KR102346864B1 - Biomarker composition for diagnosing or prognostic analysis of bladder cancer, kit comprising the same and method for diagnosing bladder cancer using the same - Google Patents

Abstract

The present invention relates to a biomarker composition and kit for bladder cancer diagnosis or prognosis analysis, and a diagnostic method using the same, and more particularly, to an agent for measuring the protein expression level of apolipoprotein A1; And MMP9 (Matrix metallopeptidase 9) relates to a biomarker composition, kit, and diagnostic method using the same for bladder cancer diagnosis or prognosis analysis comprising a formulation for measuring the expression level of the protein as an active ingredient.

Description

Biomarker composition for diagnosing or prognostic analysis of bladder cancer, kit comprising the same and method for diagnosing bladder cancer using the same

The present invention relates to a biomarker composition, kit for bladder cancer diagnosis or prognosis analysis, and a diagnostic method using the same, and more particularly, to a multiple diagnosis of two combinations of apolipoprotein A1 and MMP9 (Matrix metallopeptidase 9). It relates to the use of predicting the diagnosis and prognosis of bladder cancer.

Bladder cancer is the most frequently occurring cancer in the urinary system, and it is reported that 4.5 cases occur in Korea, compared to 16.5 cases per 100,000 people in the West every year. As such, the incidence rate is low compared to the Western countries, but the incidence rate is increasing every year, and it is known that it is the cancer with the highest incidence among cancers of the urinary system in Korea.

Bladder cancer is largely divided into non-muscle invasive bladder cancer and invasive bladder cancer according to the degree of invasiveness. Noninvasive bladder cancer is a lesion in which the cancer is confined to the mucous membrane without invasion of the muscle layer. recurrence and progression to invasive cancer are problematic. On the other hand, invasive bladder cancer refers to a state in which the cancer has penetrated to the muscle layer, and for its treatment, it is necessary to perform complex urinary diversion along with radical cystectomy, and it may lead to fatal results for the patient. Therefore, it is very important to predict, early detection and prevention of recurrence and progression after primary treatment. Therefore, such frequent recurrence and stage progression are problems frequently raised in bladder cancer, and it is necessary to find an indicator or develop a treatment method that can effectively predict the recurrence and invasiveness of bladder cancer (Korean Patent No. 10-1557183, bladder cancer prognostic diagnostic marker).

Since bladder cancer has a higher chance of recurrence after treatment compared to other cancers, regular reexamination is necessary after surgery (JB Shah, DJ McConkey, CP Dinney, New strategies in muscle-invasive bladder cancer: on the road to personalized medicine, Clin. Cancer Res. 17 (9) (2011) 2608-2612. KN Rayn, GR Hale, GP Grave, PK Agarwal, New therapies in nonmuscle invasive bladder cancer treatment, Indian J. Urol. 34 (1) (2018) 11-19 .). Currently used cystoscopy has limitations in that it is expensive and the method is invasive and can cause pain to the patient (AS Brems-Eskildsen, K. Zieger, H. Toldbod, C. Holcomb, R. Higuchi, F. Mansilla). , L. Dyrskjot, Prediction and diagnosis of bladder cancer recurrence based on urinary content of hTERT, SENP1, PPP1CA, and MCM5 transcripts, BMC Cancer 10 (2010) 646. SF Shariat, JA Karam, Y. Lotan, PI Karakiewizc, Critical evaluation of urinary markers for bladder cancer detection and monitoring, Rev. Urol. 10 (2) (2008) 120-135).

In order to overcome these limitations, there is a growing need to provide a combination of biomarkers for bladder cancer diagnosis that can be conveniently tested non-invasively and has high specificity and sensitivity.

Republic of Korea Patent Registration No. 10-1557183 (2015.09.24)

Accordingly, it is an object of the present invention to provide a biomarker composition, kit for bladder cancer diagnosis or prognosis analysis, and a diagnostic method using the same, which can be predicted with high accuracy by improving the accuracy of diagnosis and prognostic examination of bladder cancer with a combination of biomarker for diagnosis of bladder cancer.

Another object of the present invention is to provide a biomarker composition, kit, and diagnostic method using the same for bladder cancer diagnosis or prognosis analysis, which can be tested conveniently and non-invasively, and has high specificity and sensitivity.

A biomarker composition for bladder cancer diagnosis or prognosis analysis according to a preferred embodiment of the present invention for achieving the above object includes an agent for measuring the protein expression level of apolipoprotein A1; and an agent for measuring the expression level of a protein of matrix metallopeptidase 9 (MMP9); may include as an active ingredient.

In addition, in the biomarker composition for bladder cancer diagnosis or prognosis analysis according to the present invention, the agent for measuring the expression level of the protein may be any one selected from the group comprising an antibody, an aptamer, an antagonist, and a combination thereof.

In addition, in the biomarker composition for bladder cancer diagnosis or prognosis analysis according to the present invention, the protein is at least one biological sample selected from the group consisting of tissues, cells, whole blood, serum, plasma, saliva, sputum and urine obtained from mammals. , and the diagnosis of bladder cancer may include diagnosis of the bladder cancer including progression and recurrence.

The kit for bladder cancer diagnosis or prognosis analysis according to a preferred embodiment of the present invention may include the composition.

In addition, in the kit for bladder cancer diagnosis or prognosis analysis according to the present invention, the kit is a microarray, an aptamer chip kit, an ELISA (enzyme linked immunosorbent assay) kit, a blotting kit, an immunoprecipitation kit , characterized in that it is selected from the group consisting of immunofluorescence kits, protein chip kits, and combinations thereof.

A method of providing information necessary for bladder cancer diagnosis or prognosis analysis according to a preferred embodiment of the present invention is (a) the expression level of apolipoprotein A1 (Apolipoprotein A1) and MMP9 (Matrix metallopeptidase 9) proteins from a biological sample measuring; (b) when the expression level of apolipoprotein A1 (Apolipoprotein A1) and MMP9 (Matrix metallopeptidase 9) protein is high compared to a normal control group, determining that it is a bladder cancer disease.

In addition, in the method of providing information necessary for bladder cancer diagnosis or prognosis analysis according to the present invention, the expression level of the protein is measured using any one selected from the group comprising an antibody, an aptamer, an antagonist, and a combination thereof. it could be

In addition, in the method of providing information necessary for bladder cancer diagnosis or prognosis analysis according to the present invention, the level measurement is, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, octeroni immunodiffusion method, rocket immunoelectrophoresis, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography Any one method selected from the group consisting of graph-mass spectrometry (liquid chromatography-Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/Mass Spectrometry), and ELISA (enzyme linked immunosorbent assay) may be using

In addition, in the method for providing information necessary for bladder cancer diagnosis or prognosis analysis according to the present invention, the biological sample is selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, sputum and urine obtained from a mammal. It is one or more, and the diagnosis of bladder cancer may include diagnosis including progression and recurrence of the bladder cancer.

According to the present invention, the biomarker composition, kit for bladder cancer diagnosis or prognosis analysis of the present invention, and a diagnostic method using the same, are a multi-diagnosis of bladder cancer by using a combination of two types of apolipoprotein A1 (A1) and matrix metallopeptidase 9 (MMP9). By improving the accuracy of diagnostic and prognostic tests, it is possible to predict with high accuracy.

In addition, while the prior art uses invasive cystoscopy and biomarkers in blood for bladder cancer diagnosis, the present invention can provide a method for non-invasively testing a urine sample.

In particular, it is possible to provide a urine biomarker suitable for POCT (point-of-care) or home health care (self-diagnosis) for prognosis management after treatment of highly recurrent bladder cancer.

1 is a graph showing a concentration measurement result according to a clinical study of MMP9 according to an embodiment of the present invention.
Figure 2 is a graph showing the concentration measurement results according to the clinical study of apolipoprotein A1 (Apolipoprotein A1) according to an embodiment of the present invention.
3 is a graph showing the results of performing ROC curve analysis of two types of combination biomarkers of apolipoprotein A1 and MMP9 for sensitivity and specificity.

Since the present invention can apply various transformations and can have various embodiments, specific embodiments will be described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Terms such as first, second, etc. may be used to describe various elements, but the elements should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from another.

Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. It is apparent to those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit and essential characteristics of the present invention.

A biomarker composition for bladder cancer diagnosis or prognosis analysis according to an embodiment of the present invention comprises: an agent for measuring the protein expression level of apolipoprotein A1; And MMP9 (Matrix metallopeptidase 9) may include an agent for measuring the protein expression level as an active ingredient.

"Apolipoprotein" is a protein that binds to a lipid to form a lipoprotein. These proteins can act as enzyme cofactors, receptor ligands and lipid transport carriers regulating the metabolism and uptake of proteins in tissues.

"Apolipoprotein A1" is a protein that is a major protein component of high-density lipoprotein (HDL) in plasma. Apolipoprotein A1 is a cofactor of lecithin cholesterol acyltransferase (LCAT), which is responsible for the formation of most plasma cholesteryl esters. In humans, Apolipoprotein A1 is encoded by the APOAI gene and can be from any origin, eg, human, bovine, murine, equine, canine, and the like.

MMP9 (Matrix metallopeptidase 9) is a type of extracellular matrix metalloproteinase (MMP), and MMP is a type IV collagenase (MMP2, 9), stromelysin (MMP-3) and interstitial collagenase ( MMP-1), type IV collagenase is divided into gelatinase A (MMP-2, molecular weight 72 kD) and gelatinase B (MMP-9, molecular weight 92 kD), and MMP-9 is produced in both normal cells and cancer cells. It is known to be closely related to the malignancy of tumor cells.

Using the biomarker composition for bladder cancer diagnosis or prognosis analysis according to the present invention, bladder cancer can be diagnosed with significantly higher accuracy than single markers of apolipoprotein A1 and MMP9 according to the measurement of the expression level change of apolipoprotein A1 and MMP9. have.

In the present invention, the term “diagnosis” refers to confirming the presence or characteristics of a pathological condition. For the purpose of the present invention, the diagnosis means predicting or confirming the prognosis of bladder cancer, and the bladder cancer diagnosis of the present invention may include diagnosis including progression and recurrence of the bladder cancer. "Prognosis" means predicting the course and outcome of a disease, and the present invention includes predicting the recurrence and progression of bladder cancer.

In the present invention, "biomarker", "marker" or "diagnostic marker" refers to a marker that can distinguish normal or pathological conditions, predict treatment response, and can objectively measure. In particular, in relation to bladder cancer in the present invention, it means that the protein expression level or the gene expression level significantly increases or decreases in an individual having a bladder cancer disease or a possibility (risk) compared to a normal control group.

In the present invention, "measuring the expression level of a protein" refers to a process of confirming the presence and expression level of a bladder cancer diagnostic marker (protein) or a gene encoding the same in a biological sample in order to diagnose bladder cancer. The agent for measuring the expression level of the above protein may be any one selected from the group comprising an antibody, an aptamer, an antagonist, and a combination thereof. Specifically, the preparation includes an antibody specific for a protein encoded by a gene, which refers to a specific protein molecule directed against an antigenic site, and includes all of polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

For measuring the expression level of the protein, quantitative and qualitative analysis methods of proteins known in the art can be used, for example, enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay (sandwich assay). ), Western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate staining, antigen-antibody aggregation method, flow cytometry (Fluorescence Activated Cell Sorter, FACS), mass spectrometry method , MRM (Multiple Reaction Monitoring) analysis, multiple amine-specific stable isotope test method (iTRAQ, Isobaric tags for relative and absolute quantitation), or a protein chip (protein chip) methods such as, but not limited thereto.

In this case, the protein is derived from at least one biological sample selected from the group consisting of tissues, cells, whole blood, serum, plasma, saliva, sputum and urine obtained from a mammal.

A kit for bladder cancer diagnosis or prognosis analysis according to an embodiment of the present invention comprises: an agent for measuring the expression level of the apolipoprotein A1 protein; And MMP9 (Matrix metallopeptidase 9) may include an agent for measuring the protein expression level as an active ingredient.

The kit according to the present invention can diagnose bladder cancer-related diseases by quantitatively or qualitatively analyzing proteins, and protein measurement methods include enzyme immunoassay (ELISA), radioimmunoassay (RIA), and sandwich assay (sandwich assay). ), Western blotting, immunoprecipitation, immunohistochemical staining, fluorescence immunoassay, enzyme substrate coloring, antigen-antibody aggregation method, flow cytometry (Fluorescence Activated Cell Sorter, FACS), mass spectrometry method , MRM (Multiple Reaction Monitoring) analysis, multiple amine-specific stable isotope testing method (iTRAQ, Isobaric tags for relative and absolute quantitation), or a protein chip (protein chip) can be used for measurement.

For example, the diagnostic kit of the present invention may include essential elements necessary for performing ELISA. The ELISA kit contains antibodies specific for these proteins. Antibodies are antibodies with high specificity and affinity for a marker protein and little cross-reactivity with other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. The ELISA kit may also include an antibody specific for a control protein. Other ELISA kits include reagents capable of detecting bound antibody, for example, labeled secondary antibodies, chromophores, enzymes (eg, conjugated with an antibody) and their substrates or capable of binding the antibody. other materials and the like.

The kit according to the present invention is not limited thereto, and microarray, aptamer chip kit, ELISA (enzyme linked immunosorbent assay) kit, blotting kit, immunoprecipitation kit, immunofluorescence test kit, protein chip kits and combinations thereof.

In the method of providing information necessary for bladder cancer diagnosis or prognosis analysis according to an embodiment of the present invention, (a) measuring the expression level of apolipoprotein A1 (Apolipoprotein A1) and MMP9 (Matrix metallopeptidase 9) proteins from a biological sample to do; And (b) when the expression level of the protein of apolipoprotein A1 (Apolipoprotein A1) and MMP9 (Matrix metallopeptidase 9) is high compared to the normal control, it may include the step of determining a bladder cancer disease.

In the present invention, the "biological sample" of step (a) includes a sample with a difference in protein expression level or gene expression level depending on bladder cancer-related diseases, specifically, tissues, cells, and It means at least one selected from the group consisting of whole blood, serum, plasma, saliva, sputum and urine, and the biological sample may be isolated from the human body.

The expression level of the protein in step (a) can be measured using any one selected from the group comprising an antibody, an aptamer, an antagonist, and a combination thereof. In addition, protein expression level measurement includes immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, Radioimmunoassay, radioimmunodiffusion, octeroni immunodiffusion, rocket immunoelectrophoresis, complement fixation assay, two-dimensional electrophoresis, liquid chromatography-Mass Spectrometry (LC-MS), LC- Any one method selected from the group consisting of liquid chromatography-Mass Spectrometry/Mass Spectrometry (MS/MS), and enzyme linked immunosorbent assay (ELISA) may be used, but is not limited thereto.

The "normal control" in step (b) includes a normal group that does not have bladder cancer or a group that does not develop or recovers from bladder cancer. According to the present invention, when protein expression levels of apolipoprotein A1 (A1) and matrix metallopeptidase 9 (MMP9) are high compared to a normal control group, the patient group can be classified as a patient group having bladder cancer.

According to the present invention, it is possible to predict with high accuracy by improving the accuracy of the diagnosis and prognostic examination of bladder cancer through multiple diagnosis of two combinations of apolipoprotein A1 (A1) and matrix metallopeptidase 9 (MMP9).

In addition, while the prior art uses invasive cystoscopy and biomarkers in blood for bladder cancer diagnosis, the present invention can provide a method for non-invasively testing a urine sample.

In particular, it is possible to provide a urine biomarker suitable for POCT (point-of-care) or home health care (self-diagnosis) for prognosis management after treatment of highly recurrent bladder cancer.

Hereinafter, the present invention will be described in more detail through examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

<Example 1>

In the bladder cancer patient group, preoperative urine samples were obtained from 47 patients including TURB (Transurethral Resection of Bladder Tumor) and radical cystectomy, and 19 kidney donors as a normal control group.

ELISA (enzyme-linked immunosorbent assay) was used to measure the concentrations of MMP9 and Apolipoprotein A1 in urine. After obtaining a standard curve by measuring the degree of color development, the concentration was measured (refer to FIGS. 1 and 2).

The concentrations of apolipoprotein A1 and MMP9 show a significant difference between the normal control group and the patient group, which corresponds to a significant statistical result. Therefore, apolipoprotein A1 and MMP9 have significance as bladder cancer biomarker candidates. For a panel combining two biomarkers, Apolipoprotein A1 and MMP9, which showed a significant difference between the normal control group and the patient group, the superiority of bladder cancer diagnosis and prognosis prediction was analyzed as follows.

The ROC curve was confirmed by using the concentration values of each of MMP9 and Apolipoprotein A1 selected as bladder cancer biomarker candidates as one panel (see FIG. 3 ). The ROC curve expresses the relationship between sensitivity and specificity on a two-dimensional plane, and the larger the AUC (area under curve) value corresponding to the area under the ROC curve, the better the diagnosis method. When set to 0.66 on the curve, the most appropriate sensitivity (84%) and specificity (93%) were shown, and AUC was 0.94.

Selection and application of significant diagnostic markers determine the reliability of diagnostic results. A significant diagnostic marker refers to a marker with high reliability so that a result obtained by diagnosis is accurate, thus having high validity and showing consistent results even during repeated measurements. Apolipoprotein A1 and MMP9 of the present invention are a highly reliable marker combination for bladder cancer diagnosis and prognosis management because the difference in expression level is very large compared to the normal control group.

On the other hand, the above detailed description should not be construed as restrictive in all aspects, but should be considered as exemplary. The scope of the present invention should be determined by a reasonable interpretation of the appended claims, and all modifications within the equivalent scope of the present invention are included in the scope of the present invention.

Claims (9)

As a biomarker composition for bladder cancer diagnosis or prognosis analysis,
an agent for measuring the expression level of apolipoprotein A1 protein; and
An agent for measuring the protein expression level of MMP9 (Matrix metallopeptidase 9); contains as an active ingredient,
The bladder cancer diagnosis or prognostic analysis is a diagnosis or prognostic analysis for bladder cancer patients related to TURB (Transurethral Resection of Bladder Tumor) or radical cystectomy bladder cancer diagnosis or A biomarker composition for prognostic analysis.
According to claim 1,
The agent for measuring the expression level of the protein is any one selected from the group comprising an antibody, an aptamer, an antagonist, and a combination thereof, a biomarker composition for bladder cancer diagnosis or prognosis analysis.
According to claim 1,
The protein is derived from at least one biological sample selected from the group consisting of tissues, cells, whole blood, serum, plasma, saliva, sputum and urine obtained from a mammal,
The bladder cancer diagnosis is a biomarker composition for diagnosis or prognosis analysis of bladder cancer, including the progress (progress) and recurrence (Recurrence) of the bladder cancer diagnosis
A kit for diagnosing or prognosing bladder cancer, comprising the composition according to any one of claims 1 to 3.
5. The method of claim 4,
The kit includes a microarray, an aptamer chip kit, an enzyme linked immunosorbent assay (ELISA) kit, a blotting kit, an immunoprecipitation kit, an immunofluorescence assay kit, a protein chip kit, and combinations thereof. A kit for bladder cancer diagnosis or prognosis analysis, characterized in that selected from.
A method of providing information necessary for bladder cancer diagnosis or prognosis analysis, the method comprising:
(a) measuring the expression level of apolipoprotein A1 (Apolipoprotein A1) and MMP9 (Matrix metallopeptidase 9) proteins from a biological sample; and
(b) determining that the expression level of the protein of apolipoprotein A1 (Apolipoprotein A1) and MMP9 (Matrix metallopeptidase 9) is high compared to the normal control as bladder cancer disease;
The bladder cancer diagnosis or prognostic analysis is a diagnosis or prognostic analysis for bladder cancer patients related to TURB (Transurethral Resection of Bladder Tumor) or radical cystectomy bladder cancer diagnosis or How to provide the necessary information for prognostic analysis.
7. The method of claim 6,
The expression level of the protein is measured using any one selected from the group comprising an antibody, an aptamer, an antagonist, and a combination thereof, a method for providing information necessary for diagnosis or prognosis analysis of bladder cancer.
7. The method of claim 6,
The level measurement is, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay , radioimmunodiffusion, octeroni immunodiffusion, rocket immunoelectrophoresis, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-Mass Spectrometry (LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry / Mass Spectrometry), and ELISA (enzyme linked immunosorbent assay) using any one method selected from the group consisting of, bladder cancer diagnosis or a method of providing information necessary for prognosis analysis.
7. The method of claim 6,
The biological sample is at least one selected from the group consisting of tissues, cells, whole blood, serum, plasma, saliva, sputum and urine obtained from a mammal,
The bladder cancer diagnosis is a method of providing information necessary for diagnosis or prognosis analysis of bladder cancer, including diagnosis including progression and recurrence of the bladder cancer.

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