CN120425051A

CN120425051A - Application of ANGPT2 as a biomarker in molecular classification and prognostic assessment of pheochromocytoma and paraganglioma

Info

Publication number: CN120425051A
Application number: CN202510941404.9A
Authority: CN
Inventors: 李玲玉; 刘阳
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2025-07-09
Filing date: 2025-07-09
Publication date: 2025-08-05

Abstract

The present invention is applicable to the field of oncology medicine and provides the use of ANGPT2 as a biomarker in the molecular typing and prognostic assessment of pheochromocytomas and paragangliomas. The present invention discloses the use of the ANGPT2 gene and its expression products as biomarkers in the molecular typing and prognostic assessment of pheochromocytomas and paragangliomas (PPGLs). Transcriptome sequencing has revealed for the first time that ANGPT2 is specifically and highly expressed in the C1 subtype of PPGLs, and its expression level is significantly correlated with tumor aggressiveness and poor prognosis. Based on this discovery, the present invention provides a diagnostic kit containing an ANGPT2 detection reagent, which can be used for the molecular typing and prognostic assessment of PPGLs. Compared with existing technologies, the technical solution of the present invention offers advantages such as simple detection, low cost, and reliable results, providing a new tool for the precise diagnosis and treatment of PPGLs.

Description

Application of ANGPT2 as biomarker in molecular typing of pheochromocytoma and paraganglioma and prognosis evaluation

Technical Field

The invention belongs to the technical field of oncology, and particularly relates to application of ANGPT2 as a biomarker in molecular typing and prognosis evaluation of pheochromocytoma and paraganglioma.

Background

Pheochromocytomas and paragangliomas (Pheochromocytoma and Paraganglioma, PPGLs) are a rare class of tumors that originate from neuroendocrine cells and have an annual incidence of about 0.8/10 ten thousand. Such tumors exhibit significant clinical and molecular heterogeneity, with about 40% of cases being associated with hereditary tumor syndromes. The present clinic mainly divides PPGLs into three groups, namely a pseudo hypoxia signaling pathway related group (including VHL, SDHx and other gene mutations), a kinase signaling pathway related group (including RET, NF1 and other gene mutations) and a Wnt signaling pathway related group according to the characteristics of the gene mutation. However, the genotyping method based on gene detection has obvious limitations that the detection technology is high, full exon sequencing or targeted sequencing is required, the detection period is long (usually 2-4 weeks is required), the detection cost is high (the single detection cost is about 5000-10000 yuan), only 30-40% of patients have the mutation of the driving gene, and the rest of patients cannot be subjected to systematic typing and accurate prognosis evaluation.

ANGPT2 (Angiopoietin-2) is an important member of the angiopoietin family, regulating angiogenesis and remodeling primarily by binding to Tie2 receptors. The prior study proves that the ANGPT2 is highly expressed in a plurality of solid tumors such as liver cancer, breast cancer and the like, and participates in the tumor angiogenesis and metastasis process. However, no study has been reported at present regarding the expression characteristics of ANGPT2 in PPGLs and its clinical significance.

Therefore, the invention provides the application of the ANGPT2 as a biomarker in molecular typing of pheochromocytoma and paraganglioma and prognosis evaluation.

Disclosure of Invention

The invention aims to provide an application of ANGPT2 as a biomarker in molecular typing and prognosis evaluation of pheochromocytoma and paraganglioma, and aims to solve the problems in the background art.

The aim of the invention is achieved by the following technical scheme:

The application of the ANGPT2 gene or the expression product thereof as a biomarker in preparing a pheochromocytoma and paraganglioma molecular typing reagent, wherein the nucleotide sequence of the ANGPT2 gene is shown as SEQ ID NO.1, and the amino acid sequence of the expression product is shown as SEQ ID NO. 2.

The application of the ANGPT2 gene or the expression product thereof as a biomarker in preparing a chromatophila and paraganglioma prognosis evaluation reagent is provided, wherein the nucleotide sequence of the ANGPT2 gene is shown as SEQ ID NO.1, and the amino acid sequence of the expression product is shown as SEQ ID NO. 2.

A pheochromocytoma and paraganglioma molecular typing kit comprising an ANGPT2 specific detection reagent.

Further, the detection reagent is an anti-ANGPT 2 antibody.

A pheochromocytoma and paraganglioma prognosis evaluation kit comprising an ANGPT2 specific detection reagent.

Further, the detection reagent is an anti-ANGPT 2 antibody.

Compared with the prior art, the invention has the beneficial effects that:

The invention discloses an ANGPT2 gene and application of an expression product thereof as a biomarker in molecular typing of Pheochromocytoma and Paraganglioma (PPGLs) and prognosis evaluation. ANGPT2 was first found to be highly expressed specifically in subtype C1 of PPGLs by transcriptome sequencing and its expression level was significantly correlated with tumor invasiveness and adverse prognosis. Based on this finding, the present invention provides a diagnostic kit comprising an ANGPT2 detection reagent that can be used for molecular typing and prognostic assessment of PPGLs. Compared with the prior art, the technical scheme of the invention has the advantages of simple detection, low cost, reliable result and the like, and provides a new tool for PPGLs accurate diagnosis and treatment.

Drawings

FIG. 1 shows the results of a multi-dimensional analysis of the PPGLs transcriptome type in example 1 of the present invention, wherein A is an overview of PPGLs cohort (n=87 patients) and TCGA cohort (n=179 patients), the number of cases is shown by anatomical classification, subtype identification is performed by clinical data, whole Genome Sequencing (WGS) and RNA-seq, and correlation analysis related to recurrence/metastasis is performed, B is a heat map showing key metabolic and biological pathways of each subtype, pathway enrichment analysis results of three subtypes of pheochromocytoma-C1 (HIF-1 type), C2 (inflammatory type) and C3 (metabolic type) are presented, each row represents a specific biological pathway, color intensity reflects the degree of enrichment of the pathway in different subtypes, C is a heat map showing gene expression change between subtypes, and D is a violin map showing the expression level of the ANGPT2 genes of three identified subtypes in the PPGLs cohort.

FIG. 2 is a graph showing the effect of ANGPT2 on the biological behavior of PPGLs tumor cells in example 2 of the present invention; wherein, A is the ROC curve which shows the diagnostic efficacy of ANGPT2 expression on PPGLs (particularly distinguishing the C1 subtype from the non-C1 subtype), the red point marks the critical point of the highest sensitivity (88.9%) and specificity (83.3%), and AUC is the area under the curve; B is a violin diagram showing the cell cycle score distribution in PPGLs of the ANGPT2 gene high expression group (ANGPT 2 ^High) and low expression group (ANGPT 2 ^Low), and the grouping cutoff value (1.445) was determined based on a in fig. 2; C is a violin graph showing the EMT score distribution of the high (ANGPT 2 ^High) and low (ANGPT 2 ^Low) groups in PPGLs, the cut-off value for the packet (1.445) was determined based on the graph A in FIG. 2, D is a violin graph showing the HIF score distribution of the high (ANGPT 2 ^High) and low (ANGPT 2 ^Low) groups in PPGLs, the cut-off value for the packet (1.445) was determined based on the graph A in FIG. 2, E is the immunoblot result comparing the expression levels of ANGPT2 and Actin in the (ANGPT 2 ^‒/‒) rat PPGL cell line PC12, F is the result of transplanting tumors in GPT2 ^WT or ANGPT2 ^‒/‒ PPGL cells in different mice, G is a line graph showing the growth of the BAGPT 2 ^WT or ANGPT2 PC12 cells 16 days after the nude mice are inoculated, and F is a graph showing the growth of the GPT2 and GPT 58 is the heat proliferation of the GPT2 in the GPT ^‒/‒ cells The method comprises the steps of (1) quantifying EMT scores of the ANGPT2 KO transplanted tumors by using a scatter diagram, quantifying tumor metastasis scores of the ANGPT2 KO transplanted tumors by using a scatter diagram, quantifying cell proliferation scores of the ANGPT2 KO transplanted tumors by using a scatter diagram, and quantifying hypoxia scores of the ANGPT2 KO transplanted tumors by using a scatter diagram. Statistical analysis uses t-test (B-D, I-L) and data are expressed as mean.+ -. Standard deviation. * P <0.001, p <0.0001.

Fig. 3 is a graph of ANGPT2 expression levels versus patient survival in example 3 of the present invention, which compares the Progression Free Survival (PFS) of ANGPT2 high expression group (n=146 patients) and ANGPT2 low expression group (n=120 patients) PPGLs patients by survival curves, integrating the PPGL cohort and TCGA cohort data.

FIG. 4 is an immunohistochemical staining chart of ANGPT2 showing the expression of three proteins of ANGPT2, PCSK1N and GPX3 in the C1, C2 and C3 subtypes PPGLs in example 4 of the present invention, the scale bar representing 50. Mu.m.

Detailed Description

The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention.

Specific implementations of the invention are described in detail below in connection with specific embodiments.

Example 1 characterization of PPGLs transcriptome typing and ANGPT2 expression;

In this study, 87 PPGLs patients diagnosed in 2015-2020 were collected, and after diagnosis by two advanced pathologists, RNA sequencing was performed using the Illumina HiSeq platform, differential expression analysis was performed using DESeq2 software, and postoperative recurrence and metastasis risk was predicted in combination with clinical data (FIG. 1A). PPGLs was divided into three molecular subtypes of C1, C2 and C3 by means of weighted gene co-expression network analysis (WGCNA), wherein the C1 subtype is enriched in hypoxia and HIF-1 signaling pathways and is critical for tumor survival and invasiveness, the C2 subtype is an inflammatory pathway subtype, and the C3 subtype is enriched in metabolic pathways (B in FIG. 1). It was found that ANGPT2, CAV1 etc. genes were up-regulated in the C1 subtype compared to the other subtypes, and ANGPT2 genes were specifically highly expressed in the C1 subtype in an amount 3.2-5.8 times (p < 0.001) that of the other subtypes (C and D in fig. 1).

Example 2 key role of angpt2 in subtype C1 and its potential as diagnostic marker;

in subtype C1, ANGPT2 is expressed at significantly higher levels than C2 and C3, suggesting that it may play an important role in angiogenesis and vascular remodeling processes, thereby promoting tumor progression. Analysis of the subject's working characteristics (ROC) curve showed that ANGPT2 had higher specificity and sensitivity in diagnosis of subtype C1 (auc=0.880, fig. 2 a). According to ROC curve threshold of ANGPT2, samples were divided into two groups, ANGPT2 high-expression (ANGPT 2 ^High) and low-expression (ANGPT 2 ^Low) in PPGLs cohorts, and cell cycle score, EMT score and HIF-1 signal score of ANGPT2 ^High were found to be significantly higher than those of ANGPT2 ^Low (B-D in fig. 2), indicating that ANGPT2 might be not only a specific biomarker of subtype C1, but also a key factor for regulating tumor progression.

To further verify the function of ANGPT2, ANGPT2 was Knocked Out (KO) using CRISPR-Cas9 technology in rat PPGL cell line PC12 and the success of gene knockdown was confirmed by Western blot detection. Western blot detection results showed (E in FIG. 2) that the band of the ANGPT2 protein disappeared in the ANGPT2 KO (ANGPT 2 ^‒/‒) sample compared to the ANGPT2 wild type (ANGPT 2 ^WT) sample. The ACTIN protein bands used as internal references are clearly shown in the samples of ANGPT2 ^WT and ANGPT2 ^‒/‒, and the band intensities are basically consistent. This indicates that the expression level of ANGPT2 protein in the knockout group was significantly reduced, and successful ANGPT2 gene knockout was confirmed from the protein level. Subsequently, comparing the tumor growth of ANGPT2 ^‒/‒ cells with that of ANGPT2 ^WT cells in a BALB/c nude mice subcutaneous engraftment tumor model, the results showed a significant decrease in tumor growth rate in ANGPT2 ^‒/‒ cells (F and G in fig. 2). RNA sequencing results further showed that the levels of key gene expression for EMT, tumor metastasis, cell proliferation and hypoxia-related pathways were significantly reduced following ANGPT2 knockout (H-L in FIG. 2).

Example 3 correlation of angpt2 expression with PPGLs postoperative risk of recurrent metastasis;

The risk of recurrence and metastasis in 87 PPGLs patients in this center and 179 PPGLs patients in the TCGA database were analyzed by Kaplan-Meier method. The results show (fig. 3) that the risk of recurrent metastasis after C1 type surgery characterized by high ANGPT2 expression is extremely high, significantly higher than in the low expression group (p=0.0061). ANGPT2 was further confirmed to be an independent prognostic factor by multifactor Cox regression analysis (hr= 2.34,95% CI: 1.56-3.51).

Example 4 immunohistochemical detection of ANGPT2 as a clinical marker was viable;

Immunohistochemical detection was performed on 87 cases of PPGLs tissue using rabbit anti-human ANGPT2 polyclonal antibody (proteintech-ANGPT 2-24613-1-AP). As shown in fig. 4, immunohistochemical representative images of ANGPT2 in the C1, C2, C3 subtypes and control protein PCSK1N, GPX are shown. Through statistical analysis, 76.5% (26/34) of cases ANGPT2 in the C1 subtype show strong positive expression (the dyeing intensity is more than or equal to 2+), the C2 subtype strong positive rate is 12.1% (4/33), and the C3 subtype strong positive rate is 15.0% (3/20). Three sets of data were processed using the chi-square test, showing statistical significance of the differences between the sets (p < 0.001), demonstrating the feasibility of detecting ANGPT2 as a clinical marker by immunohistochemistry.

Example 5 preparation of angpt2 kit;

A kit comprising the following components was prepared:

anti-ANGPT 2 antibody (1:400 dilution), secondary antibody-HRP complex, DAB color development solution, harris hematoxylin complex, positive control section (PPGLs tissue with high expression of ANGPT 2), negative control section (normal adrenal tissue).

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that modifications and improvements can be made without departing from the spirit of the invention, and it should also be considered that the scope of the invention is not affected by the practice of the invention or the utility of the patent.

Claims

1. Use of the ANGPT2 gene or its expression product as a biomarker in the preparation of a molecular typing reagent for pheochromocytoma and paraganglioma, wherein the nucleotide sequence of the ANGPT2 gene is shown in SEQ ID NO. 1, and the amino acid sequence of its expression product is shown in SEQ ID NO. 2.

2. Use of the ANGPT2 gene or its expression product as a biomarker in the preparation of a prognosis assessment reagent for pheochromocytoma and paraganglioma, characterized in that the nucleotide sequence of the ANGPT2 gene is shown in SEQ ID NO.1, and the amino acid sequence of its expression product is shown in SEQ ID NO.2.

3. A molecular typing kit for pheochromocytoma and paraganglioma, characterized by comprising an ANGPT2 -specific detection reagent.

4. The kit according to claim 3, wherein the detection reagent is an anti-ANGPT2 antibody.

5. A prognosis assessment kit for pheochromocytoma and paraganglioma, characterized by comprising an ANGPT2- specific detection reagent.

6. The kit according to claim 5, wherein the detection reagent is an anti-ANGPT2 antibody.