CN117425827A - Kit for diagnosing cancer and use thereof - Google Patents

Abstract

A kit for diagnosing cancer, a kit for determining prognosis of a cancer patient, a method for determining a biological sample, a method for collecting data for determining whether a subject has cancer, and a method for predicting prognosis of a cancer patient are provided. A kit for diagnosing cancer comprising: specific binding substances for SOCS box protein 2 (SPSB 2) protein containing SPRY domain, primer set for amplifying cDNA of SPSB2 gene or probe specifically hybridized with mRNA of SPSB2 gene.

Description

Kits for diagnosing cancer and their uses

Technical field

The present invention relates to a kit for diagnosing cancer and its use. More specifically, the present invention relates to a kit for diagnosing cancer, a kit for determining the prognosis of a cancer patient, a method for determining a biological sample, a method for collecting and determining whether a subject A method for data on patients with cancer, a method for predicting prognosis in cancer patients, and a method for screening anti-cancer agents. Priority is claimed for Japanese Patent Application No. 2021-097456 filed in Japan on June 10, 2021, and Japanese Patent Application No. 2021-201094 filed in Japan on December 10, 2021, the contents of which are incorporated by reference. Enter this article.

Background technique

Among cancers, the incidence and mortality of bladder cancer have been increasing (see, for example, Non-Patent Document 1). Examples of treatments for bladder cancer include surgery, chemotherapy, and radiation therapy. However, the effectiveness of treating bladder cancer is not impressive. Examples of reasons for this include the absence of useful cancer markers and follow-up markers.

[citation list]

[Non-patent literature]

[Non-patent document 1]

Fitzmaurice C. et al. Global, regional and national cancer incidence, mortality, years of life lost, years lived with disability and years lived with disability adjusted for 29 cancer groups, 1990 to 2017: a systematic analysis from the Global Burden of Disease Study ("Global, Regional, and National Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life-Years for 29 Cancer Groups, 1990 to 2017: A Systematic Analysis for the Global Burden of Disease Study"), "American Medicine JAMA Oncol., Volume 5, Issue 12, Pages 1749-1768, 2019.

Contents of the invention

[technical problem]

The purpose of the present invention is to provide a technology for diagnosing cancer.

[Problem solution]

The present invention includes the following aspects.

[1] A kit for diagnosing cancer, including: a specific binding substance for the SPRY domain-containing SOCS box protein 2 (SPSB2) protein; a primer set for amplifying the cDNA of the SPSB2 gene; or a combination with the SPSB2 gene Probes for specific hybridization of mRNA.

[2] The kit for diagnosing cancer according to [1], wherein the cancer is bladder cancer, pancreatic cancer, or hepatocellular carcinoma.

[3] A kit for determining the prognosis of cancer patients, including: a specific binding substance for the SPSB2 protein, a primer set for amplifying the cDNA of the SPSB2 gene, or a probe that specifically hybridizes to the mRNA of the SPSB2 gene.

[4] The kit for determining prognosis according to [3], wherein the cancer is bladder cancer, pancreatic cancer, or hepatocellular carcinoma.

[5] A method for determining a biological sample, comprising: a step of measuring the expression level of SPSB2 protein or SPSB2 gene in the biological sample, wherein the measured expression level of the protein or gene is higher than the expression level of a control The facts indicate that the biological sample came from a cancer patient.

[6] The determination method according to [5], wherein the cancer is bladder cancer, pancreatic cancer, or hepatocellular carcinoma.

[7] A method for collecting data for determining whether a subject has cancer, the method comprising: measuring the expression level of SPSB2 protein or SPSB2 gene in a biological sample from the subject, wherein , the measured expression level of the protein or gene is data used to determine whether the subject has cancer, provided that the medical practice of the physician is not included.

[8] The method according to [7], wherein the cancer is bladder cancer, pancreatic cancer, or hepatocellular carcinoma.

[9] A method for predicting the prognosis of a cancer patient, the method comprising: measuring the expression level of SPSB2 protein or SPSB2 gene in a biological sample from the cancer patient, wherein the measured protein Or the fact that the expression level of the gene is higher than that of the control indicates that the prognosis of the cancer patient is poor.

[10] The method according to [9], wherein the cancer is bladder cancer, pancreatic cancer, or hepatocellular carcinoma.

[11] A method for screening an anticancer agent, the method comprising: a step of measuring the expression level of SPSB2 protein or SPSB2 gene in cancer cells cultured in the presence of a test substance, wherein, compared with the absence of the test substance The fact that the expression level is significantly reduced compared to the expression level of the SPSB2 protein or the SPSB2 gene when the substance is used indicates that the test substance is an anticancer agent.

[12] The method according to [11], wherein the cancer cells are cancer cells derived from bladder cancer, pancreatic cancer, or hepatocellular carcinoma.

[Beneficial effects of the invention]

According to the present invention, a new technology for diagnosing cancer can be provided.

Description of the drawings

FIG. 1 is a graph showing the results of Experimental Example 2.

2(a) to 2(c) are photographs showing representative results of immunostaining in Experimental Example 3.

FIG. 3 is a graph showing the results of Experimental Example 5.

FIG. 4 is a graph showing the results of Experimental Example 5.

5(a) to 5(d) are photographs showing representative results of immunostaining in Experimental Example 6.

FIG. 6 is a graph showing the results of Experimental Example 7.

FIG. 7 is a graph showing the results of Experimental Example 7.

FIG. 8 is a photograph showing the results of Western blotting in Experimental Example 8.

FIG. 9 is a photograph showing the results of Western blotting in Experimental Example 8.

FIG. 10 is a graph showing the quantitative value of SPSB2 protein in the urine sample measured in Experimental Example 9.

FIG. 11 is a graph showing the quantitative value of SPSB2 protein in the urine sample measured in Experimental Example 9.

Fig. 12 is a graph showing the quantitative value of SPSB2 protein in the urine sample measured in Experimental Example 9.

Fig. 13 is a graph showing the quantitative value of SPSB2 protein in the urine sample measured in Experimental Example 9.

Figure 14 is the ROC curve created in Experimental Example 10.

Figure 15 is the ROC curve created in Experimental Example 10.

FIG. 16 is a graph showing the results of analyzing the cancer-specific survival rate in Experimental Example 10.

FIG. 17 is a graph showing the results of analyzing the deterioration-free survival rate in Experimental Example 10.

FIG. 18(a) is a photograph showing the results of Western blotting in Experimental Example 11. In addition, FIG. 18(b) is a graph showing the results of FIG. 18(a).

Detailed ways

[Kit for diagnosing cancer, kit for determining prognosis of cancer patients]

In one embodiment, the present invention provides a kit for diagnosing cancer, including: a substance that specifically binds to the SPSB2 protein, a primer set capable of amplifying the cDNA of the SPSB2 gene, or a probe that specifically hybridizes to the mRNA of the SPSB2 gene. Needle.

As described in subsequent examples, the inventors have identified that SPSB2 protein or SPSB2 gene can be used as a cancer marker. In addition, the inventors have clarified that the SPSB2 protein or SPSB2 gene can be used as a marker not only for bladder cancer but also for pancreatic cancer and hepatocellular carcinoma. Therefore, in the kit for diagnosing cancer of the present embodiment, examples of cancer include bladder cancer, pancreatic cancer, and hepatocellular carcinoma.

Using the kit of this embodiment, the expression level of SPSB2 protein or SPSB2 gene in a biological sample from a subject can be measured to determine whether the subject has cancer. Examples of biological samples include serum, plasma, urine, and tissue. In addition, particularly when the cancer is bladder cancer, examples of biological samples include urine, tissue, and the like. Here, regarding urine, exosomes in urine can be extracted and used as biological samples.

In addition, as described in subsequent examples, the inventors analyzed bladder cancer patients and hepatocellular carcinoma patients, and clarified that when the SPSB2 protein or SPSB2 gene is highly expressed, the prognosis is often poor. Therefore, it can also be said that the kit for diagnosing cancer in this embodiment is a kit for determining prognosis. In this specification, a poor prognosis may mean poor survival, short progression-free survival, etc.

That is, it can be said that the present invention provides a kit for determining the prognosis of cancer patients, including: a substance that specifically binds to the SPSB2 protein, a primer set that can amplify the cDNA of the SPSB2 gene, or specifically hybridizes with the mRNA of the SPSB2 gene. probe. In the kit for determining the prognosis of a cancer patient (a kit for predicting the prognosis of a cancer patient), examples of cancer include bladder cancer, pancreatic cancer, and hepatocellular carcinoma.

The NCBI accession numbers of human SPSB2 protein are NP_001139788.1, NP_001306599.1 and NP_116030.1, etc. In addition, the NCBI accession numbers of the human SPSB2 gene mRNA are NM_001146316.2, NM_001146317.1, NM_001319670.2 and NM_032641.4, etc.

[Substance that binds specifically]

The kit of this embodiment may include substances that specifically bind to the SPSB2 protein. Examples of specifically binding substances include antibodies, antibody fragments, aptamers, and the like. Examples of antibody fragments include F(ab') ₂ , Fab', Fab, Fv, scFv, and the like. Antibodies or antibody fragments may be polyclonal or monoclonal. The aptamer is not particularly limited as long as it has specific binding ability to the SPSB2 protein, and examples thereof may include nucleic acid aptamers, peptide aptamers, and the like.

For example, SPSB2 protein expression levels can be determined by immunostaining fixed tissue sections using specifically binding substances. The determination of the expression level of SPSB2 protein is not limited to immunostaining, and can also be performed by extracting the protein from the test sample and using Western blotting or ELISA.

The fact that the expression level of SPSB2 protein in the test sample was higher than that of the control indicates that the test sample came from a cancer patient. In this specification, "higher than the control" preferably means statistically significantly higher than the control. Here, examples of controls include expression levels of SPSB2 protein measured using samples from normal tissues.

(primer set)

The kit for diagnosing cancer of this embodiment may include a primer set for amplifying cDNA of the SPSB2 gene. The sequence of the primer set is not particularly limited as long as it can amplify at least part of the cDNA of the SPSB2 gene.

The fact that the expression level of the SPSB2 gene in the test sample was higher than that of the control indicates that the test sample came from a cancer patient. In addition, examples of controls include expression levels of the SPSB2 gene measured using samples from normal tissues.

(probe)

The kit of this embodiment may include a probe that specifically hybridizes to the mRNA of the SPSB2 gene.

The probe may be, for example, a nucleic acid fragment having a base sequence complementary to the base sequence of at least part of the mRNA of the SPSB2 gene. In addition, probes may have various chemical modifications for the purpose of improving stability and specificity during hybridization. For example, to prevent degradation by hydrolytic enzymes such as nucleases, phosphate residues can be replaced with chemically modified phosphate residues such as phosphorothioate PS, methylphosphonate, and phosphorodithioate. base. Additionally, at least a portion thereof may be composed of nucleic acid analogs such as peptide nucleic acid (PNA).

The probe can be immobilized on a solid phase. Examples of solid phases include beads, plate-like substrates, membranes, and the like. For example, probes can be immobilized on the surface of a plate-like substrate to form a microarray. In this case, for example, one can detect RNA in the test sample by extracting RNA from the test sample, labeling the RNA with a fluorescent substance, and hybridizing the RNA to the microarray to detect RNA bound to the probes on the microarray. Expression of SPSB2 gene.

The fact that the expression level of the SPSB2 gene in the test sample was higher than that of the control indicates that the test sample came from a cancer patient. Here, examples of the control include the expression level of the SPSB2 gene measured using samples from normal tissues.

[Methods for determining a biological sample, methods for collecting data to determine whether a subject has cancer, and methods for predicting the prognosis of a cancer patient]

In one embodiment, the present invention provides a method for determining a biological sample, including the step of determining the expression level of the SPSB2 protein or gene in the biological sample, wherein the measured expression level of the protein or gene is higher than that of the control The fact that the expression levels are present in biological samples comes from cancer patients.

As described in subsequent examples, the inventors have identified that SPSB2 protein or SPSB2 gene can be used as a cancer marker. In addition, the inventors have clarified that the SPSB2 protein or SPSB2 gene can be used as a marker not only for bladder cancer but also for pancreatic cancer and hepatocellular carcinoma. Therefore, whether the biological sample comes from a cancer patient can be determined by the method of this embodiment. In the determination method of this embodiment, examples of cancer include bladder cancer, pancreatic cancer, and hepatocellular carcinoma.

As mentioned above, serum, plasma, urine, tissue, etc. from a subject can be used as biological samples. In addition, particularly when the cancer is bladder cancer, examples of biological samples include urine, tissue, and the like. Here, regarding urine, exosomes in urine can be extracted and used as biological samples. In addition, similar to the above, "higher than the control" preferably means statistically significantly higher than the control. In addition, examples of controls include expression levels of SPSB2 protein or SPSB2 gene measured using samples from normal tissues.

It can also be said that the determination method of this embodiment is a method for collecting data that determines whether a biological sample comes from a cancer patient. The methods used to collect data did not include physicians' medical practices.

That is, it can be said that the present invention provides a method for collecting data for determining whether a biological sample is from a cancer patient, the method including the step of measuring the expression level of the SPSB2 protein or gene in the biological sample, wherein the measured protein Or the expression level of a gene is data used to determine whether a biological sample is from a cancer patient. The fact that the expression level of the SPSB2 protein or gene in the biological sample is higher than that of the control indicates that the biological sample is from a cancer patient.

Alternatively, it can be said that the present invention provides a method for collecting data for determining whether a subject has cancer, the method comprising determining the expression level of the SPSB2 protein or gene in a biological sample from the subject. Step, wherein the measured expression level of the protein or gene is data used to determine whether the subject has cancer. The fact that the expression level of the SPSB2 protein or gene in the biological sample from the subject is higher than the expression level of the control indicates that the subject has cancer. In the method used to collect data, examples of cancer include bladder cancer, pancreatic cancer, and hepatocellular carcinoma.

In addition, as described in subsequent examples, the inventors analyzed bladder cancer patients and hepatocellular carcinoma patients, and clarified that when the SPSB2 protein or SPSB2 gene is highly expressed, the prognosis is often poor. Therefore, it can be said that the determination method of this embodiment is a method for predicting prognosis.

That is, it can be said that the present invention provides a method for predicting the prognosis of a cancer patient, which method includes the step of measuring the expression level of the SPSB2 protein or the SPSB2 gene in a biological sample from the cancer patient, wherein the measured protein Or the fact that the gene's expression level is higher than that of the control indicates a poor prognosis for the cancer patient. In the method of predicting the prognosis of a cancer patient, examples of cancer include bladder cancer, pancreatic cancer, and hepatocellular carcinoma.

[Method for screening anticancer agents]

In one embodiment, the present invention provides a method for screening anticancer agents, which method includes the step of determining the expression level of SPSB2 protein or SPSB2 gene in cancer cells cultured in the presence of a test substance, wherein, with The fact that the expression levels are significantly reduced compared to the expression levels of the SPSB2 protein or SPSB2 gene in the absence of the test substance indicates that the test substance is an anticancer agent.

As described in subsequent examples, the higher the malignancy of cancer cells, the higher the expression level of SPSB2 protein or SPSB2 gene. Therefore, it can be said that a test substance that reduces the expression level of SPSB2 protein or SPSB2 gene is a candidate anticancer agent.

In the screening method of this embodiment, the test substance is not particularly limited, and examples thereof include natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, and the like.

In the screening method of this embodiment, the cancer cells may be cancer cells derived from bladder cancer, pancreatic cancer, hepatocellular carcinoma, etc.

Alternatively, the cancer cells may be cisplatin-resistant strains obtained by culturing in the presence of cisplatin while gradually increasing the cisplatin concentration. Cisplatin-resistant strains are often resistant to anticancer agents other than cisplatin and often have a high degree of malignancy.

[Other embodiments]

In one embodiment, the present invention provides a method for treating cancer, comprising the step of determining the expression level of SPSB2 protein or SPSB2 gene in a biological sample from a subject, wherein the expression level is higher than the expression level of a control The facts indicate that the subject has cancer; and, if the subject has cancer, the step of surgically removing cancerous tissue from the subject or treating the subject with an anti-cancer agent.

In the treatment method of this embodiment, examples of cancer include bladder cancer, pancreatic cancer, hepatocellular carcinoma, and the like. In addition, examples of biological samples include serum, plasma, urine, tissue, and the like. In particular, when the cancer is bladder cancer, examples of biological samples include urine, tissue, and the like. Here, regarding urine, exosomes in urine can be extracted and used as biological samples.

As described in the subsequent examples, in the case of using urine as a biological sample, there are cases where the expression level of the SPSB2 protein or SPSB2 gene may be high not only in cancer patients but also in urinary tract infection patients. In this case, a urine culture test can be used to diagnose whether the subject has a urinary tract infection. That is, subjects with high expression levels of SPSB2 protein or SPSB2 gene and no urinary tract infection can be diagnosed as having cancer.

In the treatment method of this embodiment, examples of anticancer agents include cisplatin, M-VAC (combination of methotrexate, vinblastine, adriamycin, and cisplatin) , GC (combination of cisplatin and gemcitabine), enfortumab vedotin as an antibody-drug complex (ADC) targeting nectin-4, enno monoclonal antibody (Padcev) and Keytruda (pembrolizumab, pembrolizumab), etc.

These anticancer agents are usually administered via intravenous drip. In addition, the dosage of these anticancer agents varies depending on the patient's symptoms, weight, age, gender, etc., but a suitable dosage can be appropriately selected by those skilled in the art.

[Example]

Next, the present invention will be described in more detail by showing experimental examples, but the present invention is not limited to the following experimental examples.

[Experimental example 1]

So far, the inventors have prepared a large number of bladder cancer-specific antibodies in research. In addition, a technique for identifying target proteins using autoantibodies in serum by dot blotting has been established, and identification of a large number of autoantibodies against bladder cancer has been achieved. The reactivity of these prepared bladder cancer-specific antibodies and bladder cancer-directed autoantibodies with a large collection of serum and tumor tissues from bladder cancer patients was examined. Therefore, the SPSB2 protein or SPSB2 gene was designated as a candidate for a new cancer marker.

[Experimental example 2]

The expression level of SPSB2 gene in bladder cancer tissues was examined using the public database The Cancer Genome Atlas (TCGA). FIG. 1 is a diagram showing inspection results. In Figure 1, the vertical axis represents the expression level of the SPSB2 gene. In addition, “normal” represents the expression level of the SPSB2 gene in normal tissue, and “primary tumor” represents the expression level of the SPSB2 gene in bladder cancer tissue. The results showed that the expression level of SPSB2 gene was significantly increased in bladder cancer tissue.

[Experimental example 3]

Tissue sections prepared from total cystectomy specimens were immunostained with anti-SPSB2 antibodies. As total cystectomy specimens, 126 specimens of total cystectomy performed at Kitasato University Hospital from 1990 to 2015 were used as subjects. Immunostaining was performed using a Bond-MAX automated immunostaining device (Leica Biosystems).

The intensity of nuclear staining of tumor cells with anti-SPSB2 antibodies in tissue sections was compared with the intensity of nuclear staining of surrounding cells with anti-SPSB2 antibodies, and the intensity was evaluated in three segments according to the following evaluation criteria. Evaluation criteria 0 to 1 were classified into the SPSB2 protein low expression group, and evaluation criterion 2 was classified into the SPSB2 protein high expression group for the following analysis.

(evaluation standard)

0: low expression

1: equal

2: High expression

Figures 2(a) to 2(c) are photographs showing representative immunostaining results. Figure 2(a) is a photograph showing the results of immunostaining of normal urothelial tissue. In addition, Fig. 2(b) is a photograph showing the immunostaining results of bladder cancer tissues classified into the SPSB2 protein low expression group. In addition, FIG. 2(c) is a photograph showing the immunostaining results of bladder cancer tissues classified into the SPSB2 protein high expression group.

[Experimental Example 4]

Based on the results of Experimental Example 3, the relationship between the expression of SPSB2 protein and clinical pathological factors was analyzed. The analysis results are shown in Table 1 below. In Table 1, "p value" indicates the p value calculated by Fisher's exact test. p<0.05 was determined as significant difference. Bold text indicates significant differences. The results showed that the expression of SPSB2 protein was related to gender, degree of vertical invasion (pT stage), degree of atypia and vascular invasion.

[Table 1]

Table 2 below shows the results of studying the correlation between the expression of S100A8 protein, S100A9 protein, Uroplakin III protein and HNRNPA3 protein, which are reported as bladder cancer markers, and the expression of SPSB2 protein. In Table 2, "p value" indicates the p value calculated by Fisher's exact test. p<0.05 was determined as significant difference. Bold text indicates significant differences.

The results showed that the expression of SPSB2 protein was related to the respective expressions of S100A8 protein, S100A9 protein, Uroplakin III protein and HNRNPA3 protein.

[Table 2]

[Experimental Example 5]

Based on the results of Experimental Example 3, the relationship between SPSB2 protein expression and prognosis was analyzed. Fig. 3 is a graph showing the results of analyzing cancer-specific survival rates by the Kaplan-Meier method. In Figure 3, "SPSB2 low" represents the results of the SPSB2 protein low expression group, and "SPSB2 high" represents the results of the SPSB2 protein high expression group. In addition, "number at risk" represents the number of survivors at each point in time. The results showed that the group with high SPSB2 protein expression had a significantly higher risk of death due to bladder cancer.

FIG. 4 is a graph showing the results of the deterioration-free survival rate analyzed by the Kaplan-Meier method based on the results of Experimental Example 3. In Figure 4, "SPSB2 low" represents the results of the SPSB2 protein low expression group, and "SPSB2 high" represents the results of the SPSB2 protein high expression group. Additionally, “number at risk” represents the number of worsening-free survivors at each time point. The results showed that in the group with high SPSB2 protein expression, the time period until bladder cancer recurred was significantly shortened.

Table 3 below shows the results of univariate analysis and multivariate analysis based on the Cox proportional hazards model on cancer-specific survival rates. In addition, Table 4 below shows the results of univariate analysis and multivariate analysis based on the Cox proportional hazards model regarding the worsening-free survival rate. In Tables 3 and 4, “HR” represents the risk ratio, and “95% CI” represents the 95% confidence interval. p<0.05 was determined as significant difference. Bold text indicates significant differences.

The results showed that SPSB2 protein expression together with lymph node metastasis were independent factors for cancer-specific survival and progression-free survival.

[table 3]

[Table 4]

[Experimental Example 6]

SPSB2 protein expression was studied in cancer types other than bladder cancer. 5(a) to 5(c) are photographs showing representative results of immunostaining with anti-SPSB2 antibodies on respective tissue sections of pancreatic cancer, hepatocellular carcinoma, and ovarian cancer. Figure 5(a) is the result of tissue sectioning of pancreatic cancer, Figure 5(b) is the result of tissue sectioning of hepatocellular carcinoma, Figure 5(c) is the result of tissue sectioning of ovarian cancer, Figure 5(d) is the result of pancreatic cancer Results of tissue sections of normal tissue.

The results showed that SPSB2 protein was confirmed to be strongly expressed in pancreatic cancer and moderately expressed in hepatocellular carcinoma and ovarian cancer. On the other hand, SPSB2 protein is weakly or not expressed in kidney cancer, prostate cancer, esophageal cancer, gastric cancer, colon cancer, breast cancer, and lung cancer.

[Experimental Example 7]

The expression level of SPSB2 gene in hepatocellular carcinoma tissues was examined using the public database The Cancer Genome Atlas (TCGA). FIG. 6 is a diagram showing inspection results. In Figure 6, the vertical axis represents the expression level of the SPSB2 gene. In addition, “normal” represents the expression level of the SPSB2 gene in normal tissue, and “primary tumor” represents the expression level of the SPSB2 gene in hepatocellular carcinoma tissue.

The results showed that the expression level of SPSB2 gene was significantly increased in hepatocellular carcinoma tissues.

Figure 7 is a graph showing the relationship between the expression level of the SPSB2 gene in hepatocellular carcinoma and the prognosis in TCGA samples. In Figure 7, "high expression" represents the results of the SPSB2 protein high expression group, and "low/moderate expression" represents the results of the SPSB2 protein low to moderate expression group. The results showed that the group with high SPSB2 gene expression in hepatocellular carcinoma had a significantly poor prognosis.

[Experimental example 8]

The abundance of SPSB2 protein in serum exosomes and urinary exosomes from healthy subjects and bladder cancer patients was studied by Western blotting.

First, exosomes were extracted from serum samples and urine samples using total exosome isolation reagent (Thermo Fisher Scientific).

Subsequently, the extracted exosomes were used to detect SPSB2 protein. In addition, it was confirmed that exosomes can be extracted by Western blotting of CD9, one of the exosome markers. The molecular weight of SPSB2 protein is approximately 26kDa, and that of CD9 is approximately 24kDa.

FIG. 8 is a photograph showing the results of Western blotting of exosomes in serum. In Figure 8, “C” represents exosomes from healthy subjects, and “T” represents exosomes from bladder cancer patients.

The results confirmed that exosomes could be extracted since CD9 could be detected. In addition, it was also clarified that the presence of SPSB2 protein was not confirmed in exosomes in the serum of both healthy subjects and bladder cancer patients.

FIG. 9 is a photograph showing the results of Western blotting of exosomes in urine. In Figure 9, “C” represents exosomes from healthy subjects, and “T” represents exosomes from bladder cancer patients.

The results confirmed that exosomes could be extracted since CD9 could be detected. In addition, the presence of SPSB2 protein was not confirmed in exosomes in serum from healthy subjects, whereas the presence of SPSB2 protein was detected in urinary exosomes from bladder cancer patients.

[Experimental Example 9]

The abundance of SPSB2 protein in urine from healthy subjects, urinary stone patients, urinary tract infection patients and bladder cancer patients was quantified and studied by enzyme-linked immunosorbent assay (ELISA).

As patients with bladder cancer, 91 patients whose urine was collected immediately before transurethral bladder tumor resection at Kitasato University Hospital from 2009 to 2015 were selected as subjects. The background of the bladder cancer patients is shown in Table 5 below. In Table 5, "NMIBC" means non-muscle invasive bladder cancer, and "MIBC" means muscle-invasive bladder cancer. The urine specific gravity of all urine samples was corrected to 1.002, and the abundance of SPSB2 protein was determined by ELISA.

[table 5]

FIG. 10 is a graph showing quantitative values of SPSB2 protein in urine samples of each group. In addition, FIG. 10 also shows the analysis results by Mann-Whitney U-test. In Figure 10, "BC" represents the results of patients with bladder cancer, "Health" represents the results of healthy subjects, "Stone" represents the results of patients with urinary tract stones, and "UTI" represents the results of patients with urinary tract infection.

Therefore, the abundance of SBSP2 protein in the urine of patients with bladder cancer is higher than that in the urine of healthy subjects and patients with urinary tract stones, but lower than that in the urine of patients with urinary tract infection. Spend. Urinary tract infections can be diagnosed through urine culture tests and other tests.

FIG. 11 is a graph showing quantitative values of SPSB2 protein in urine samples of non-muscle-invasive bladder cancer patients and muscle-invasive bladder cancer patients. In addition, FIG. 11 also shows the analysis results by the Mann-Whitney U test. In Figure 11, "NMIBC" indicates non-muscle invasive bladder cancer, and "MIBC" indicates muscle-invasive bladder cancer.

The results showed that the abundance of SBSP2 protein in the urine of patients with bladder cancer showed a higher value than the abundance of SBSP2 protein in the urine of patients with muscle-invasive bladder cancer.

Figure 12 is a graph showing quantitative values of SPSB2 protein in urine samples from patients with pathological grade 1 and 2 bladder cancer and patients with pathological grade 3 bladder cancer. In addition, FIG. 12 also shows the analysis results by the Mann-Whitney U test.

The results showed that the abundance of SBSP2 protein in the urine of patients with pathological grade 3 bladder cancer tended to show higher values than the abundance of SBSP2 protein in the urine of patients with pathological grade 1 and 2 bladder cancer.

Figure 13 is a graph showing quantitative values of SPSB2 protein in urine samples of muscle-invasive bladder cancer patients and urinary tract infection patients. In addition, FIG. 13 also shows the analysis results by the Mann-Whitney U test. In Figure 13, “MIBC” indicates muscle invasive bladder cancer, and “UTI” indicates the results of patients with urinary tract infection.

Therefore, no significant difference was confirmed in the abundance of SBSP2 protein in the urine of patients with muscle-invasive bladder cancer and urinary tract infection. Urinary tract infections can be diagnosed through urine culture tests and other tests.

[Experimental Example 10]

Based on the results of Experimental Example 9, the relationship between the expression of SPSB2 protein and prognosis was analyzed. FIG. 14 is a ROC curve prepared based on the quantitative values of SPSB2 protein in urine samples of bladder cancer patients and healthy subjects measured in Experimental Example 9. The results show that the area under the ROC curve (AUC: area under the curve, area under the curve) is 0.7791.

FIG. 15 is a ROC curve prepared based on the quantitative values of SPSB2 protein in urine samples of muscle-invasive bladder cancer patients and healthy subjects measured in Experimental Example 9. The results show that the area under the ROC curve (AUC) is 0.8699.

Subsequently, based on the ROC curve, the cutoff value was set to 162.8ng/mL (sensitivity: 58.2%, specificity: 80.0%), and survival analysis was performed, in which the abundance of SPSB2 protein in urine was lower than 162.8ng/mL. mL of SPSB2 protein is a low-expression group, and patients whose SPSB2 protein abundance in urine is 162.8ng/mL or greater than 162.8ng/mL is a high-SPSB2 protein expression group.

Fig. 16 is a graph showing the results of analyzing cancer-specific survival rates by the Kaplan-Meier method. In Figure 16, "SPSB2 low" represents the results of the SPSB2 protein low expression group, and "SPSB2 high" represents the results of the SPSB2 protein high expression group. In addition, "number at risk" represents the number of survivors at each point in time. The results showed that the group with high SPSB2 protein expression had a significantly higher risk of death due to bladder cancer.

FIG. 17 is a graph showing the results of analysis of worsening-free survival rate by the Kaplan-Meier method. In Figure 17, "SPSB2 low" represents the results of the SPSB2 protein low expression group, and "SPSB2 high" represents the results of the SPSB2 protein high expression group. Additionally, “number at risk” represents the number of worsening-free survivors at each time point. Therefore, no significant difference was confirmed in the time period until bladder cancer recurrence between the SPSB2 protein low expression group and the SPSB2 protein high expression group.

Table 6 below shows the results of univariate analysis and multivariate analysis based on the Cox proportional hazards model on cancer-specific survival rates. In Table 6, "NMIBC" means non-muscle invasive bladder cancer, "MIBC" means muscle invasive bladder cancer, "G3" means grade 3, "G1,2" means grade 1 or 2, "HR" means hazard ratio, " "95% CI" represents the 95% confidence interval. p<0.05 was determined as significant difference. Bold text indicates significant differences.

The results indicate that SPSB2 protein expression, together with the degree of vertical invasion of the cancer, is a prognostic factor for cancer-specific survival.

[Table 6]

[Experimental Example 11]

T24 and 5637, which are human bladder cancer cell lines, were cultured in the presence of cisplatin while gradually increasing the concentration to obtain T24CDDPR and 5637CDDPR, both of which are cisplatin-resistant lines. T24CDDPR and 5637CDDPR are considered to have a higher degree of malignant cancer than T24 and 5637.

Subsequently, the expression level of SPSB2 protein was determined by performing Western blotting on each cell line of T24, T24CDDPR, 5637 and 5637CDDPR. In addition, the expression level of β-actin was measured as a control.

Figure 18(a) is a photograph showing the results of Western blotting. In addition, Fig. 18(b) shows the results of Fig. 18(a) in a graph. The vertical axis in Fig. 18(b) represents the expression level of SPSB2 relative to the expression level of β-actin. “*” indicates that there is a significant difference at p<0.05, and “**” indicates that there is a significant difference at p<0.01. significant difference.

The results showed that the expression level of SPSB2 in T24CDDPR and 5637CDDPR was significantly higher than that in each of T24 and 5637.

This result shows that the higher the malignancy of the cancer, the higher the expression level of SPSB2 in the cancer cells.

[Industrial Applicability]

According to the present invention, a new technology for diagnosing cancer can be provided.

Claims (12)

1. A kit for diagnosing cancer, comprising:

specific binding substances for SOCS box protein 2 (SPSB 2) proteins containing SPRY domains;

primer set for amplifying cDNA of SPSB2 gene; or (b)

Probes specifically hybridizing with mRNA of SPSB2 gene.

2. The kit for diagnosing cancer according to claim 1, wherein the cancer is bladder cancer, pancreatic cancer or hepatocellular carcinoma.

3. A kit for determining prognosis of a cancer patient, comprising:

specific binding substances for the SPSB2 protein;

primer set for amplifying cDNA of SPSB2 gene; or (b)

Probes specifically hybridizing with mRNA of SPSB2 gene.

4. The kit for determining prognosis as claimed in claim 3, wherein the cancer is bladder cancer, pancreatic cancer or hepatocellular carcinoma.

5. A method for determining a biological sample, comprising:

a step of measuring the expression level of SPSB2 protein or SPSB2 gene in the biological sample,

wherein the fact that the expression level of the protein or gene is determined to be higher than the expression level of the control indicates that the biological sample is from a cancer patient.

6. The method for determining according to claim 5, wherein the cancer is bladder cancer, pancreatic cancer or hepatocellular carcinoma.

7. A method for collecting data for determining whether a subject has cancer, the method comprising: a step of determining the expression level of an SPSB2 protein or an SPSB2 gene in a biological sample from the subject, wherein the determined expression level of the protein or gene is data for determining whether the subject has cancer, provided that medical practice by a doctor is excluded.

8. The method of claim 7, wherein the cancer is bladder cancer, pancreatic cancer, or hepatocellular cancer.

9. A method for predicting prognosis of a cancer patient, the method comprising: a step of determining the expression level of SPSB2 protein or SPSB2 gene in a biological sample from the cancer patient,

wherein the fact that the expression level of the protein or gene determined is higher than the expression level of the control indicates that the cancer patient has poor prognosis.

10. The method of claim 9, wherein the cancer is bladder cancer, pancreatic cancer, or hepatocellular cancer.

11. A method for screening for an anticancer agent, the method comprising: a step of measuring the expression level of SPSB2 protein or SPSB2 gene in cancer cells cultured in the presence of a test substance,

wherein the fact that the expression level is significantly reduced compared to the expression level of the SPSB2 protein or the SPSB2 gene in the absence of the test agent indicates that the test agent is an anti-cancer agent.

12. The method of claim 11, wherein the cancer cell is a cancer cell from bladder cancer, pancreatic cancer, or hepatocellular carcinoma.