CN111500721B

CN111500721B - Application of TACE in diagnosis of endoleak after abdominal aortic aneurysm endoluminal repair

Info

Publication number: CN111500721B
Application number: CN202010313974.0A
Authority: CN
Inventors: 王曰伟
Original assignee: Affiliated Hospital of University of Qingdao
Current assignee: Affiliated Hospital of University of Qingdao
Priority date: 2020-04-20
Filing date: 2020-04-20
Publication date: 2022-11-29
Anticipated expiration: 2040-04-20
Also published as: CN111500721A

Abstract

The invention discloses an application of TACE in diagnosis of endoleak after abdominal aortic aneurysm endoluminal repair, which discovers that the remarkable difference of the endoleak and non-endoleak groups of TACE after the abdominal aortic aneurysm endoluminal repair is shown, and further performs ROC curve analysis to discover that TACE has higher area under the curve and higher sensitivity and specificity as a detection variable, thereby indicating that TACE has higher diagnosis efficiency as a diagnosis marker of the endoleak after the abdominal aortic aneurysm endoluminal repair.

Description

Application of TACE in diagnosis of endoleak after abdominal aortic aneurysm endoluminal repair

Technical Field

The invention belongs to the field of biological medicine, and relates to application of TACE in diagnosis of endoleak after abdominal aortic aneurysm intracavity repair.

Background

Abdominal aortic aneurysms are common diseases in vascular surgery, and generally mean that the diameter of abdominal aorta is 3cm, or the lumen is larger than 50% of the normal artery, for example, the diameter of a tumor body is not treated, the tube wall is expanded continuously and becomes thinner gradually, and finally the tumor body is cracked, and the crack can cause heavy bleeding and death. With the development of the intracavity technology, the repair of the abdominal aortic aneurysm has become a main treatment means for the abdominal aortic aneurysm, but the durability thereof is worthy of attention because the incidence rate of postoperative complications is high, the main complications thereof comprise stent displacement fracture or endoleak, and the like, and the endoleak is the most common. These patients need to undergo long-term enhanced CT or ultrasound examination to detect endoleaks as early as possible after surgery. Enhancing CT requires repeated exposure to ionizing radiation and contrast agents, which is contraindicated for patients with iodine allergy or kidney disease. Ultrasound examination is another method of assessing endoleaks, but not every center has specialized infrastructure and trained personnel, and Digital Subtraction Angiography (DSA), although the gold standard for diagnosing vasculopathy, cannot be used as a routine follow-up, since it is an invasive examination, requires comprehensive assessment to be followed, and is expensive. To overcome these disadvantages, a simple, accurate biological marker is needed to diagnose endoleaks.

There are several previous reports of the correlation of biomarker concentrations in blood with endoleaks. The meta-analysis found that there was a significant positive correlation between plasma Matrix Metalloproteinase (MMP) -9 concentration and endoleaks 3 months after intraluminal repair of abdominal aortic aneurysms. However, recent reports indicate that MMP-9 concentrations are not associated with endoleaks. Patients with type I endoleaks had significantly elevated plasma D-dimer concentrations. Yet another study found that post-operative endoleak patients had lower plasma D-dimer levels than patients without endoleaks, but plasma D-dimer concentrations were also reported to be independent of endoleaks. At present, no effective biomarker exists in clinic to assist in judging the endoleak after abdominal aortic aneurysm intracavity repair.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, the invention aims to provide a biomarker related to endoleak after abdominal aortic aneurysm endoluminal repair and a diagnostic product prepared from the biomarker, and to screen patients needing further enhanced CT based on the biomarker, and reduce the influence of contrast agents and ionizing radiation on the patients.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an application of a reagent for detecting the expression level of TACE in a sample in preparing a product for diagnosing endoleak after abdominal aortic aneurysm endoluminal repair.

Further, detecting the expression level of TACE comprises measuring the level of a polynucleotide of TACE by means of a method using a primer and/or probe capable of specifically hybridizing to the polynucleotide of TACE or one or more regions within said polynucleotide.

Further, detecting the expression level of TACE comprises measuring the protein level of the TACE gene product by means of a bioassay using a binding agent, an antibody or a fragment thereof for the TACE protein, its precursor form or its metabolite.

Further, a level of TACE expression detected as compared to a reference level, higher than the reference level is indicative of endoleak following endoluminal repair of an abdominal aortic aneurysm.

Further, the sample serum, plasma or whole blood.

The invention provides a product for diagnosing endoleak after abdominal aortic aneurysm intracavity repair, which comprises a reagent for detecting the expression level of TACE in a sample.

Further, the reagent includes a reagent for detecting the expression level of TACE in the sample by a sequencing technique, a nucleic acid hybridization technique, a nucleic acid amplification technique, a protein immunoassay technique, and preferably, the protein immunoassay technique is an enzyme-linked immunoassay.

Further, the product comprises an instruction for use, which describes the use and method of use of the product, the use being selected from any one of the following: 1) Evaluating the risk of endoleak after abdominal aortic aneurysm endoluminal repair; 2) Diagnosis of endoleaks after endoluminal repair of abdominal aortic aneurysms.

Further, the specification describes a threshold value for evaluating or diagnosing the endoleak after the abdominal aortic aneurysm endoluminal repair, and when the expression level of TACE is detected to be higher than the threshold value, the endoleak after the abdominal aortic aneurysm endoleak is indicated.

In a specific embodiment of the invention, the threshold for detecting the expression level of TACE in a sample using an enzyme-linked immunoassay is 3674.5pg/ml.

Further, the product comprises a chip and a kit.

The invention provides an application of TACE in constructing a model or a device for calculating internal leakage after abdominal aortic aneurysm endoluminal repair.

Detailed Description

Through a large number of experiments and repeated researches, the significant difference of TACE in the population with internal leakage and the population without internal leakage after abdominal aortic aneurysm intracavity repair is found, and the effectiveness of TACE in diagnosing the internal leakage after the abdominal aortic aneurysm intracavity repair is further verified on the basis, so that the invention is completed.

The present invention may utilize any method known in the art to determine the expression level of a gene. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the invention, as long as the level of expression of the biomarker can be detected at the transcriptional or translational level. Methods for detecting the expression level of a gene in the present invention include, but are not limited to, nucleic acid sequencing techniques, nucleic acid hybridization techniques, nucleic acid amplification techniques, protein immunization techniques.

Illustrative, non-limiting examples of nucleic acid sequencing techniques in the present invention include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments. Another illustrative, non-limiting example of a nucleic acid sequencing technique includes next generation sequencing (deep sequencing/high throughput sequencing), which is a unimolecular cluster-based sequencing-by-synthesis technique based on proprietary reversible termination chemical reaction principles. Random fragments of genomic DNA are attached to the surface of optically transparent glass during sequencing, hundreds of millions of clusters are formed on the surface of the glass after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.

Illustrative, non-limiting examples of nucleic acid hybridization techniques in the present invention include, but are not limited to, in Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The location and quantification of the radioactively, fluorescently or antigenically labeled base-labeled probes in the tissue is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

Illustrative, non-limiting examples of nucleic acid amplification techniques in the present invention include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription Mediated Amplification (TMA), ligase Chain Reaction (LCR), strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that some amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while others amplify RNA directly (e.g., TMA and NASBA).

Illustrative, non-limiting examples of protein immunization techniques in the present invention include, but are not limited to, sandwich immunoassays, such as sandwich ELISA, in which detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), enzyme Immunoassay (EIA), fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunization can be carried out, for example, in the form of microtiter plates or strips.

In the present invention, the term "primer" refers to 7 to 50 nucleic acid sequences capable of forming a base pair (base pair) complementary to a template strand and serving as a starting point for copying the template strand. The primers are generally synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer does not necessarily need to be completely identical to the sequence of the template, and may be sufficiently complementary to hybridize with the template. Additional features that do not alter the basic properties of the primer may be incorporated. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of more than one nucleic acid with a homolog, and modification between nucleic acids.

In the present invention, the term "probe" refers to a molecule that is capable of binding selectively to a specifically intended target biomolecule, for example a nucleotide transcript or protein encoded by or corresponding to an intrinsic gene. Probes may be synthesized by one skilled in the art, or may be derived from a suitable biological preparation. Probes can be specifically designed to label them. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

In the present invention, the term "antibody" is used in the broadest sense and specifically covers, for example, monoclonal antibodies, polyclonal antibodies, antibodies with polyepitopic specificity, single chain antibodies, multispecific antibodies and antibody fragments. Such antibodies can be chimeric, humanized, human and synthetic.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, such variants typically being present in minor amounts, except for possible variants that may arise during the course of production of the monoclonal antibody. Such monoclonal antibodies typically include an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from a collection of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the selected target binding sequence may be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

Monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remaining portion of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

An "antibody fragment" comprises a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, fab ', F (ab') 2 and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

"functional fragments" of an antibody of the invention refer to those fragments that retain the binding of the polypeptide with substantially the same affinity as the intact full chain molecule from which they are derived and that are active in at least one assay, such as in a mouse model, or in vitro, the biological activity of an antigen to which the antibody fragment binds.

In the present invention, the term "chip", also referred to as "array", refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.

The invention provides application of TACE in preparation of a model for calculating internal leakage after abdominal aortic aneurysm endoluminal repair. As the skilled artisan will appreciate, the measurement of two or more markers may be used to improve the diagnostic question in the survey. The biochemical markers may be determined individually, or in one embodiment of the invention, they may be determined simultaneously, for example using a chip or bead-based array technology. The concentration of the biomarkers is then interpreted independently, for example using individual retention of each marker, or a combination thereof.

In the present invention, the step of associating a marker level with a certain likelihood or risk may be implemented and realized in different ways. Preferably, the measured concentrations of the gene and one or more other markers are mathematically combined and the combined value is correlated to the underlying diagnostic problem. The determination of marker values may be combined by any suitable prior art mathematical method.

Drawings

FIG. 1 is a graph comparing the diameters, notch1 and TACE before and after the operation between the endoleak group and the group without endoleak, wherein Panel A is a graph comparing diameters, panel B is a graph comparing Notch1, and Panel C is a graph comparing TACE.

FIG. 2 is a ROC plot of TACE as the sensed variable.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example study of markers associated with endoleak following endoluminal repair of abdominal aortic aneurysm

1. Is taken into patients

55 patients with elective abdominal aortic aneurysm intraluminal repair were included in this study. The study protocol was approved by the ethical committee of the affiliated hospital of the Qingdao university. To remove confounding factors that affect post-operative endoleaks, we collected a number of clinical data including gender, age, hypertension, diabetes, hyperlipidemia, coronary heart disease, peripheral arterial disease, chronic obstructive pulmonary disease, and history of smoking.

2. Experimental methods

Computed Tomography Angiography (CTA) (LightSpeed VCT 64, milwaukee, wisconsin, usa) was performed before and 1 month after abdominal aortic aneurysm endoluminal repair to detect possible endoleaks. The maximum diameter of the infrarenal abdominal aorta was measured by CTA 1 month before and after surgery, respectively. Endoleaks were determined by CTA assessment and there was contrast enhancement outside the stent graft but inside the aortic aneurysm wall.

Venous blood samples were collected pre-and post-operatively for 1 month, respectively. Blood samples were collected in EDTA anticoagulation tubes and immediately centrifuged at 1000rpm for 15 minutes. The separated plasma was stored at-80 ℃ until a subsequent batch analysis. Plasma concentrations of Norch1 and TACE were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit (CUSABIO, wuhan Huamei Biotech, inc.) according to the manufacturer's instructions. Plasma samples were routinely diluted 1: 4. The coefficient of variation is less than 10%.

Statistical analysis: all consecutive data are expressed as mean ± sd. Continuous variables were compared using paired t-test or Mann-Whitney U-test to classify variablesBy means of x ² Or Fisher's test. Logistic regression analysis removed the potential confounding effects and determined the relationship of plasma Notch1 or TACE concentration to endoleak. The ability of the patient to determine the presence of endoleaks following surgery was studied using the Receiver Operating Characteristic (ROC) curve and the area under the curve (AUC) for plasma Notch1 or TACE concentrations. All statistical tests are bi-directional, P<0.05 was considered statistically significant. All analyses were performed using Windows' SSPS 16.0 (SPSS inc, chicago, il).

3. As a result, the

The mean age of the patients was 69.8 ± 1.4 years (47-83 years), of which 48 (81.25%) were males. Combined with 40 cases of hypertension, 7 cases of peripheral artery disease, and 22 smokers (table 1). There were 39 cases (70.9%) without endoleaks after the operation, and 16 cases (29.1%) with endoleaks 1 month after the operation.

There was no significant difference in preoperative and postoperative diameters for patients with and without endoleaks (5.7 ± 0.2cm vs 5.8 ± 0.1cm, p =0.488, 6.3 ± 0.2cm vs 6.2 ± 0.2cm, p = 0.257) (table 2, fig. 1A). Patients with and without endoleaks had no difference in plasma Notch1 concentration both before and after surgery (218.8 ± 4.8pg/ml vs 195.9 ± 4.0pg/ml, P =0.061 215.0 ± 4.6pg/ml vs 235.7 ± 7.8pg/ml, P = 0.065) (table 2, fig. 1B). For patients without endoleaks, there was no significant difference between pre-and post-operative plasma TACE concentrations (2358.1 ± 47.9pg/ml versus 2283.5 ± 60.9pg/ml, P = 0.073). In patients with endoleaks following surgery, plasma TACE concentrations increased (2467.0. + -. 54.7pg/ml vs 5376.2. + -. 49.5pg/ml, P < 0.01) (Table 2, FIG. 1C).

The age between the endoleak group and the nonleak group (69.9 ± 1.7 years of vs 69.4 ± 2.3 years, P = 0.573), preoperative diameter (5.8 ± 0.2mm vs 6.2 ± 0.2mm, P = 0.11) and postoperative plasma Notch1 concentration (195.9 ± 4.0pg/ml vs 235.7 ± 7.8pg/ml, P = 0.067) were not significantly different. Male, diabetes, hyperlipidemia, coronary heart disease, peripheral artery disease, chronic obstructive pulmonary disease and history of smoking are not associated with postoperative endoleaks. The prevalence of hypertension was higher in the endoleak group (93.8% vs 64.1%, P = 0.043), and was considered as a confounding factor in the subsequent analysis (table 1). Patients with endoleaks had higher post-operative plasma TACE concentrations than patients without endoleaks (5376.2 ± 49.5pg/ml vs 2467.0 ± 54.7pg/ml, P < 0.01) (table 1). In determining the presence or absence of endoleaks, ROC curve analysis of plasma TACE concentrations yielded an AUC of 0.824 (95% CI of 0.713-0.935) (FIG. 2). The optimal critical value for the presence of endoleaks after plasma TACE diagnosis is 3674.5pg/ml, at which the diagnosis of endoleaks is most accurate with a sensitivity of 87.5% and a specificity of 71.8%.

TABLE 1 patient data

TABLE 2 comparison of plasma Notch1, TACE concentrations and aneurysm diameters

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. Use of a reagent for detecting the level of TACE expression in a sample selected from the group consisting of serum, plasma and whole blood for the preparation of a product for the diagnosis of endoleaks following endoluminal repair of an abdominal aortic aneurysm.

2. Use according to claim 1, wherein detecting the expression level of TACE comprises measuring the level of a polynucleotide of TACE by means of a method using a primer and/or probe capable of specifically hybridizing to a polynucleotide of TACE or to one or more regions within said polynucleotide.

3. Use according to claim 1, wherein detecting the expression level of TACE comprises measuring the protein level of the TACE gene product by means of a bioassay.

4. The use of claim 1, wherein a detected level of TACE expression, compared to a reference level, above the reference level is indicative of endoleak following endoluminal repair of an abdominal aortic aneurysm.

5. The use of claim 1, wherein the reagent comprises a reagent for detecting the expression level of TACE in the sample by sequencing, nucleic acid hybridization, nucleic acid amplification, or protein immunization techniques.

6. Use according to claim 5, wherein the protein immunization technique is an enzyme-linked immunoassay.

7. The use according to any one of claims 1 to 6, wherein the product comprises instructions for use which describe the use of the product and the method of use, said use being selected from any one of: 1) Evaluating the risk of endoleak after abdominal aortic aneurysm endoluminal repair; 2) Diagnosis of endoleaks after endoluminal repair of abdominal aortic aneurysms.

8. The use of claim 7, wherein the instructions recite a threshold value for assessing or diagnosing endoleak following endoluminal repair of an abdominal aortic aneurysm, wherein detection of a level of TACE expression above the threshold value is indicative of endoleak following endoleak.

9. The use of claim 8, wherein the threshold for detecting the expression level of TACE in a sample using an enzyme-linked immunoassay is 3674.5pg/ml.