year AUC = 0.82, 8-year AUC = 0.84), indicating that the nomogram method for predicting PFS has high sensitivity and specificity. Furthermore, after including intratumoral TLS, pT stage, pM stage, and ISUP grade, an increased OS predictive nomogram risk score in 290 ccRCC patients was significantly associated with shortened OS. The AUC values ​​of the time-dependent ROC curves also showed that the 2-year AUC was 0.78, the 5-year AUC was 0.82, and the 8-year AUC was 0.83. Furthermore, we performed a nomogram analysis involving intratumoral TLS on 105 patients with ccRCC from an external validation set, finding that higher risk scores significantly predicted progressive disease-free survival (PFS) and poorer overall survival (OS). In summary, these results demonstrate that intratumoral TLS has a sensitive and independent prognostic efficacy for renal cell carcinoma patients and reveal that a nomogram incorporating intratumoral TLS can significantly distinguish between patients at high risk of long-term tumor recurrence and those with poor clinical prognosis, enabling the establishment of a precise prognostic assessment system for renal cell carcinoma patients.

[0021] Fifthly, TLS plays a positive role in the anti-tumor process, and there are numerous cases regarding the mechanism and clinical application of TLS in enhancing tumor immunity. For example, HEV recruits various immune cells, including T cells and B cells; B cells also recruit various immune cells: T cells, NK cells, and dendritic cells (DCs). Mature DCs promote antigen presentation to T cells and B cells. However, there are also reports that TLS can be used by tumor cells under certain conditions, promoting lymphatic infiltration of tumor cells and leading to tumor lymphatic metastasis. To explore the specific mechanism by which TLS alters the tumor immune microenvironment, we performed quantitative multiplex immunohistochemical staining on renal cell carcinoma tissue to assess the distribution of various tumor-infiltrating lymphocytes in the tumor tissue and their relationship with the tertiary lymphoid structure. We defined a series of lymphocytes infiltrating tumors and the stroma, and quantitatively calculated their proportions per 100 cells using an algorithm. These included Tregs, exhausted T cells, M2 macrophages, tumor-associated macrophages, and tumor cells. We found that as TLS maturity increased, anti-tumor immune activation-related lymphocyte aggregation increased, and Treg cells were significantly suppressed. This suggests that TLS in tumor tissues responding to immunotherapy exhibits a mature secondary follicle-like TLS structure. This indicates that a mature TLS structure can improve the tumor immunosuppressive microenvironment by activating effector T cells while simultaneously reducing Treg cell recruitment.

[0022] The above studies indicate that intratumoral TLS, especially mature TLS, can be used for adjuvant treatment decisions and prognostic assessment in patients with intermediate-to-advanced or high-risk renal cell carcinoma who have failed first-line therapy, as well as in patients with surgically resectable renal cell carcinoma.

[0023] Based on the above research, the first aspect of the present invention provides the application of tertiary lymphoid structures in the preparation of reagents or kits for the subtyping diagnosis or prognostic assessment of renal cell carcinoma.

[0024] Preferably, the reagent for subtyping diagnosis or prognostic assessment of renal cell carcinoma is a reagent for intratumoral localization and maturity detection of tertiary lymphoid structures in renal cell carcinoma tissue samples; the kit contains reagents for intratumoral localization and maturity heterogeneity detection of tertiary lymphoid structures.

[0025] Further optimization involves using HE staining reagents for intratumoral localization of tertiary lymphoid structures. HE staining allows for the detection of the localization of TLS within the tumor and peritumoral tissues. Reagents for detecting the maturity heterogeneity of tertiary lymphoid structures include immunohistochemical or multiplex fluorescent immunohistochemical reagents. Immunohistochemical detection detects characteristic signals within TLS at different maturity levels, enabling differentiation between TLS at different maturity stages; multiplex fluorescent immunohistochemistry is used to obtain the composition of multiple cells and to detect cell function.

[0026] Further optimization involves using immunohistochemical detection reagents or multiplex fluorescent immunohistochemical detection reagents containing antibodies that detect characteristic signals of secondary follicles in tertiary lymphoid structures, specifically CD20 and CD21 antibodies.

[0027] A second aspect of the present invention provides a kit for the classification and diagnosis or prognostic assessment of renal cell carcinoma, the kit comprising reagents for intratumoral localization and maturity detection of tertiary lymphoid structures in biological samples. Preferably, the reagent for localization detection of tertiary lymphoid structures is an HE staining reagent, and the reagent for maturity detection of tertiary lymphoid structures includes at least CD20 and CD21 antibodies for detecting secondary follicle-like characteristic signals.

[0028] A third aspect of the invention provides the use of an agent that promotes the formation of tertiary lymphoid structures or the formation of secondary follicles therein in the preparation of a drug for treating renal cell carcinoma.

[0029] A fourth aspect of the present invention provides a pharmaceutical composition for treating renal cell carcinoma, comprising an active component and pharmaceutically or medically acceptable excipients, wherein the active component comprises an agent that promotes the formation of tertiary lymphoid structures or promotes the formation of secondary follicular TLS therein.

[0030] In a fifth aspect, the present invention provides a system for the classification diagnosis or prognostic assessment of renal cell carcinoma, characterized in that it includes a reagent kit and an analysis system installed on a terminal carrier.

[0031] The kit is the renal cell carcinoma subtyping diagnostic or prognostic assessment kit described above; the analysis system includes an image acquisition component, a positive signal counting system, and a cell density calculation system. The image acquisition component is independent of or integrated into the microscope and is used to capture immunohistochemical or immunofluorescence images; the positive signal counting system displays and counts positive signals in the images; the cell density calculation system calculates the area of ​​the tissue section and distinguishes cell density based on the cutoff value.

[0032] The beneficial protections and effects of this invention are as follows:

[0033] This invention utilizes HE staining, IHC staining, and multiplex immunohistochemical staining (mIHC) for histopathological examination of renal cell carcinoma (RCC) tissues. It incorporates samples and clinicopathological data from 395 patients with ccRCC from two cohorts, confirming the existence of stable, differently mature tumor-resistant structures (TLS) in both RCC and peritumoral tissues. Furthermore, the location and maturation heterogeneity of TLS significantly predict overall survival and progression-free survival in RCC patients. The results show that intratumoral TLS has a sensitive and independent prognostic efficiency for RCC patients, and can significantly distinguish high-risk areas for long-term tumor recurrence. By leveraging TLS location heterogeneity, a precise prognostic assessment system for RCC patients can be established.

[0034] TLS can induce more peripheral B cells and T cells to enter tumor tissue during immunotherapy, exerting a stronger adaptive anti-tumor immune response, and can effectively predict the benefit of ICIs in renal cell carcinoma patients. This suggests that TLS can effectively improve the tumor immune microenvironment, and this effect is mainly achieved by stimulating and recruiting effector T cells, especially CD8+. + T lymphocytes exert anti-tumor immune killing efficacy, which helps improve the efficacy of immune checkpoint inhibitor therapy in renal cell carcinoma patients and assists clinicians in establishing a comprehensive and accurate prognostic assessment system for renal cell carcinoma patients. With increased TLS maturity, the aggregation of anti-tumor immune activation-related lymphocytes increases, while Treg cells are significantly suppressed, indicating that TLS in tumor tissues responding to immunotherapy exhibits a mature secondary follicle-like TLS structure. This suggests that a mature TLS structure can improve the tumor immunosuppressive microenvironment by activating effector T cells while simultaneously reducing Treg cell recruitment.

[0035] Therefore, this invention provides new and precise pathological monitoring indicators for the clinical treatment and prognostic assessment of renal cell carcinoma. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the components of a three-tiered lymphatic system.

[0037] Figure 2 This is a schematic diagram illustrating the formation mechanism of the three-tiered lymphatic structure.

[0038] Figure 3 This is a HE staining feature diagram of renal cell carcinoma tissue and adjacent normal tissue: three-level lymphoid structures (Intra-TLS and Peri-TLS) are present in both renal cell carcinoma tissue and adjacent normal renal tissue.

[0039] Figure 4 This is an IHC staining image of E-TLS and PFL-TLS structures in renal cell carcinoma tissue and adjacent normal tissue. Antibodies against CD3, CD20, and CD21 bound to tumor-infiltrating lymphocytes were used to define TLS maturity. Due to the significant differences in maturity among the three levels of lymphoid structures, representing different growth stages of TLS under pathological conditions, immunohistochemical staining of T cells (CD3), B cells (CD20), and FDCs (CD21) was performed on sections containing TLS. The TLS maturity stage was assessed for each HPF image as follows: early TLS (E-TLS), dense lymphocyte aggregates lacking CD20 and CD21 signaling; primary follicle-like TLS (PFL-TLS), dense lymphocyte aggregates, CD21, but no CD20 signaling.

[0040] Figure 5This is an IHC staining image of SFL-TLS structures in renal cell carcinoma tissue and adjacent normal tissue. Antibodies against CD3, CD20, and CD21, combined with tumor-infiltrating lymphocytes, were used to define TLS maturity. Significant differences in maturity exist among the three levels of lymphoid structures, representing different growth stages of TLS under pathological conditions. Immunohistochemical staining of T cells (CD3), B cells (CD20), and FDCs (CD21) was performed on sections containing TLS. Secondary follicle-like TLS (SFL-TLS), with dense lymphocyte aggregation and CD20 and CD21 signaling, indicates the presence of germinal centers (GCs).

[0041] Figure 6 This is a survival analysis plot showing the predictive risk of recurrence and prognosis in renal cell carcinoma patients based on the heterogeneity of TLS location in the two cohorts. In 290 Chinese patients with ccRCC from the FUSCC cohort, the presence of intratumoral TLS significantly predicted better long-term survival (OS: P < 0.001, HR = 0.456) and suppressed progression (PFS: P = 0.002, HR = 0.387). Conversely, peritumoral TLS positivity was significantly associated with poor long-term survival (OS: P = 0.021, HR = 1.444) and progressive disease (PFS: P = 0.020, HR = 1.486) in ccRCC. Similarly, in the external validation set, the presence of intratumoral TLS was significantly associated with prolonged OS (P = 0.012, HR = 0.263) and suppressed PFS (P = 0.042, HR = 0.167). However, positive extratumoral TLS in 105 patients with ccRCC was also significantly associated with poor OS (P=0.009, HR=2.024) and progressive PFS (P=0.043, HR=1.993). These results indicate that the local heterogeneity of TLS in renal cell carcinoma can effectively differentiate risk stratification and prognostic differences among renal cell carcinoma patients.

[0042] Figure 7This is a diagram of the quantitative multiplex immunofluorescence histochemistry staining results. To better characterize TLS in heterogeneous ccRCC samples, we investigated the cellular composition of TLS using multispectral immunofluorescence immunohistochemistry (mIHC) in 58 ccRCC tissues and aimed to identify the heterogeneity of TLS maturation and its impact on local TME. We detected 12 cellular markers of adaptive immune responses involved in recognizing, responding to, and eliminating tumor cells during tumor progression in the TME activity of renal cell carcinoma. In group 1, we detected CD8+ T cells, PD-1+CD8+ exhausted T cells, CD68+CD163-M1 macrophages, CD68+CD163+M2 macrophages, CK-surrounded CD68+CD163+ TAMs, PD-L1+CD68+CD163+M2 macrophages, and PD-L1-expressing CPS, and performed further analysis. In group 2, the abundance of CD3+ T cells, CD4+ T cells, CD4+FoxP3+ Treg cells, CD20+ B cells, and CD56bright / dim NK cells was assessed in the TME. In summary, the cellular composition of the mature TLS stage exhibited significant heterogeneity in tumor-infiltrating lymphocytes.

[0043] Figure 8This is a nomogram based on Cox regression analysis to include the presence of intratumoral TLS. In univariate Cox regression analysis, traditional prognostic predictors, such as pathological TNM stage, ISUP nuclear grade, AJCC stage, and the presence or absence of intratumoral and peritumoral TLS, were significantly associated with overall survival (OS) and progression-free survival (PFS) in renal cell carcinoma patients. In multivariate Cox regression analysis, pT stage, pM stage, ISUP grade, and the presence or absence of intratumoral TLS remained associated with both PFS and OS; in the FUSCC cohort, similarly, in an external validation set including 105 renal cell carcinoma patients, pT stage, pM stage, ISUP grade, and the presence of intratumoral TLS were also significantly associated with PFS and OS. Multivariate Cox regression analysis showed that, in the FUSCC training set, a nomogram was constructed based on the heterogeneity of intratumoral TLS localization and maturity to predict 2-, 5-, and 8-year overall survival (OS) and progression-free survival (PFS) in patients with renal cell carcinoma (ccRCC). Including these four variables, the intratumoral TLS-related risk score in 290 ccRCC patients was significantly associated with abnormally aggressive PFS, indicating that the nomogram method for predicting PFS has high sensitivity and specificity. Furthermore, after incorporating intratumoral TLS, pT stage, pM stage, and ISUP grade, an increased OS predictive nomogram risk score was significantly associated with shortened OS in the 290 ccRCC patients. The AUC values ​​of the time-dependent ROC curves also showed that the 2-year AUC was 0.78, the 5-year AUC was 0.82, and the 8-year AUC was 0.83. Furthermore, we performed a nomogram involving intratumoral TLS in 105 patients with ccRCC from an external validation set, and found that higher risk scores significantly predicted progressive disease-free survival (PFS) and poor overall survival (OS). In summary, these results demonstrate that intratumoral TLS has a sensitive and independent prognostic efficacy for renal cell carcinoma patients, and reveal that a nomogram incorporating intratumoral TLS can significantly distinguish between patients at high risk of long-term tumor recurrence and those with poor clinical prognosis, enabling the establishment of a precise prognostic assessment system for renal cell carcinoma patients.

[0044] Figure 9This diagram illustrates how the heterogeneity of TLS in renal cell carcinoma effectively predicts the therapeutic efficacy of immune checkpoint inhibitors in patients. Results show that with increasing TLS maturity, the abundance of tumor-killing CD8+ T cells significantly increases within the tumor tissue, while the proportion of CD4+FoxP3+ Treg cells significantly decreases, indicating a highly active anti-tumor immune microenvironment. Furthermore, to investigate the impact of TLS on patients receiving immunosuppressant therapy, we collected data from patients in the BIONIKK clinical trial with advanced clear cell renal cell carcinoma undergoing immunotherapy, validating the relationship between intratumoral TLS staining and immunotherapy efficacy. Results showed that patients with high intratumoral TLS expression had longer progression-free survival (PFS); and this group of patients with high intratumoral TLS expression also had a higher objective response rate to immunotherapy. Detailed Implementation

[0045] The present invention will now be described in detail with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0046] All reagents and raw materials used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed according to standard procedures or the manufacturer's recommendations. Unless otherwise stated, percentages and parts are by volume.

[0047] I. Identification of heterogeneity in the location and maturity of tertiary lymphoid structures in renal cell carcinoma and adjacent tissues

[0048] I. Experimental Methods:

[0049] The presence of TLS, as well as its heterogeneity in location and maturity, was confirmed in renal cell carcinoma tissue and adjacent normal renal tissue. Tumor-infiltrating lymphocytes can be bound by antibodies such as CD3, CD20, and CD21, so HE and IHC were used to detect it.

[0050] 1.1 Hematoxylin-eosin staining (HE)

[0051] A. Sectioning: The formalin-fixed-paraffin-embedded samples were serially sectioned by Severn, with a section thickness of 4 micrometers.

[0052] B. Dewaxing and hydration of paraffin sections: Place the paraffin sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, and anhydrous ethanol II for 5 minutes in sequence.

[0053] C. Hematoxylin staining: After immersing the section in hematoxylin staining solution for 4 minutes, differentiate it with differentiation solution for 30 seconds, rinse with tap water for 5 minutes; then immerse it in blue solution for 30 seconds, and rinse with tap water for 5 minutes.

[0054] D. Eosin staining: First dehydrate with 85% alcohol for 5 minutes, then dehydrate with 95% alcohol for 5 minutes; stain with eosin solution for 5 minutes.

[0055] E. Dehydration and mounting: Immerse the slides in anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 10 minutes, xylene I for 5 minutes, and xylene II for 5 minutes in sequence. After the slides are slightly dry, add an appropriate amount of neutral resin to the tissue area, cover with a glass slide, and allow to air dry naturally until images are acquired.

[0056] 1.2 Immunohistochemistry (IHC)

[0057] A. Dewaxing and hydration of paraffin sections: Place the paraffin sections in xylene I for 15 minutes, xylene II for 15 minutes, xylene III for 15 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol for 5 minutes, and rinse with tap water.

[0058] B. Antigen retrieval: Place the slide in a water bath filled with citrate antigen retrieval buffer (pH 6.0), microwave for 8 minutes (100°C), then microwave again for 7 minutes (80°C) after a 10-minute interval; cool to room temperature.

[0059] C. Blocking: Rinse the sections with PBS for 5 minutes, repeat 3 times; use a histochemical pen to draw circles along the tissue edge to prevent reagent loss (be careful to avoid damaging the tissue), add 50 μL of blocking solution, and let stand at room temperature for 30 minutes.

[0060] D. Add primary antibody: Rinse the slides with PBS for 5 minutes, repeat 3 times; add CD3 (1:200), CD20 (1:200) and CD21 (1:200) diluted with primary antibody dilution buffer, place the slides in a humidified chamber, and incubate overnight at 4°C.

[0061] E. Add secondary antibody: Return the section temperature to room temperature, rinse the section with PBS for 5 minutes, repeat 3 times. Add secondary antibody diluted in PBS (1:800) and incubate at room temperature for 60 minutes.

[0062] F. DAB staining: Rinse sections with PBS for 5 minutes, repeat 3 times. Add freshly prepared DAB staining solution (1:20) to the circle, and stain for 5 minutes. Rinse sections with tap water to stop staining.

[0063] G. Counterstaining cell nuclei: Place the sections in hematoxylin staining solution for 1 minute, wash with tap water for 5 minutes, differentiate with differentiation solution for 30 seconds, rinse with tap water for 5 minutes, re-blue with blue solution for 30 seconds, and rinse with running water for 5 minutes.

[0064] H. Dehydration and mounting: Place the paraffin sections sequentially in 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, anhydrous ethanol for 10 minutes, xylene I for 15 minutes, xylene II for 15 minutes, and xylene III for 15 minutes. After removing the sections from the xylene, allow them to air dry slightly, add a small amount of neutral resin, and cover with a coverslip. Place in a ventilated area to dry, awaiting image acquisition.

[0065] For results analysis, at least one TLS was confirmed as positive in each hematoxylin-eosin stained section. This process was performed by three pathologists. TLSs were categorized as Intra-TLS and Peri-TLS based on their location and extent. Immunohistochemical images were captured using an Olympus DP20 camera attached to a CX31 microscope. Immunofluorescence images were captured using a fluorescence microscope. All TLS regions in the entire section were recorded under 20X magnification. Positive signals within TLSs were manually counted using ImageJ software, a process performed collaboratively by the three pathologists as described above. A Nanozoomer scanner was used to scan the sections, and nanoscale digital pathology view software was used to calculate the tissue area of ​​the sections. Cell density was recorded as cells / mm². 2 Cell densities were divided into high-cell-density groups (represented by High-CD20 and High-CD20) and low-cell-density groups (represented by Low-CD20 and Low-CD20) based on cutoff values. Kaplan-Meier survival analysis used the log-rank test, and Cox proportional hazards regression analysis was used to test for independent prognostic factors. Cutoff values ​​for each group in the survival analysis were calculated using X-title software.

[0066] 1.3 Multiplexed immunohistochemical assay (mIHC)

[0067] Currently, the main technologies for detecting the tumor immune microenvironment include gene sequencing, traditional immunohistochemistry, and flow cytometry. These three technologies cannot simultaneously obtain information on cellular composition, cell location, and cell function within the tumor immune microenvironment. Gene sequencing and flow cytometry require lysing tumor tissue samples to obtain relevant genes and cells before quantitative detection can be performed, thus failing to obtain information on cell location within the tumor immune microenvironment. While traditional immunohistochemistry can obtain cell location information for detection indicators, it cannot obtain information on the composition of multiple cells or detect cell function.

[0068] Multiplex fluorescence immunohistochemistry (MFI) can image and analyze 5-9 target sites in tissue samples. It uses a flow cytometry-like method to quantitatively assess cell phenotype and activity, while also providing information on the tissue microenvironment and spatial location between cells—something other technologies currently cannot achieve. MFI can more comprehensively and accurately stratify patients to the optimal treatment pathway. The specific detection steps are as follows:

[0069] 1) Sampling: After tumor formation in mice, treatment was administered. Two groups were established: an NC control group, a VG160 monotherapy group, and a VG161 monotherapy group. Mice were sacrificed on days 3, 7, and 15 after administration, and tumor specimens were collected. The tissues were fixed in formalin, embedded in paraffin, cut into 4μm thick paraffin sections, and fixed onto detachable glass slides.

[0070] 2) Staining: Based on the TSA staining principle, horseradish peroxidase (HRP)-labeled secondary antibody is used to activate the fluorescent dye, covalently binding the dye to the antigen. By utilizing different fluorescent dyes, in situ multi-target staining of tissues or cells is achieved through multiple staining cycles. The specific staining procedure is as follows:

[0071] (a) Sample slide preparation: Bake slides at 60-65 degrees Celsius for 2 hours or overnight; soak slides in Xylene for 10 minutes, repeat 3 times; soak slides in a gradient of ethanol: 100% for 5 minutes; 95% for 5 minutes; 70% for 5 minutes; rinse slides with deionized water for 3 minutes.

[0072] (b) Microwave retrieval: After boiling the antigen retrieval solution, place the slide into the antigen retrieval box, adjust the microwave power to low and continue retrieval for 15-20 minutes, then allow it to cool naturally at room temperature (15-30 minutes); rinse the slide with deionized water, treat the sample with 3% H2O2 water for 10-15 minutes to remove endogenous peroxidase; wash the sample slide with 1×TBST for 3 minutes.

[0073] (c) Blocking: Remove the TBST buffer from the slide, place it in a staining humidifier, and circle the tissue sample area on the slide with an immunohistochemistry pen; add blocking buffer to cover the tissue area on the slide and incubate for 10 min in a humidified environment.

[0074] (d) Primary antibody incubation: Remove the blocking buffer from the slide, add diluted primary antibody working solution, incubate at room temperature for 1 h, wash with 1X TBST, shake at room temperature for 3 min, and repeat 3 times;

[0075] (e) HRP secondary antibody incubation: Remove TBST buffer from the slide, add secondary antibody to the slide, incubate at room temperature for 10 min, wash with 1X TBST, shake at room temperature for 3 min, and repeat 3 times.

[0076] (f) Staining signal development: Remove the TBST buffer from the slide, add 1×TSA staining working solution to the slide, incubate in a moist environment for 5-10 min, then soak the slide in 1×TBST solution, shake at room temperature for 3 min, and repeat 3 times. This completes the staining of one protein marker.

[0077] (g) Counterstain subsequent protein markers and repeat the above steps (af).

[0078] (h) DAPI staining and mounting: Remove TBST buffer from the slide, add DAPI working solution to the slide, incubate at room temperature for 3-5 min, soak the slide in 1×TBST, shake at room temperature for 5 min, remove TBST buffer; add fluorescent anti-quenching mounting medium to the slide and mount it, then fix the cover slide with nail polish.

[0079] 3) Imaging and Analysis: The KR-HT5 multispectral imaging system from Kuoran Biomedical (Shanghai) Co., Ltd. was used for high-speed scanning and acquisition of all fluorescence signals on paraffin sections stained in the above steps. High-precision spectral overlap and separation technology was employed to subtract tissue autofluorescence signals and separate individual monochromatic fluorescence signals from the mixed signals, establishing a corresponding monochromatic fluorescence spectral library. Tissue regions were identified and separated sequentially according to standards set by pathologists or experts. Based on the cell nuclei identified by DAPI staining, the cell nucleus, cytoplasm, and cell membrane were separated. Cell types were classified based on the markers of various proteins on the cells. Finally, the percentages of single-positive, double-positive, and triple-positive cells for any protein in the tumor solid and stromal regions were statistically analyzed.

[0080] (4) Statistical analysis and model building

[0081] The primary endpoint was progression-free survival (PFS), and the secondary endpoint was overall survival (OS). Survival analyses were performed using the Kaplan-Meier survival method. Criterion values ​​were determined using the "survminer" R package in R software and evaluated using a log-rank test with 95% confidence intervals (95% CI). To identify independent predictors, hazard ratio (HR) estimates and 95% CIs were performed using univariate and multivariate Cox logistic regression models. As a supplement to survival analysis, Cox logistic regression analyses were performed on patients from FUSCC and external validation cohorts using the "rms" and "survival" R packages to predict PFS and OS. This plot, based on multivariate Cox regression analysis, integrates the presence of intratumoral and extratumoral TLS with multiple conventional clinicopathological predictors, then scales the complex regression equations into a visual graph on the same plane. Nomograms were used to display the relationships between variables in the predictive model, accurately assessing the 2-year, 5-year, and 8-year recurrence rates or survival rates for the corresponding ccRCC patients.

[0082] II. Experimental Results

[0083] (1) HE staining results showed that tertiary lymphoid structures (Intra-TLS and Peri-TLS) were present in both renal cell carcinoma tissue and adjacent normal renal tissue. Figure 3 ).

[0084] (2) IHC staining results showed that CD3, CD20, and CD21 antibodies bound to tumor-infiltrating lymphocytes were used to define TLS maturity. Due to the significant differences in maturity among the three levels of lymphoid structures, representing different growth stages of TLS under pathological conditions, we assessed the TLS maturity stages using each HPF image as follows: Early TLS (E-TLS), characterized by dense lymphocyte aggregates lacking CD20 and CD21 signals; Primary follicle-like TLS (PFL-TLS), characterized by dense CD21-signaled lymphocyte aggregates but lacking CD20 signals; Secondary follicle-like TLS (SFL-TLS), characterized by dense lymphocyte aggregates with CD21 and dense CD20 signals, indicating the presence of germinal centers (GCs). Different maturities of TLS were present in renal cell carcinoma tumor tissue, tumor stroma tissue, and adjacent normal tissue, indicating that, similar to other solid tumors such as liver and lung cancer, TLS is widely present in tumor tissue and peritumoral tissue and can initiate an invasive anti-tumor immune response. Figure 4 , Figure 5 ).

[0085] (3) Survival analysis showed that in 290 Chinese patients with ccRCC from the FUSCC cohort, the presence of positive intratumoral TLS significantly predicted better long-term survival (OS: P < 0.001, HR = 0.456) and suppressed progression (PFS: P = 0.002, HR = 0.387). Conversely, positive peritumoral TLS was significantly associated with poor long-term survival (OS: P = 0.021, HR = 1.444) and progressive progression (PFS: P = 0.020, HR = 1.486) in ccRCC. Similarly, in the external validation set, the presence of intratumoral TLS was significantly associated with prolonged OS (P = 0.012, HR = 0.263) and suppressed PFS (P = 0.042, HR = 0.167). However, positive peritumoral TLS in 105 patients with ccRCC was also significantly associated with poor overall survival (OS) (P = 0.009, HR = 2.024) and progressive disease-free survival (P = 0.043, HR = 1.993). These results indicate that the local heterogeneity of TLS in renal cell carcinoma can effectively differentiate risk stratification and prognostic differences among renal cell carcinoma patients. Figure 6 ).

[0086] (4) Quantitative multiplex immunofluorescence staining results suggest that a more comprehensive description of TLS heterogeneity may help define a “TLS status” profile based on cellular composition, location, maturity, and anti-tumor immune response function. To better characterize TLS in heterogeneous ccRCC samples, we investigated the cellular composition of TLS using multispectral immunofluorescence (mIHC) in 58 ccRCC tissues and aimed to identify the heterogeneity of TLS maturation and its impact on local TME. We detected 12 adaptive immune response cellular markers involved in recognizing, responding to, and eliminating tumor cells during tumor progression in the TME activity of ccRCC. In group 1, CD8 was detected. + T cells, PD-1 + CD8 + Exhausted T cells, CD68 + CD163 - M1 macrophages, CD68 + CD163 + M2 macrophages, CD68 surrounded by CK + CD163 + TAMs, PD-L1 + CD68 + CD163 + CPS expressed in M2 macrophages and PD-L1 were analyzed further. In group 2, CD3 was assessed in the TME. + T cells, CD4 + T cells, CD4 + FoxP3 + Treg cells, CD20 + B cells and CD56 bright / dim NK cell abundance. In summary, the cellular composition of the mature TLS stage showed significant heterogeneity in tumor-infiltrating lymphocytes. Figure 7 ).

[0087] (5) Figure 8Univariate and multivariate Cox regression analyses showed that, in univariate Cox regression analysis, traditional prognostic predictors, such as pathological TNM stage, ISUP nuclear grade, AJCC stage, and the presence or absence of intratumoral and peritumoral TLS, were significantly associated with overall survival (OS) and progression-free survival (PFS) in renal cell carcinoma patients. In multivariate Cox regression analysis, pT stage, pM stage, ISUP grade, and presence or absence of intratumoral TLS remained associated with both types of PFS (pT stage: P = 0.005; pM stage: P < 0.001; ISUP grade: P = 0.001; intratumoral TLS: P = 0.029) and OS (pT stage: P = 0.004; pM stage: P < 0.001; ISUP grade: P = 0.027; intratumoral TLS: P = 0.030). In the FUSCC cohort... Similarly, in an external validation set including 105 patients with renal cell carcinoma, the pT stage, pM stage, ISUP grade, and presence of intratumoral TLS were significantly associated with PFS (pT stage: P = 0.036; pM stage: P = 0.002; ISUP grade: P = 0.029; intratumoral TLS: P = 0.029) and OS (pT stage: P < 0.001; pM stage: P = 0.011; ISUP grade: P = 0.088; intratumoral TLS: P = 0.039).

[0088] Multivariate Cox regression analysis showed that, in the FUSCC training set, a nomogram based on the constructed intratumoral TLS localization and maturity heterogeneity was used to predict 2-, 5-, and 8-year overall survival (OS) and progression-free survival (PFS) in patients with renal cell carcinoma (ccRCC). Including these four variables, a high intra-atls risk score was significantly associated with abnormally aggressive PFS in 290 ccRCC patients (P = 7.4e-23, HR = 5.92). The AUC values ​​of the time-dependent ROC curves were higher than 0.80 (2-year AUC = 0.82, 5-year AUC = 0.82, 8-year AUC = 0.84), indicating that the nomogram method for predicting PFS has high sensitivity and specificity.

[0089] Furthermore, after incorporating intratumoral TLS, pT stage, pM stage, and ISUP grade, we established a prognostic prediction nomogram for ccRCC patients. Risk score (PFS) = 1.728 * Pathological T stage (ref. T1 / T2) + 4.189 * Pathological M stage (ref. M0) + 1.696 * ISUP grade (ref. G1 / G2) - 0.426 * Intra-TLS (ref. Absence); Risk score (OS) = 1.807 * Pathological T stage (ref. T1 / T2) + 4.659 * Pathological M stage (ref. M0) + 1.463 * ISUP grade (ref. G1 / G2) - 0.33 * Intra-TLS (ref. Absence)

[0090] On the one hand, the Riskscore (OS) was significantly associated with shortened OS (P = 2.8e-23, HR = 6.14). The AUC values ​​of the time-dependent ROC curves also showed a significant correlation: 0.78 at 2 years, 0.82 at 5 years, and 0.83 at 8 years. Furthermore, we performed a nomogram on 105 patients with ccRCC from an external validation set, involving intratumoral TLS, and found that a higher PFS-related risk score significantly predicted progressive PFS (P = 3.2e-13, HR = 6.47, 5-year AUC = 0.82) and poorer OS (P = 1.4e-9, HR = 6.32, 8-year AUC = 0.83).

[0091] In summary, these results demonstrate that intratumoral TLS has a sensitive and independent prognostic efficiency for patients with renal cell carcinoma, and reveal that nomograms incorporating intratumoral TLS can significantly distinguish between patients at high risk of long-term tumor recurrence and those with poor clinical prognosis, enabling the establishment of a precise prognostic assessment system for renal cell carcinoma patients.

[0092] (5) Figure 9 This study demonstrates that the heterogeneous properties of TLS in renal cell carcinoma effectively predict the therapeutic efficacy of immune checkpoint inhibitors in patients with renal cell carcinoma. The results indicate that with increasing TLS maturity, the number of CD8+ tumor-killing cells within the tumor tissue increases. + T cell abundance was significantly increased, and CD4 + FoxP3 +The significantly downregulated proportion of Treg cells suggests that the immunosuppressive state of the tumor microenvironment was relieved, and a more active anti-tumor immune microenvironment was observed. Furthermore, to investigate the impact of intratumoral TLS on patients receiving immunosuppressive therapy, we collected data from patients in the BIONIKK clinical trial with advanced clear cell renal cell carcinoma who underwent immunotherapy, validating the relationship between intratumoral TLS staining and immunotherapy efficacy. Results showed that patients with high intratumoral TLS expression had longer progression-free survival (PFS) (median PFS = 16.3 months vs. 6.4 months, p = 0.039); and these patients with high intratumoral TLS expression also had higher objective responses (ORR) to immunotherapy.

[0093] In summary, this invention is the first to identify the heterogeneity of the three-level lymphoid structure in renal cell carcinoma, providing a new pathological monitoring indicator for the clinical efficacy detection and recurrence risk assessment of renal cell carcinoma, and can effectively assist clinicians in establishing a precise prognostic assessment system for renal cell carcinoma patients throughout the entire process.

[0094] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. The application of reagents used for localization and maturity detection of tertiary lymphoid structures in the preparation of a prognostic assessment kit for clear cell renal cell carcinoma, characterized in that, The reagent used for intratumoral localization of tertiary lymphoid structures is HE staining reagent; the reagent used for maturity detection of tertiary lymphoid structures is immunohistochemical detection reagent or multiplex fluorescent immunohistochemical detection reagent. The immunohistochemical detection reagents or multiplex fluorescent immunohistochemical detection reagents all contain antibodies that detect secondary follicle-like characteristic signals in tertiary lymphoid structures. The antibodies that detect secondary follicle-like characteristic signals are CD20 antibodies and CD21 antibodies. Positive intratumoral grade 3 lymphoid structures were significantly associated with prolonged overall survival and progression-free survival, while positive intratumoral grade 3 lymphoid structures were significantly associated with shortened overall survival and progression-free survival. The secondary follicular tertiary lymphoid structures contain CD21 and CD20 signals, and these structures can relieve the immunosuppressive state of the tumor microenvironment.

2. A prognostic assessment system for clear cell renal cell carcinoma, characterized in that, This includes the reagent kit and the analysis system installed on the terminal carrier. The kit contains reagents for intratumoral localization and maturity detection of tertiary lymphoid structures in biological samples. The reagent for intratumoral localization of tertiary lymphoid structures is an HE staining reagent, and the reagent for maturity detection of tertiary lymphoid structures includes at least CD20 and CD21 antibodies for detecting secondary follicle-like characteristic signals. Positive intratumoral grade 3 lymphoid structures were significantly associated with prolonged overall survival and progression-free survival, while positive intratumoral grade 3 lymphoid structures were significantly associated with shortened overall survival and progression-free survival. Secondary follicular tertiary lymphoid structures contain CD21 and CD20 signals, and these structures can relieve the immunosuppressive state of the tumor microenvironment. The analysis system includes an image acquisition component, a positive signal counting system, and a cell density calculation system. The image acquisition component is independent of or integrated into the microscope and is used to capture immunohistochemical or immunofluorescence images. The positive signal counting system displays and counts positive signals in the images. The cell density calculation system calculates the area of ​​the tissue section and distinguishes cell density based on the cutoff value.

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