Biomarker for prognosis evaluation of recurrent nasopharyngeal carcinoma patient and application thereof

Abstract

The invention provides a prognosis biomarker for a recurrent nasopharyngeal carcinoma patient and application of the prognosis biomarker. The biomarker is CD8 + ZNF683 + cells and / or SIRP alpha + myeloid cells and / or a three-level lymph structure. Compared with a relapse nasopharyngeal carcinoma patient with a low CD8 + ZNF683 + cell proportion, the relapse nasopharyngeal carcinoma patient with a high CD8 + ZNF683 + cell proportion is poorer in OS and LRRFS and has a poorer survival outcome. In addition, the expression of CD47 in rNPC malignant cells is higher than that of pNPC, and the expression of SIRP alpha in myeloid cells closer to CD47 + tumor cells in a recurrent nasopharynx cancer sample is obviously higher. In addition, compared with patients with nasopharyngeal carcinoma primarily treated, patients with recurrent nasopharyngeal carcinoma have fewer three-level lymphatic structures, patients with recurrent nasopharyngeal carcinoma with high-density three-level lymphatic structures have better survival results, and the density of the three-level lymphatic structures is remarkably related to prognosis of the patients with recurrent nasopharyngeal carcinoma. Therefore, the CD8 < + > ZNF683 < + > cell, the SIRP alpha < + > myeloid cell and the three-level lymph structure can be used as specific biomarkers for prognosis of the recurrent nasopharyngeal carcinoma, and the immune escape state and survival outcome of a patient with the recurrent nasopharyngeal carcinoma can be evaluated.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor prognosis assessment, and specifically relates to biomarkers for prognosis assessment of patients with recurrent nasopharyngeal carcinoma and their applications. Background Art

[0002] Nasopharyngeal carcinoma (NPC) is highly prevalent in my country, and a large number of infiltrating immune cells have been found around tumor foci, suggesting the presence of a specific tumor microenvironment (TME) in NPC. The TME is a complex microecological system characterized by spatiotemporal interactions between heterogeneous cell types, including malignant cells, immune cells, and stromal cells. The TME and its heterogeneity are closely associated with resistance to tumor therapy and tumor recurrence.

[0003] Immune checkpoint blockade (ICB) has demonstrated clinical efficacy by enhancing immune activation to counteract immune evasion and has become an important salvage therapy for rNPC, although response rates remain suboptimal. While the TME and immune escape mechanisms can influence the therapeutic efficacy of ICB, the underlying mechanisms of immune escape in recurrent NPC remain unclear. Therefore, studying the immune microenvironment of recurrent NPC and analyzing the mechanisms of immune escape in recurrent NPC may provide new targets for prognosis and treatment. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide a biomarker for evaluating the prognosis of patients with recurrent nasopharyngeal carcinoma and its application. The biomarker is related to the immune escape of recurrent nasopharyngeal carcinoma and can evaluate the prognosis of patients with recurrent nasopharyngeal carcinoma.

[0005] To achieve the above objectives, the present invention adopts the following technical solutions.

[0006] The first aspect of the present invention is to provide the use of CD8+ZNF683+ cells and / or SIRPα+ myeloid cells and / or tertiary lymphoid structures as biomarkers in the prognosis assessment of recurrent nasopharyngeal carcinoma.

[0007] The second aspect of the present invention is to provide a reagent for detecting the content of biomarkers in a biological sample for use in preparing a prognosis assessment product for recurrent nasopharyngeal carcinoma, wherein the biomarkers include at least one of CD8+ZNF683+ cells, SIRPα+ myeloid cells and tertiary lymphoid structures.

[0008] In some embodiments, the reagents include reagents for detection using IHC staining, flow cytometry, HE staining, or immunofluorescence staining.

[0009] In some embodiments, the reagents include antibodies specific for detecting CD8+ZNF683+ cells and / or SIRPα+ myeloid cells and / or tertiary lymphoid structures.

[0010] In some embodiments, the product is a kit.

[0011] The third aspect of the present invention is to provide a kit for evaluating the recurrence of nasopharyngeal carcinoma, wherein the kit includes a reagent for detecting the content of a biomarker in a biological sample; the biomarker includes at least one of CD8+ZNF683+ cells, SIRPα+ myeloid cells and tertiary lymphoid structures.

[0012] In some embodiments, the reagents include reagents for detection using IHC staining, flow cytometry, HE staining, or immunofluorescence staining.

[0013] In some embodiments, the reagent comprises a specific detection antibody against CD8+ZNF683+ cells and / or SIRPα+ myeloid cells and / or tertiary lymphoid structures.

[0014] The present invention has discovered biomarkers associated with the prognosis of recurrent nasopharyngeal carcinoma through research. The biomarkers are CD8+ZNF683+ cells and / or SIRPα+ myeloid cells and / or tertiary lymphoid structures. Compared with newly treated samples, the proportion of CD8+ZNF683+ T cells in recurrent nasopharyngeal carcinoma samples is significantly increased. The immune escape of patients with recurrent nasopharyngeal carcinoma is associated with the conversion of T cells to more tissue-resident, low-toxic ZNF683+ T cells. Compared with patients with recurrent nasopharyngeal carcinoma with a low proportion of CD8+ZNF683+ cells, patients with recurrent nasopharyngeal carcinoma with a high proportion of CD8+ZNF683+ cells have worse OS and LRRFS, and have worse survival outcomes. In addition, through ligand receptor analysis, it was found that in recurrent nasopharyngeal carcinoma samples, tumor cells and myeloid cells interact through CD47-SIRPα, and CD47 expression in rNPC malignant cells is higher than that in pNPC. In recurrent nasopharyngeal carcinoma samples, SIRPα expression is significantly higher in myeloid cells closer to CD47+ tumor cells. In addition, compared with patients with newly diagnosed NPC, patients with recurrent NPC had fewer tertiary lymphatic structures. Compared with patients with recurrent NPC with low-density tertiary lymphatic structures, patients with recurrent NPC with high-density tertiary lymphatic structures had better survival outcomes. The density of tertiary lymphatic structures was significantly correlated with the prognosis of patients with recurrent NPC.

[0015] Therefore, CD8+ZNF683+ cells, SIRPα+ myeloid cells and tertiary lymphoid structures can be used as specific biomarkers for the prognosis of recurrent NPC and evaluate the immune escape status and survival outcomes of patients with recurrent NPC. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 These are the experimental results showing that CD8+ZNF683+T cells are associated with immune escape in patients with recurrent nasopharyngeal carcinoma.

[0017] Figure 2 These are the experimental results showing that CD8+ZNF683+T cells are associated with the prognosis of patients with recurrent nasopharyngeal carcinoma.

[0018] Figure 3 These are experimental results showing that SIRPα+ myeloid cells are associated with immune escape in patients with recurrent nasopharyngeal carcinoma.

[0019] Figure 4 These are the experimental results on the correlation between tertiary lymphatic structure and the prognosis of patients with recurrent nasopharyngeal carcinoma. DETAILED DESCRIPTION

[0020] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0021] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0022] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0023] The "and / or" mentioned in the present invention describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0024] The following describes the method in conjunction with specific embodiments.

[0025] Example 1

[0026] We collected fresh tissue samples from 11 newly diagnosed and 13 recurrent NPC patients for single-cell sequencing. We also collected samples from 8 newly diagnosed and 7 recurrent patients for spatial transcriptome sequencing. Single-cell and spatial transcriptome sequencing were performed by Guangzhou Yuanxin Biotechnology Co., Ltd.

[0027] By constructing a global tumor ecosystem map, we found that relapse samples had higher immune surveillance and immune escape characteristics.

[0028] In the process of further comparing the differences in tumor microenvironment between relapsed and newly treated samples, we found 18 types of T / NK cell subsets, including 4 types of NK cell subsets, 6 types of CD4+T cell subsets, and 8 types of CD8+T cell subsets. Among them, CD8+T cells can also be divided into naive type, cytotoxic type and exhausted type according to their corresponding gene characteristics ( Figure 1 A~ Figure 1 C, Figure 1 E), where we found that the proportion of CD8+ZNF683+T cells in recurrent NPC samples was significantly increased compared with that in newly treated samples ( Figure 1 D). We further used Monocle 3 to perform evolutionary trajectory analysis to explore the dynamic immune status and cell transformation of infiltrating CD8+ T cells in nasopharyngeal carcinoma ( Figure 1 F and Figure 1 G). Based on pseudo-chronic analysis, we found that this transition begins with CD8+TCF7+T cells, passes through the intermediate toxic state of CD8+FOSB+T cells, CD8+ITM2C+T cells, and finally reaches two endpoints, one of which is significantly exhausted CD8+TIGIT+T cells and the other is CD8+ZNF683+T cells with a memory phenotype ( Figure 1 E- Figure 1 G).

[0029] We further explored the cells and found that the main characteristics of CD8+ZNF683+T cells were low cytotoxicity and low expression of granzyme fraction memory T cell phenotype, indicating that they were in a relatively static state and had weak cytotoxicity. The evolutionary trajectory analysis also showed that CD8+ZNF683+T cells were tissue-resident phenotype ( Figure 1 E- Figure 1 G).

[0030] In addition, there was no significant difference in exhaustion scores between relapsed and newly treated samples, while the costimulation score of T cells in relapsed NPC was significantly lower than that in newly treated samples ( Figure 1 H and Figure 1 This suggests that the immune escape of tumor cells is not due to the transformation of T cells from a naive to an exhausted phenotype in rNPCs, but may be caused by the conversion of T cells to more tissue-resident, low-toxic CD8+ZNF683+T cells.

[0031] The above results suggest that CD8+ZNF683+T cells are associated with immune escape in patients with recurrent nasopharyngeal carcinoma, and patients with recurrent nasopharyngeal carcinoma with a high proportion of CD8+ZNF683+T cells may be more likely to achieve immune escape and thus have a worse prognosis.

[0032] Example 2

[0033] We further verified the results of Example 1.

[0034] 1. Verification queue 1

[0035] Forty paired samples from patients with newly diagnosed and relapsed nasopharyngeal carcinoma were collected as validation cohort 1, and the proportion of CD8+ZNF683+T cells in the samples was detected by IHC staining.

[0036] The results showed that the proportion of CD8+ZNF683+T cells in patients with recurrent nasopharyngeal carcinoma was significantly higher than that in patients with initial treatment ( Figure 2 A and 2B), which is consistent with the results of Example 1.

[0037] 2. Verification queue 2

[0038] We further collected 86 previously radiotherapy-naive nasopharyngeal carcinoma samples as validation cohort 2. We measured the proportion of CD8+ZNF683+ T cells in these samples and divided them into a high-CD8+ZNF683+ T cell proportion group and a low-CD8+ZNF683+ T cell proportion group based on the results. Patients in the top 50% of MCAM expression levels were designated as the high-MCAM expression group, while those in the bottom 50% were designated as the low-MCAM expression group. OS and LRRFS were analyzed for both groups.

[0039] The results showed that compared with the low CD8+ZNF683+T cell ratio group, the high CD8+ZNF683+T cell ratio group was significantly associated with worse survival outcomes such as OS and LRRFS ( Figure 2 C).

[0040] 3. Detection of Cellular Granzyme B Expression Levels

[0041] To better verify the above results, we obtained fresh nasopharyngeal carcinoma tissue samples, dissociated them, separated CD8+ZNF683+ and CD8+ZNF683- T cells by flow cytometry, and detected their granzyme B expression levels respectively.

[0042] Flow cytometric sorting and granzyme B expression assay were performed as follows: ZNF683 T cells were first identified and sorted. Fresh tumor tissue was dissected, stored in serum-free RPMI 1640 medium (Cat. C11875500BT, GIBCO), and minced with scissors. Cells were digested at 37°C for 1 hour using a Tumor Dissociation Kit (Cat. 130-095-929, Miltenyi Biotec). Digestion was stopped by adding 1640 medium supplemented with 5% FBS (Cat. FSP500, ExCell Bio). Cells were filtered through a 40 μm cell strainer (Cat. 258369, NEST), centrifuged, and washed. Cells were incubated with 5% BSA for 10 minutes, stained with a CD8 antibody (1:1000, 301014, BioLegend), and fixed with 4% paraformaldehyde for 20 minutes. The cells were resuspended in 1x Permeabilization Medium, centrifuged at 720 g for 7 minutes, and the supernatant discarded. The above steps were repeated once. The cells were then incubated with an antibody against ZNF683 (1:400, ZRB1736-4X25UL, Sigma-Aldrich) at 4°C overnight, washed in the dark, centrifuged at 720 g for 7 minutes, and the supernatant discarded. The washed cells were incubated with an antibody against Granzyme B (1:1000, 396413, biolgend) at room temperature for 30 minutes, washed with PBS, centrifuged at 720 g for 7 minutes, and the supernatant discarded. After antigen retrieval and washing, the cells were incubated with a secondary antibody (1:100, A11008, Invitrogen) for 30 minutes in the dark. The cells were washed and centrifuged three times. The results were analyzed using a flow cytometer FC 500 (Beckman Coulter).

[0043] The results showed that the expression level of granzyme B in CD8+ZNF683+T cells was significantly decreased ( Figure 2 D) indicates that a type of CD8+ZNF683+T cells in T cells of patients with recurrent nasopharyngeal carcinoma is significantly increased, mainly memory-type low-toxic tissue-resident T cells with reduced cytotoxicity, which may be one of the immune escape mechanisms of recurrent nasopharyngeal carcinoma.

[0044] In general, we found that the CD8+T cell population of rNPC was enriched in memory T cells, and a type of CD8+ZNF683+T cells was significantly increased with weak cytotoxicity, which may contribute to the immune escape of rNPC and is significantly correlated with the prognosis of rNPC.

[0045] Example 3

[0046] Based on the sequencing results of Example 1, we also explored the differences in myeloid cells between newly treated and relapsed samples. The results showed that there was a significant increase in a type of FCN1+ monocytes ( Figure 3 In addition, multicolor immunofluorescence staining confirmed that the expression ratio of FCN1+ monocytes in recurrent NPC samples was increased compared with the newly diagnosed NPC samples in validation cohort 1 ( Figure 3 C and 3D), suggesting that it may be involved in immune escape.

[0047] Furthermore, based on spatial transcriptome analysis, we found that myeloid cells were located closer to tumor cells than other immune cells and their interactions with tumor cells were significantly enhanced in relapse samples ( Figure 3 E). Further analysis of ligand receptors revealed that tumor cells and myeloid cells in recurrent NPC samples interacted through CD47-SIRPα ( Figure 3 F). We observed that CD47 expression in rNPC malignant cells was higher than that in pNPC ( Figure 3 G). In addition, we found that SIRPα expression was significantly higher in myeloid cells closer to CD47+ tumor cells in recurrent NPC samples, but not in PT (naive NPC) samples ( Figure 3 H and 3I). Furthermore, through multiple immunohistochemical staining, we confirmed that SIRPα+CD1C+ dendritic cells were located near CD47+ recurrent malignant cells and inhibited antigen presentation through CD47-SIRPα interaction ( Figure 3 I).

[0048] These data explore the CD47-SIRPα interaction, highlighting its potential role in inhibiting DC maturation and antigen presentation in rNPCs.

[0049] Example 4

[0050] Based on the sequencing results of Example 1, we observed that the proportion of B cells in rNPC was significantly reduced, which was further confirmed in the validation cohort 1 ( Figure 4 D). In addition to T cells, the adaptive immune system is mainly composed of B cells. We further detected 23,394 B cells, which can be divided into five clusters, including B IGHM, B MCM4, B NEIL1, B FCRL4 and B memory ( Figure 4 A- Figure 4 C). In addition, we observed a significant decrease in the proportion of memory B cells and an increase in the proportion of B IGHM cells in rNPCs ( Figure 4E). B cell localization to so-called tertiary lymphoid structures (TLSs) has been found in several types of cancer and may improve antigen presentation, increase cytokine-mediated signaling, release tumor-specific antibodies, and lead to better survival outcomes. Based on spatial transcriptome co-localization analysis, we found that in pNPCs, CD4+ T, CD8+ T, Treg, and cycling T cells were generally located closer to B cells ( Figure 4 F).

[0051] We further performed H&E staining on eight pNPC and seven rNPC samples. The method was as follows: fresh NPC tissue samples obtained by biopsy or surgery were fixed in 10% formalin for 12 hours, embedded in paraffin, and sectioned, ensuring integrity. The sections used for H&E staining were 4-5 μm thick. The positive or negative TLs were determined based on the staining results. An experienced pathologist selected representative sections under a microscope to observe the presence of tertiary lymphoid structures surrounded by lymphocytes and counted the number of tertiary lymph nodes within the field of view.

[0052] After H&E staining, the overall tissue of each tumor and the presence and location of TLS were annotated by a trained pathologist (WMH). We found 5 of 8 pNPC samples to be TLS positive (62.5%), while no TLS positive samples were found in 7 rNPC samples (0.0%) ( Figure 4 G). Compared with other tumor tissues, the TLS region had a higher distribution of B lineage and T cell scores; NK cells were significantly reduced in the TLS (p = 5.91e-05 and p = 3.77e-10), while pDC cells and cycling T cells were evenly distributed and scored higher in the TLS region. In addition, we found that all B cell subtypes except B FCRL4 cells were enriched in the TLS region. In contrast, CD8+ZNF683+ cells and CD8+HAVCR2+ cells were significantly reduced in the TLS region. TLS-negative tumors did not show this organized pattern, showing very low and diffusely distributed B lineage scores, as well as scattered localization of other immune and stromal features ( Figure 4 G).

[0053] The above results indicate that the tertiary lymphoid structure in patients with recurrent nasopharyngeal carcinoma is significantly reduced or even disappears compared with patients with initial treatment, which may be one of the reasons for the occurrence of immune escape.

[0054] Based on the results of gene expression profiling, we next performed histological assessment of tumor samples to gain deeper insights into the density and distribution of B cells and their association with TLS and survival outcomes. We validated the correlation between TLS and immune escape and prognosis in recurrent NPC in validation cohorts 1 to 3. Validation cohorts 1 to 2 were the same as those in Example 2, while validation cohort 3 was a prospective trial cohort reported by our team, which included 52 patients with recurrent or metastatic NPC who received treatment with a PD-1 inhibitor and an anti-VEGFR antibody.

[0055] First, we performed immunofluorescence staining structural analysis on the sections of the validation cohort 2 samples and showed that CD20+ B cells co-localized with CD4+ T cells, CD8+ T cells, FOXP3+ T cells, and CD21+ follicular dendritic cells in the TLS area ( Figure 4 H).

[0056] H&E staining was used to detect the TLS density in the validation cohort 1 samples. The density was calculated as follows: experienced pathologists counted the number of TLS in representative fields of view for PT and RT. The results showed that the TLS density of PT samples in the validation cohort 1 was confirmed to be higher than that of RT samples ( Figure 4 I).

[0057] We detected the TLS density of samples in the validation cohort 2 and further divided them into a high TLS density group and a low TLS density group. The grouping criteria were as follows: the median value of TLS density in the sample was selected, and the top 50% density group was defined as the high TLS density group, and the bottom 50% was defined as the low density group. We found that in the validation cohort 2, the high TLS density group had a better OS ( Figure 4 J) and LRRFS( Figure 4 K). Therefore, TLS density was significantly associated with the survival outcome of patients with recurrent NPC.

[0058] Interestingly, in validation cohort 3, patients with negative TLS status had worse PFS outcomes after treatment with anti-VEGFR and PD-1 inhibitors ( Figure 1 L).

[0059] These results indicate that TLS are significantly reduced in rNPCs, are associated with the promotion of rNPC immune escape, and are significantly correlated with poor survival outcomes.

[0060] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0061] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Application of CD8+ZNF683+ cells and / or SIRPα+ myeloid cells and / or tertiary lymphoid structures as biomarkers in the prognostic assessment of recurrent nasopharyngeal carcinoma.

2. The use of a reagent for detecting the content of a biomarker in a biological sample in the preparation of a product for evaluating the prognosis of recurrent nasopharyngeal carcinoma, characterized in that: The biomarkers include at least one of CD8+ZNF683+ cells, SIRPα+ myeloid cells, and tertiary lymphoid structures.

3. The use according to claim 2, characterized in that The reagents include IHC staining, immunofluorescence staining, flow cytometry 、 Reagents for HE staining.

4. The use according to claim 3, characterized in that The reagents include specific detection antibodies against CD8+ZNF683+ cells and / or SIRPα+ myeloid cells and / or tertiary lymphoid structures.

5. The use according to any one of claims 2 to 4, characterized in that: The product is a test kit.

6. A kit for evaluating the recurrence of nasopharyngeal carcinoma, characterized in that: The kit includes reagents for detecting the content of biomarkers in a biological sample; the biomarkers include at least one of CD8+ZNF683+ cells, SIRPα+ myeloid cells and tertiary lymphoid structures.

7. The kit according to claim 6, wherein The reagents include IHC staining, immunofluorescence staining, flow cytometry 、 Reagents for HE staining.

8. The kit according to claim 7, wherein The reagents include specific detection antibodies against CD8+ZNF683+ cells and / or SIRPα+ myeloid cells and / or tertiary lymphoid structures.

Images