Peripheral blood marker combination for predicting immune efficacy of nasopharyngeal carcinoma and dynamic monitoring

Abstract

The application discloses a peripheral blood marker combination for predicting the immunotherapy effect of nasopharyngeal carcinoma and dynamic monitoring, belongs to the technical field of tumor immunotherapy, and comprises the following steps: predicting the immunotherapy effect by detecting the CD3, CD4, CD8, CD45RA, CCR7 and PD-1 molecule levels in the peripheral blood before immunotherapy; and dynamically monitoring the immunotherapy effect by detecting the CD3, CD4, CD8, CD45RA, CCR7, CD69 and CXCR5 molecule levels in the peripheral blood after immunotherapy. In the application, the CD3, CD4, CD8, CD45RA, CCR7 and PD-1 molecule levels in the peripheral blood of nasopharyngeal carcinoma patients before immunotherapy are detected to predict the immunotherapy effect, potential benefit patients are screened for immunotherapy; the CD3, CD4, CD8, CD45RA, CCR7, CD69 and CXCR5 molecule levels in the peripheral blood of nasopharyngeal carcinoma patients after immunotherapy are detected to dynamically monitor the immunotherapy effect, and then the subsequent treatment scheme is guided. It is worth large-scale promotion and application in the clinic.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, specifically a combination of peripheral blood biomarkers for predicting the efficacy of immunotherapy in nasopharyngeal carcinoma and for dynamic monitoring. Background Technology

[0002] Immunotherapy, represented by PD1 / PDL1 inhibitors, has become a major treatment for nasopharyngeal carcinoma. Currently, common biomarkers for immunotherapy in clinical practice include PD-L1 expression levels, tumor mutational burden (TMB), and microsatellite instability (MSI) / mismatch repair deficiency (dMMR). However, the predictive performance of these biomarkers in nasopharyngeal carcinoma is currently poor, and there are no unified judgment criteria or industry standards. For example, PD-L1 expression levels are as high as 90% in nasopharyngeal carcinoma, and multiple previous studies have reported that PD-L1 expression levels cannot distinguish between sensitive and resistant patients; while MSI / dMMR are only applicable to specific tumors such as colorectal cancer and are not applicable to nasopharyngeal carcinoma.

[0003] Immunotherapy differs from surgery, radiotherapy, and chemotherapy in its mechanism of action, primarily by activating and mobilizing T cells to induce anti-tumor immunity. Therefore, in recent years, experts in the field have proposed that the functional status of immune cells in peripheral blood circulation is closely related to tumor treatment outcomes, and that the phenotypic characteristics of peripheral blood immune cells can serve as potential predictive biomarkers for immunotherapy. Furthermore, compared to tumor tissue biomarkers, peripheral blood immune biomarkers offer advantages such as less invasiveness, higher safety, and the ability to be dynamically monitored, thus their role and predictive value are receiving increasing clinical attention.

[0004] Current research in melanoma and lung cancer has confirmed that peripheral blood biomarkers can effectively predict the efficacy of immunotherapy. For example, in non-small cell lung cancer patients receiving PD-1 / PD-L1 inhibitor therapy, a higher baseline proportion of well-differentiated CD4+ T cells (CD27-CD28 low / -) (>40%) in peripheral blood can predict the objective response rate with 100% specificity and 70% sensitivity. However, there are currently no effective peripheral blood biomarkers for predicting immunotherapy efficacy in nasopharyngeal carcinoma. Therefore, a combination of peripheral blood biomarkers for predicting and dynamically monitoring immunotherapy efficacy in nasopharyngeal carcinoma has been proposed.

[0005] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0006] This invention aims to at least address one of the technical problems existing in the prior art. Therefore, one object of this invention is to propose a combination of peripheral blood biomarkers for predicting and dynamically monitoring the immunotherapy efficacy of nasopharyngeal carcinoma.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A combination of peripheral blood biomarkers used to predict the efficacy of immunotherapy in nasopharyngeal carcinoma and for dynamic monitoring includes:

[0009] Molecular levels of CD3, CD4, CD8, CD45RA, CCR7, and PD-1 in peripheral blood before immunotherapy can predict the efficacy of immunotherapy.

[0010] Dynamic monitoring of immunotherapy efficacy was conducted by measuring the molecular levels of CD3, CD4, CD8, CD45RA, CCR7, CD69, and CXCR5 in peripheral blood after immunotherapy.

[0011] Methods for predicting and dynamically monitoring the efficacy of nasopharyngeal carcinoma immunotherapy based on peripheral blood immune cell phenotypic characteristics:

[0012] By performing spectral flow cytometry analysis on peripheral blood of patients enrolled in a prospective clinical trial before treatment, and conducting comparative analysis based on efficacy (treatment response group R vs. treatment resistance group NR), a total of 8 major cell groups, including T cells, B cells, monocytes, and dendritic cells, were identified. Subsequently, T cells were further subdivided and subpopulations were screened, and the proportion of T cell subpopulations that were significantly correlated with efficacy were identified.

[0013] Subsequently, the preliminary results were validated using two separate cohorts of recurrent and metastatic nasopharyngeal carcinoma (validation set 1) and locally advanced nasopharyngeal carcinoma (validation set 2). Immunotherapy-sensitive individuals were screened using a combination of six indicators: CD3, CD4, CD8, CD45RA, CCR7, and PD-1. An integrated T-cell score was derived by calculating four indicators: CD4+Tem / CD4T and CD8+Tem / CD4T ratios, and PD1+ / CD4+Tem and PD1+ / CD8+Tem, serving as a predictive indicator of immunotherapy efficacy.

[0014] Further comparison of peripheral blood marker changes before and after immunotherapy in two validation cohorts revealed that the proportions of CD69+Tem cells and CXCR5+Tfh cells increased only in the treatment response group after treatment, while no significant changes were observed in the resistance group. This suggests that the efficacy of immunotherapy can be dynamically monitored by detecting the proportions of peripheral blood CD69+Tem cells and CXCR5+Tfh cells, i.e., by using a combination of seven indicators: CD3, CD4, CD8, CD45RA, CCR7, CD69, and CXCR5.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] In this invention, the efficacy of immunotherapy is predicted by detecting the molecular levels of CD3, CD4, CD8, CD45RA, CCR7, and PD-1 in the peripheral blood of nasopharyngeal carcinoma patients before immunotherapy, and potential beneficiaries are screened for immunotherapy. This method is worthy of large-scale clinical application.

[0017] In this invention, the efficacy of immunotherapy is dynamically monitored by detecting the molecular levels of CD3, CD4, CD8, CD45RA, CCR7, CD69, and CXCR5 in the peripheral blood of nasopharyngeal carcinoma patients after immunotherapy, thereby guiding subsequent treatment plans.

[0018] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0019] Figure 1 This is a diagram of the flow cytometry detection gate method of the present invention;

[0020] Figure 2 This is a diagram showing the classification and content analysis of the main cell groups in blood samples in this invention, as well as the inter-group comparison of different therapeutic effects.

[0021] Figure 3 This is a graph showing the intergroup comparison of major T cell subsets in different efficacy groups in the validation set 1 of this invention, and the changes in the expression of activating molecules and immune checkpoint molecules of effector CD4+ and CD8+ T cells before and after treatment.

[0022] Figure 4 This invention presents a survival analysis graph showing the correlation between four indicators—CD4+Tem / CD4T and CD8+Tem / CD4T ratios, PD1+ / CD4+Tem, and PD1+ / CD8+Tem—and plasma EBV DNA, as well as the prediction of progression after nasopharyngeal carcinoma treatment.

[0023] Figure 5 This is a graph showing the changes in PD-1 and CD69 molecular levels expressed in CD4+Tem and CD8+Tem before and after treatment in validation set 2 of this invention, as well as the inter-group comparison of different efficacy groups;

[0024] Figure 6 This invention provides a comparison of the various Th cell subsets and Treg cells in validation set 2 before and after treatment in different efficacy groups, and a trend graph of Tfh in the CR group after treatment.

[0025] Figure 7 This is a diagram showing the effects of pretreatment and posttreatment monitoring according to the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1

[0028] This invention provides a technical solution:

[0029] The study utilized blood samples from patients enrolled in the prospective clinical trial (Jupiter-02 study) for the training set. Patients in the experimental group of the Jupiter-02 study received 6 cycles of GP chemotherapy combined with immunotherapy, followed by follow-up. Based on whether tumor progression occurred during the follow-up period, patients were divided into a treatment response group (R) and a treatment resistance group (NR) for comparative analysis.

[0030] As attached Figure 1 As shown, eight major cell groups in peripheral blood were identified by spectroscopic flow cytometry, as shown in the attached figure. Figure 2 As shown, the cells include T cells, B cells, monocytes, dendritic cells, etc. A comparison of the main cellular components between the two groups revealed that the treatment response group had a higher number of CD8+ T cells before treatment.

[0031] Subsequently, T cells were further subdivided, with CD4+ and CD8+ T cells classified into naive T cells (Tn), central memory T cells (Tcm), effector T cells (Tem), and terminal effector T cells (Temra), as shown in the appendix. Figure 3 As shown, the proportion of CD4+Tem and CD8+Tem cells in the response group was significantly higher than that in the drug-resistant group before treatment. Furthermore, the CD4+Tem and CD8+Tem cells in the response group highly expressed PD-1, and the expression level of PD-1 molecule decreased significantly after receiving immunotherapy, suggesting that CD4+Tem and CD8+Tem cells are targets of immunotherapy.

[0032] A correlation analysis was performed between the pre-treatment proportions of CD4+Tem and CD8+Tem and the pre-treatment plasma EBV DNA level, as shown in the attached figure. Figure 3 As shown, the ratio of CD4+Tem and CD8+Tem is significantly negatively correlated with EBV DNA, and patients with heavier nasopharyngeal carcinoma disease burden have a lower proportion of effector T cells in peripheral blood.

[0033] Example 2

[0034] As attached Figure 4As shown, patient survival was analyzed based on the proportions of CD4+Tem and CD8+Tem cells in the blood. Patients with higher proportions of CD4+Tem and CD8+Tem cells in peripheral blood showed significantly longer progression-free survival after immunotherapy, suggesting that CD4+Tem and CD8+Tem levels can serve as important indicators for predicting patient survival prognosis. Modeling was performed on four indicators: the ratios of CD4+Tem / CD4T and CD8+Tem / CD4T, and the ratios of PD1+ / CD4+Tem and PD1+ / CD8+Tem, to obtain the T-cell immune score (integrated score). Patients with higher scores were more likely to benefit from immunotherapy, and their progression-free survival was significantly longer than that of patients with lower scores.

[0035] Example 3

[0036] Blood samples were analyzed after immunotherapy to dynamically assess phenotypic or functional changes in peripheral blood T cells during the immunotherapy process. (See attached image) Figure 5 As shown, the levels of CD69 molecules expressed by CD4+Tem and CD8+Tem were significantly increased in patients with a treatment response (CR) after immunotherapy, while no significant change was observed in resistant patients (NCR). Meanwhile, as shown in the attached... Figure 6 As shown, patients with a treatment response generated an increase in CXCR5+Tfh cells after treatment, while the proportion of Tfh cells remained unchanged in resistant patients.

[0037] In summary, the efficacy of immunotherapy can be inferred by detecting the levels of CD69 molecules expressed by CD4+Tem and CD8+Tem, and the proportion of CXCR5+Tfh after immunotherapy, as shown in the attached figure. Figure 7 As shown.

[0038] All parts not described in this invention are the same as or can be implemented using existing technology. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A combination of peripheral blood biomarkers for predicting the immunotherapy efficacy of nasopharyngeal carcinoma, characterized in that, Predictive biomarkers for immunotherapy efficacy: CD4+Tem / CD4+T, CD8+Tem / CD8+T, PD-1+CD4+Tem / CD4+Tem and PD-1+CD8+Tem / CD8+Tem.

2. The peripheral blood biomarker combination according to claim 1, characterized in that: The biomarker combination samples were collected from the prospective clinical trial Jupiter-02.

3. A method for constructing a model to predict the efficacy of immunotherapy in nasopharyngeal carcinoma patients, characterized in that, Includes the following steps: Modeling was performed based on four indicators: CD4+Tem / CD4+T ratio, CD8+Tem / CD8+T ratio, PD-1+CD4+Tem / CD4+Tem ratio, and PD-1+CD8+Tem / CD8+Tem ratio.

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