CN108949982B

CN108949982B - Method for evaluating clinical prognosis of glioma by utilizing immune co-stimulatory molecules

Info

Publication number: CN108949982B
Application number: CN201810794978.8A
Authority: CN
Inventors: 邹存义; 吴安华; 程文
Original assignee: First Hospital of China Medical University
Current assignee: First Hospital of China Medical University
Priority date: 2018-07-09
Filing date: 2018-07-09
Publication date: 2021-10-26
Anticipated expiration: 2038-07-09
Also published as: CN108949982A

Abstract

The invention discloses an application of SLAMF8 in preparation of a tumor tissue marker or a serum marker for prognosis evaluation of human glioma, and an application of the SLAMF8 in preparation of a marker for prognosis evaluation of human glioma. The invention also provides a method for evaluating clinical prognosis of glioma by utilizing immune co-stimulatory molecules. According to the invention, the study finds that the SLAMF8 has high expression in the malignant phenotype of glioma and glioblastoma, and is closely related to the poor prognosis of glioma, and the high-expression SLAMF8 is an important marker of the poor prognosis of glioma. Moreover, high expression of SLAMF8 was associated with resistance to chemotherapy. The invention suggests that SLAMF8 influences glioma microenvironment mainly through monocytes and dendritic cells and is closely related to immune response. SLAMF8 can affect glioma immune function, suppress glioma immune response and promote inflammatory response. In conclusion, SLAMF8 can be used as an important clinical prognosis prediction marker for glioma.

Description

Method for evaluating clinical prognosis of glioma by utilizing immune co-stimulatory molecules

Technical Field

The invention relates to a method for evaluating clinical prognosis of glioma by utilizing immune co-stimulatory molecules, relates to application of SLAMF8 in preparation of a tumor tissue marker or a serum marker for human glioma prognosis evaluation and application of SLAMF8 as a marker for human glioma prognosis evaluation, and belongs to the technical field of biological detection.

Background

Glioma is the most common primary tumor of central nervous system, and has high malignancy degree, fast progress and easy recurrence. The current main treatment method is combined treatment of surgical excision and postoperative chemoradiotherapy. Despite advances in treatment, the prognosis for the most malignant Glioblastoma (GBM) patient remains poor. Recently, immunotherapy, including Chimeric Antigen Receptor (CAR) T cell therapy, Dendritic Cell (DC) therapy, and immune site blockade, brought new eosin to patients with brain gliomas. The immune research of glioma is possible to provide new ideas and methods for treating glioma.

The immunomodulating sites include costimulatory and cosuppressive molecules required to generate an immune response. Blocking co-inhibitory checkpoint molecules, such as CTLA-4, PD-1 and PD-L1, have made breakthrough progress in the treatment of a variety of malignancies, including brain gliomas. However, treatment with blockade of the site of immune regulation is beneficial only to a subset of patients with brain gliomas, and a large subset of patients with gliomas fail to benefit. Site-blocking therapy is often associated with immune-inflammatory and other related side effects, such as dermatitis, colitis, inflammatory endocrinopathies, and even fatal cerebral edema. The consequences of inflammatory injury to the brain parenchyma are often severe due to poor regeneration of the central nervous system. Therefore, balancing immune activation and inflammation suppression is an important part of the immunotherapy of brain glioma. In this regard, studies of immune regulatory sites involved in inflammation may be suggestive of immune regulatory site treatment of gliomas.

Family 8 of signaling molecules (SLAMF8, CD353), which is the eighth member of the SLAMF co-stimulatory receptor family, can modulate the development and function of a number of immune cells, including T lymphocytes, B cells, neutrophils, dendritic cells, macrophages, and eosinophils. SLAMF8 activates macrophages during inflammation, which are an important component of gliomas. Tumor-associated macrophages (TAMs) provide a favorable local microenvironment for glioma cell growth, contribute to glioma invasion expansion, and affect local immune and inflammatory responses. SLAMF8 is overexpressed in autoimmune or inflammatory diseases, such as inflammatory bowel disease, in renal transplant patients, suggesting that SLAMF8 plays an important role in immune-related inflammatory responses. However, no study has been reported to date on the role of SLAMF8 in cancer.

Disclosure of Invention

Aiming at the prior art, the invention discovers through research that the immune co-stimulatory molecule SLAMF8 plays an important role in the immune and inflammatory reactions of glioma, and can be used as a potential serological and histological index for evaluating the clinical prognosis of human glioma so as to be used for clinical application.

The invention is realized by the following technical scheme:

the application of SLAMF8 in preparing the marker for the prognosis evaluation of glioma.

The SLAMF8 is used as a marker for the prognosis evaluation of human glioma.

Further, the SLAMF8 is applied to preparing a preparation of a tumor tissue marker or a serum marker for the prognosis evaluation of human glioma.

Still further, the preparation of the tumor tissue marker or the serum marker for the human glioma prognostic evaluation is a reagent or a kit of the tumor tissue marker or the serum marker for the human glioma prognostic evaluation.

A method for evaluating clinical prognosis of glioma by using immune co-stimulatory molecules comprises the following steps: the expression condition of the immune co-stimulatory molecule SLAMF8 in the blood or tumor tissue of a person to be detected is detected, if the expression condition is high, poor prognosis is suggested, and the expression condition can be used as one of clinical diagnosis bases of poor glioma prognosis for reference of doctors.

According to the invention, the study finds that the SLAMF8 has high expression in the malignant phenotype of glioma and glioblastoma, and is closely related to the poor prognosis of glioma, and the high-expression SLAMF8 is an important marker of the poor prognosis of glioma. Moreover, high expression of SLAMF8 was associated with resistance to chemotherapy. The invention suggests that SLAMF8 influences glioma microenvironment mainly through monocytes and dendritic cells and is closely related to immune response. SLAMF8 can affect glioma immune function, suppress glioma immune response and promote inflammatory response. In conclusion, SLAMF8 can be used as an important clinical prognosis prediction marker for glioma.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.

Drawings

FIG. 1: expression and prognostic value of SLAMF8 in glioma, wherein,

a: SLAMF8 expression levels were correlated with grade, histological grade, molecular subtype, and TCGA subtype.

B: SALMF8 was enriched for high expression in the interstitial subtype in CGGA.

C-F: in CGGA, the expression level of SLAMF8 affects the prognosis of patients, and high expression indicates poor prognosis.

G-J: in TCGA, SLAMF8 expression levels affected the patient prognosis, and high expression indicated poor prognosis.

K-M: the prediction of 1-year, 3-year and 5-year survival rates by using the expression level of SLAMF8 in CGGA has good value.

FIG. 2: SLAMF8 prognoses in TCGA expression and in low-grade glioma (LGG), where,

a: the expression level of SLAMF8 was correlated with grade, histopathological classification, molecular typing and TCGA subtype.

B: SLAMF8 was specifically enriched in the mesenchymal subtype of TCGA.

C: SLAMF8 has a prognostic evaluation effect in LGG of CGGA.

D: SLAMF8 has a prognostic evaluation effect in LGG of TCGA.

FIG. 3: the prognostic value of SLAMF8 in different molecular subtypes of CGGA, among which,

a: in LGG, SLAMF8 has predictive significance for IDH wild type.

B: in LGG, SLAMF8 had predictive significance for the IDH mutant group.

C. D: SLAMF8 had no prognostic value in 1P19q co-deficient patients, but had prognostic value in non-co-deficient patients.

E-H: high SLAMF8 has a poor prognosis compared to low SLAMF8 in both IDH wild-type and MGMT methylated states of GBM, whereas there is no significant difference in IDH mutations and MGMT unmethylated states.

FIG. 4: the prognostic value of SLAMF8 in TCGA different molecular subtypes and radiotherapy and chemotherapy, among them,

A-D: in LGG of TCGA, SLAMF8 had predictive value in the 1p19q non-deleted, IDH wild-type and mutant states, while 1p19q co-deleted states had no predictive value.

E-H: for GBM of TCGA, high SLAMF8 was poor prognostic in IDH wild-type and MGMT methylated states, but had no prognostic value in IDH mutant and MGMT unmethylated states.

I-M: SLAMF8 has predictive prognostic value in patients receiving radiation or chemotherapy, but no predictive value in patients not receiving radiation or chemotherapy.

FIG. 5: SLAMF8 had an effect on the response to chemotherapy, where,

A. b: the prognosis of patients receiving chemotherapy was better in the low SLAMF8 group than patients receiving radiotherapy alone, but was not significantly different in the high SLAMF8 group.

C: patients with MGMT methylation who are highly SLAMF8 have a similar prognosis as patients with MGMT non-methylation. Whereas patients with tumors with low SLAMF8 survived significantly longer than patients with MGMT unmethylated.

D: similar C results were present in GBM patients receiving chemotherapy.

E: for GBM patients with MGMT methylation low SLAMF8, the prognosis of chemotherapy was superior to that of pure radiation therapy.

F: for GBM patients with MGMT methylation high SLAMF8, the prognosis of chemotherapy is not significantly different from that of pure radiation therapy.

FIG. 6: SLAMF8 is associated with a tumor microenvironment and immune response, wherein,

a: high SLAMF8 correlates with low glioma purity, monocyte and myeloid dendritic cell and fibroblast enrichment.

B: DAVID analysis indicated that biological functions such as immune response were associated with SLAMF 8.

C-H: GSEA verified biological processes associated with SLAMF8 in CGGA.

FIG. 7: SLAMF8 was correlated with immune score, interstitial score, and glioma purity, wherein,

a to F: high levels of SLAMF8 were associated with high immune scores, high interstitial scores, and low glioma purities in each of the CGGA and TCGA stages.

G-L: SLAMF8 correlates well with immune score, interstitial score, and glioma purity.

FIG. 8: SLAMF 8-associated transcriptome signatures were analyzed with GSEA, where,

A. b: in CGGA and TCGA, SLAMF8 affected the expression profile of the entire transcriptome of glioma.

C: GSEA verified biological functions associated with SLAMF8 at CGGA.

D-J: GSEA verified biological functions associated with SLAMF8 at TCGA.

FIG. 9: the immune inflammatory response is associated with SLAMF8, wherein,

A-D: there was a clear difference between the low and high groups of SLAF8 in CGGA and TCGA in the T cell mediated immune response and the immune response phenotype to tumor cells.

E-H: the classical immune regulatory point is closely and positively correlated with SLAMF 8.

I: the inflammatory response related gene is closely and positively related to SLAMF8 in CGGA.

J: inflammatory processes, including acute and chronic inflammation, are affected by the level of SLAMF8 expression at CGGA.

FIG. 10: SLAMF8 has been implicated in related immunity and inflammation, where,

A-C: gliomas with higher expression levels of SLAMF8 present a stronger immune response phenotype in CGGA.

D-F: glioma with higher expression level SLAMF8 has a stronger immune response phenotype in TCGA.

G-H: the level of SLAMF8 expression in TCGA was closely and positively correlated with the inflammatory response.

Detailed Description

The present invention will be further described with reference to the following examples.

The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.

Example 1 study of the use of SLAMF8 in the prognosis evaluation of human gliomas

The method comprises the following steps:

sample of the patient: the study of the present invention relates to RNA sequencing results and detailed clinical information data of 946 glioma patients, wherein 310 is derived from Chinese glioma genomic map (CGGA) and 636 is derived from American tumor genomic map (TCGA) (https:// TCGA-data. Overall Survival (OS) is the time from diagnosis to death or end of final follow-up.

Bioinformatics analysis: glioma stromal score, immune score, and glioma purity were calculated using the R language to assess the proportion of non-tumor cell components in the tumor. The R-bag calculation microenvironment cell subset is used for calculating the absolute enrichment degree of eight immune cells and two interstitial cells. The corresponding gene is screened by utilizing the Pearson correlation analysis for functional analysis. DAVID was used for gene ontology functional analysis. GSEA was used to analyze the different functional phenotypes between high and low expression groups of SLAMG 8. Principal Component Analysis (PCA) and Gene Set Variation Analysis (GSVA) were used to differentiate genomic, immune function and inflammatory responses caused by different expression of SLAMF 8.

Statistical analysis: SPSS, Graphpad Prism 7 and R3.3.3 (HTTPS:///www.r-project. org) software for statistical analysis. the t-test was used to evaluate the differential expression between the different groups, and the pearson correlation was used to calculate the correlation. The low expression group and the high expression group were grouped according to median expression level. The prognosis results were evaluated using Kaplan-Meier survival analysis, and the log-rank test was used to evaluate the difference in prognosis between the different groups. Cox single factor and multivariate regression analysis were used to determine whether it was an independent prognostic factor. Receiver Operating Curves (ROCs) were plotted using Medcalc software to determine the accuracy of the prediction method. Other statistical calculations and statistical maps are plotted using an R-package (ggplot2, corrplot, pheatmap, etc.). A two-tailed P value of < 0.05 was defined as statistically significant.

And (4) conclusion of results: the description is made in the following eight aspects.

High expression of SLAMF8 in glioblastoma and interstitial subtypes

Based on the expression profile drawn by Graphpad Prism 7, the invention finds that the expression of SLAMF8 is increased along with the malignant progression of glioma, and high-grade glioma expresses higher level of SLAMF8 in CGGA. The level of SLAMF8 expression was best in GBM based on histopathological classification. Among the five molecular typing, SALMF8 was highly expressed in IDH wild-type low-grade glioma (LGG) and IDH wild-type GBM (fig. 1A). The same expression trend was verified in TCGA (fig. 2A).

The TCGA typing protocol classified gliomas into four subtypes, with high interstitial malignancy and poor prognosis. The present inventors found that expression of SLAMF8 was significantly higher in the mesenchymal subtype compared to other subtypes in CGGA and TCGA (fig. 1A and 2A). To validate this finding, we tested SLAMF8 for the ability to predict interstitial patterns using ROC curves. The area under the curve (AUC) was as high as 94.2% and 93.1% in the CGGA data (fig. 1B) and TCGA data (fig. 2B), respectively, indicating that SLAMF8 is more accurate for interstitial subtype prediction than PD-1, TIM-3, CD44, and VIM. These results indicate that SLAMF8 is highly expressed in gliomas with an aggressive malignant phenotype.

2. High SLAMF8 expression indicates poor prognosis of glioma

To test the prognostic value of SLAMF8, groups were expressed according to median and survival curves were plotted. Patients with high SLAMF8 generally survived less than patients with low SLAMF8 (fig. 1C). After hierarchical survival analysis based on glioma grading, although the prognosis of grade II is not statistically significant, in most analyses it was shown that high expression SLAMF8 suggests poor outcome and a shortened overall survival. (FIGS. 1D-F and 2C). Similar analysis was verified in TCGA, with high expression SLAMF8 suggesting poor prognosis (fig. 1G-J and 2D). Considering the prognostic significance of SLAMF8, we used the ROC curve to evaluate the predictive value of SLAMF8 for 1-year, 3-year, and 5-year survival in CGGA. The AUC survival rate was 76.7% at1 year, 80.7% at 3 years, and 85.1% at 5 years, which is more accurate than prediction using age or IDH status (fig. 1K-M).

We then examined SLAMF8 for prognostic independence using Cox regression analysis. Single and multifactorial analyses showed SLAMF8 expression levels to be an independent prognostic factor for glioma (HR ═ 1.074, P ═ 0.008) (table 1). SLAMF8 also independently suggested poor prognosis in TCGA (HR 1.195, P0.001) (table 2). In conclusion, SLAMF8 plays an important role in judging prognosis of glioma, high-expression SLAMF8 indicates poor prognosis, and SLAMF8 can be used as a marker of poor prognosis of glioma.

TABLE 1 Single and Multi-factor regression analysis of clinical prognosis in CGGA

TABLE 2 Single and Multi-factor regression analysis of clinical prognosis in TCGA

SLAMF8 with different prognostic value for glioma of different molecular subtypes

IDH mutations, MGMT promoter methylation and 1P19q co-deletions have important biological and clinical value in gliomas. We investigated the prognostic value of SLAMF8 in different subtypes of LGG and GBM using survival analysis. High expression of SLAMF8 in LGG, whether IDH mutant or wild-type, suggested shorter survival (CGGA fig. 3A and B, TCGA fig. 4A and B). For LGG patients not co-deficient in 1p19q, higher SLAMF8 expression levels meant shorter survival times. However, the prognosis of SLAMF8 was not significant in LGG patients with 1p19q co-deficiency (fig. 3C and D, fig. 4C and D).

High expression of SLAMF8 in IDH wild-type GBM patients suggests a poor prognosis, but no difference in high-low expression of SLAMF8 in IDH mutant patients (CGGA fig. 3E and F, TCGA fig. 4E and F). In MGMT promoter unmethylated GBM, no difference in survival was observed between high and low SLAMF8 expression. However, for MGMT methylated GBM, the level of SLAMF8 expression significantly affected the prognosis (CGGA FIGS. 3G and H, TCGA FIGS. 4G and H).

SLAMF8 associated with GBM chemotherapy

Radiotherapy and chemotherapy are the standard adjunctive treatment for GBM. In CGGA GBM, we investigated the relationship between SLAMF8 and prognosis for different treatment regimens. The prognosis for the low and high SLAMF8 groups was similar for patients without radiotherapy or chemotherapy. However, SLAMF8 had a clear prognostic significance in patients receiving radiation or chemotherapy (fig. 4I-M). We grouped GBM patients according to SLAMF8 expression levels. We found that in the low SLAMF8 group, the chemotherapy patients survived significantly longer than those who received radiotherapy alone. However, this therapeutic benefit was not significant in patients with higher SLAMF8 (fig. 5A and B).

GBM methylated from the reporter MGMT promoter is more likely to have a better prognosis and benefit from chemotherapy. We performed a grouping of survival curves based on MGMT promoter status, treatment modality and SLAMF8 expression and found that for all GBM patients or GBM patients receiving chemotherapy, only patients with MGMT promoter methylation and low SLAMF8 expression had survival advantage over unmethylated patients, whereas patients with high expression of SLAMF8 with MGMT promoter methylation had a prognosis close to that of patients with MGMT promoter unmethylated. (FIGS. 5C and D). Further analysis showed that even with GBM with MGMT promoter methylation, patients only benefited from chemotherapy when SLAMF8 was under expressed (fig. 5E and F). These results indicate that high SLAMF8 is closely associated with chemotherapy resistance.

Effect of SLAMF8 on glioma purity and local immune cell subpopulation

To determine the effect of SLAMF8 on the tumor microenvironment, we calculated the immune score, the interstitial score, and the glioma purity of the CGGA and TCGA. SLAMF8 was positively correlated with immune score, interstitial score, and negatively correlated with glioma purity (fig. 6A and 7G-L). Even when ranked, their relationship is still significant (fig. 7A-F).

To explore the compositional differences of non-tumor cells in different microenvironments, we used the method of microenvironment cell subpopulation counting described by Becht in the literature. The results suggest that SLAMF8 expression correlates well with the degree of enrichment of monocytes (R0.462), myeloid dendritic cells (R0.490), and fibroblasts (R0.444) (fig. 6A). This suggests that high expression SLAMF8 glioma has a more complex matrix composition and recruits more monocytes macrophages and dendritic cells, thereby reducing glioma purity and shaping a more complex tumor microenvironment to promote malignant progression.

SLAMF8 involved in immune and inflammation-related functions

To explore the function of SLAFF8, we performed principal component analysis to investigate the transcriptome profile associated with SLAMF 8. The overall transcriptome expression profile varied significantly between tumor patients with high and low SLAMF8 (fig. 8A, B), suggesting that glioma patients with different levels of SLAMF8 expression have different biological phenotypes.

To elucidate the differences in biological function due to different expression levels of SLAMF8, we screened 1365 related genes with an absolute value of greater than 0.4 in relation to SLAMF8 in CGGA and TCGA for functional analysis (DAVID). These genes were significantly enriched in the IFN-. gamma./TNF/TLR-mediated signaling pathway involved in immune and inflammatory responses, in antigen processing and presentation (FIG. 6B). GSEA was used to further validate the biological function of SLAMF8, and the results indicated that the highly expressed SLAMF8 group had more active antigen processing and presenting capacity and a more active IFN- γ/TNF/TLR-mediated signaling pathway phenotype compared to the less expressed SLAMF8 group (fig. 6C-H and 8C-J). These suggest that SLAMF8 plays an important role in glioma immune and inflammatory responses.

SLAFF8 for enhancing immunosuppressive effects of brain gliomas

To distinguish between cases of immune responses associated with SLAMF8 expression, we performed PCA analysis of the gene set of T cell-mediated immune responses and immune responses to tumor cells. The results showed that the immune response status was significantly different between the high and low SLAMF8 groups based on the SLAMF8 expression status (fig. 9A-D). GSEA demonstrated that the high level SLAMF8 group had a stronger T cell-mediated immunophenotype (fig. 10A-F).

To further elucidate the relationship between SLAMF8 and the anti-tumor immune response, we analyzed the association between SLAMF8 and co-inhibitory regulatory points, including PD-L1, CTLA-4, PD-1, PD-L2, B7-H3, and TIM-3. SLAMF8 was strongly positively correlated with CTLA-4, PD-1, PD-L2, B7-H3 and TIM-3 in the CGGA and TCGA groups. However, SLAMF8 has little correlation with PD-L1. Similar results were obtained for GBM (FIGS. 9E-H). These results suggest that SLAMF8 interacts with a variety of co-inhibitory regulatory molecules, inhibiting an effective anti-tumor immune response, but functions differently from PD-L1.

High expression of SLAMF8 aggravates the inflammatory activity of gliomas

Since the inflammatory response enhances immune suppression of gliomas and promotes tumor progression, we investigated the relationship of different expression levels of SLAMF8 to the inflammatory response. We analyzed its relationship to SLAMF8 using the immunoinflammation-associated gene set and plotted as a heat map. In gliomas, most gene sets, including HCK, LCK, MHC-I, MHC-II, STAT1, and interferon-related genes, are over-expressed in high SLAMF8 patients. Only the IgG gene set associated with B lymphocyte activity was enriched in low SLAMF8 glioma (CGGA fig. 9I, TCGA fig. 10G). We then performed GSVA analysis using the inflammatory response-associated gene ontology, gene set in G0, to more clearly determine the relationship between SLAMF8 and inflammation. The results suggest that SLAMF8 is closely related to both acute and chronic inflammatory responses (CGGA fig. 9J, TCGA fig. 10H). In conclusion, expression of SLAMF8 is crucial for promoting the inflammatory response of gliomas.

The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each such publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

Claims

1. The application of the reagent for detecting the expression level of SLAMF8 in preparing a kit of tumor tissue markers or serum markers for the prognosis evaluation of human glioma.