Cancer diagnostic kit and its use

Abstract

According to the present invention, a kit for diagnosis of cancer, a kit for determining prognosis of a patient with cancer, a method for assessing a biological sample, a method for collecting data for determining whether or not a subject is affected by cancer, and a method for predicting prognosis of a patient with cancer, use a substance that specifically binds to the SPRY domain-containing SOCS box 2 (SPSB2) protein, a primer set that is capable of amplifying cDNA of the SPSB2 gene, or a probe that specifically hybridizes with mRNA of the SPSB2 gene.

Description

[Technical Field]

[0001] The present invention relates to a cancer diagnostic kit and its use. More specifically, the present invention relates to a cancer diagnostic kit, a kit for determining the prognosis of cancer patients, a method for determining a biological sample, a method for collecting data for determining whether a subject has cancer, a method for predicting the prognosis of a cancer patient, and a method for screening anticancer drugs. This application claims priority based on 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 herein by reference. [Background technology]

[0002] Among cancers, the incidence and mortality rates of bladder cancer are steadily increasing (see, for example, Non-Patent Document 1). Treatments for bladder cancer include surgery, chemotherapy, and radiotherapy. However, the response rate to bladder cancer treatment is not satisfactory. One reason for this is the lack of useful cancer markers and follow-up markers. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Fitzmaurice C., et al., 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, JAMA Oncol., 5 (12), 1749-1768, 2019. Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a new cancer diagnostic technique. [Means for solving the problem]

[0005] The present invention includes the following aspects. [1] A cancer diagnostic kit comprising a substance that specifically binds to SPRY domain-containing SOCS box protein 2 (SPSB2) protein, a primer set capable of amplifying cDNA of the SPSB2 gene, or a probe that specifically hybridizes to mRNA of the SPSB2 gene. [2] The cancer diagnostic kit according to [1], wherein the cancer is bladder cancer, pancreatic cancer, or hepatocellular carcinoma. [3] A kit for determining the prognosis of a cancer patient, comprising 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. [4] The prognosis determination kit according to [3], wherein the cancer is bladder cancer, pancreatic cancer, or hepatocellular carcinoma. [5] A method for evaluating a biological sample, comprising a step of measuring the expression level of SPSB2 protein or SPSB2 gene in the biological sample, wherein a higher expression level of the measured protein or gene compared to a control indicates that the biological sample is derived from a cancer patient. [6] The method of determining cancer according to [5], wherein the cancer is bladder cancer, pancreatic cancer, or hepatocellular carcinoma. [7] A method for collecting data to determine whether a subject has cancer, comprising a step of measuring the expression level of SPSB2 protein or SPSB2 gene in a biological sample derived from the subject, wherein the measured expression level of the protein or gene is data for determining whether the subject has cancer (excluding medical procedures by a physician). [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, comprising a step of measuring the expression level of SPSB2 protein or SPSB2 gene in a biological sample derived from the cancer patient, wherein a higher expression level of the measured protein or gene compared to a control indicates a poor prognosis for the cancer patient.

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

[11] A method for screening anticancer drugs, 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 a significant decrease in the expression level compared to the expression level of SPSB2 protein or SPSB2 gene in the absence of the test substance indicates that the test substance is an anticancer drug.

[12] The method according to

[11] , wherein the cancer cells are cancer cells derived from bladder cancer, pancreatic cancer, or hepatocellular carcinoma. [Effects of the Invention]

[0006] The present invention can provide a new cancer diagnostic technique. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a graph showing the results of Experimental Example 2. [Figure 2] 2(a) to (c) are photographs showing representative immunostaining results in Experimental Example 3. FIG. [Figure 3] FIG. 3 is a graph showing the results of Experimental Example 5. [Figure 4] FIG. 4 is a graph showing the results of Experimental Example 5. [Figure 5] 5(a) to (d) are photographs showing representative immunostaining results in Experimental Example 6. FIG. [Figure 6] FIG. 6 is a graph showing the results of Experimental Example 7. [Figure 7] FIG. 7 is a graph showing the results of Experimental Example 7. [Figure 8] FIG. 8 is a photograph showing the results of Western blotting in Experimental Example 8. [Figure 9] FIG. 9 is a photograph showing the results of Western blotting in Experimental Example 8. [Figure 10] FIG. 10 is a graph showing the quantitative values ​​of SPSB2 protein in urine samples measured in Experimental Example 9. [Figure 11] FIG. 11 is a graph showing the quantitative values ​​of SPSB2 protein in urine samples measured in Experimental Example 9. [Figure 12] FIG. 12 is a graph showing the quantitative values ​​of SPSB2 protein in urine samples measured in Experimental Example 9. [Figure 13] FIG. 13 is a graph showing the quantitative values ​​of SPSB2 protein in urine samples measured in Experimental Example 9. [Figure 14] FIG. 14 shows the ROC curve created in Experimental Example 10. [Figure 15] FIG. 15 shows the ROC curve created in Experimental Example 10. [Figure 16] FIG. 16 is a graph showing the results of analyzing the cancer-specific survival rate in Experimental Example 10. [Figure 17] FIG. 17 is a graph showing the results of analyzing the progression-free survival rate in Experimental Example 10. [Figure 18] Figure 18(a) is a photograph showing the results of Western blotting in Experimental Example 11. Figure 18(b) is a graph showing the results of Figure 18(a). DETAILED DESCRIPTION OF THE INVENTION

[0008] [Cancer diagnostic kits, kits for determining the prognosis of cancer patients] In one embodiment, the present invention provides a cancer diagnostic kit comprising a substance that specifically binds to SPSB2 protein, a primer set capable of amplifying cDNA of the SPSB2 gene, or a probe that specifically hybridizes to mRNA of the SPSB2 gene.

[0009] As described later in the Examples, the inventors have demonstrated that the SPSB2 protein or SPSB2 gene can be used as a cancer marker. The inventors have also demonstrated that the SPSB2 protein or SPSB2 gene is useful not only for bladder cancer, but also for pancreatic cancer and hepatocellular carcinoma. Therefore, in the cancer diagnostic kit of this embodiment, examples of cancer include bladder cancer, pancreatic cancer, and hepatocellular carcinoma.

[0010] The kit of this embodiment can be used to measure the expression level of SPSB2 protein or SPSB2 gene in a biological sample derived from a subject, and determine whether the subject is suffering from cancer. Examples of biological samples include serum, plasma, urine, tissue, etc. Furthermore, particularly when the cancer is bladder cancer, examples of biological samples include urine and tissue. Regarding urine, exosomes may be extracted from urine to serve as the biological sample.

[0011] Furthermore, as described later in the Examples, the inventors analyzed bladder cancer patients and hepatocellular carcinoma patients and found that high expression of the SPSB2 protein or SPSB2 gene tends to be associated with a poor prognosis. Therefore, the cancer diagnostic kit of this embodiment can also be referred to as a prognosis determination kit. In this specification, a poor prognosis may refer to a low survival rate, a short progression-free survival time, or the like.

[0012] In other words, the present invention provides a kit for determining the prognosis of cancer patients, which comprises a substance that specifically binds to the SPSB2 protein, a primer set capable of amplifying cDNA of the SPSB2 gene, or a probe that specifically hybridizes to mRNA of the SPSB2 gene. In the kit for determining the prognosis of cancer patients (kit for predicting the prognosis of cancer patients), examples of cancers include bladder cancer, pancreatic cancer, and hepatocellular carcinoma.

[0013] The NCBI accession numbers for the human SPSB2 protein are NP_001139788.1, NP_001306599.1, NP_116030.1, etc. The NCBI accession numbers for the mDNA of the human SPSB2 gene are NM_001146316.2, NM_001146317.1, NM_001319670.2, NM_032641.4, etc.

[0014] (specific binding substance) The kit of this embodiment may contain a substance that specifically binds to the SPSB2 protein. Examples of specific binding substances include antibodies, antibody fragments, and aptamers. Examples of antibody fragments include F(ab')2, Fab', Fab, Fv, and scFv. The above-mentioned antibodies or antibody fragments may be polyclonal or monoclonal. The aptamer is not particularly limited as long as it is a substance that has the ability to specifically bind to the SPSB2 protein, and examples include nucleic acid aptamers and peptide aptamers.

[0015] For example, the expression level of SPSB2 protein can be measured by immunostaining a fixed tissue section with the above-mentioned specific binding substance. Measurement of the expression level of SPSB2 protein is not limited to immunostaining, and may also be performed by extracting the protein from a test sample and subjecting it to Western blotting or ELISA.

[0016] A higher expression level of SPSB2 protein in a test sample compared to a control indicates that the test sample is derived from a cancer patient. As used herein, "higher than the control" preferably means a statistically significantly higher level compared to the control. Examples of the control include the expression level of SPSB2 protein measured using a sample derived from normal tissue.

[0017] (primer set) The cancer diagnostic kit of this embodiment may include a primer set for amplifying cDNA of the SPSB2 gene. The primer set is not particularly limited in terms of its sequence, as long as it can amplify at least a portion of the cDNA of the SPSB2 gene.

[0018] A higher expression level of the SPSB2 gene in the test sample compared to the control indicates that the test sample is derived from a cancer patient. Examples of the control include the expression level of the SPSB2 gene measured using a sample derived from normal tissue.

[0019] (probe) The kit of this embodiment may contain a probe that specifically hybridizes to the mRNA of the SPSB2 gene.

[0020] The probe may be, for example, a nucleic acid fragment having a base sequence complementary to at least a portion of the base sequence of the mRNA of the SPSB2 gene. The probe may also be chemically modified in various ways to improve stability, specificity during hybridization, etc. For example, to suppress degradation by hydrolases such as nucleases, phosphate residues may be substituted with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. Furthermore, at least a portion of the probe may be composed of a nucleic acid analog such as peptide nucleic acid (PNA).

[0021] The probe may be immobilized on a solid phase. Examples of the solid phase include beads, a plate-like substrate, and a membrane. The probe may be immobilized on the surface of a plate-like substrate to form a microarray. In this case, for example, RNA is extracted from the test sample, labeled with a fluorescent substance, and hybridized with the microarray to detect the RNA bound to the probe on the microarray, thereby detecting the expression of the SPSB2 gene in the test sample.

[0022] A higher expression level of the SPSB2 gene in the test sample compared to the control indicates that the test sample is derived from a cancer patient. Examples of the control include the expression level of the SPSB2 gene measured using a sample derived from normal tissue.

[0023] [Method for assessing biological samples, method for collecting data for assessing whether a subject has cancer, and method for predicting the prognosis of a cancer patient] In one embodiment, the present invention provides a method for assessing a biological sample, comprising a step of measuring the expression level of SPSB2 protein or gene in the biological sample, wherein a higher expression level of the measured protein or gene compared to a control indicates that the biological sample is derived from a cancer patient.

[0024] As described later in the Examples, the inventors have demonstrated that the SPSB2 protein or SPSB2 gene can be used as a cancer marker. Furthermore, the inventors have demonstrated that the SPSB2 protein or SPSB2 gene is useful not only for bladder cancer but also for pancreatic cancer and hepatocellular carcinoma. Therefore, the method of this embodiment can be used to determine whether a biological sample is derived from a cancer patient. Examples of cancers that can be detected by the method of this embodiment include bladder cancer, pancreatic cancer, and hepatocellular carcinoma.

[0025] As described above, serum, plasma, urine, tissue, etc. derived from a subject can be used as a biological sample. Furthermore, particularly when the cancer is bladder cancer, examples of the biological sample include urine and tissue. Regarding urine, exosomes extracted from urine may be used as the biological sample. Furthermore, as described above, "higher than the control" preferably means statistically significantly higher than the control. Examples of the control include the expression level of the SPSB2 protein or SPSB2 gene measured using a sample derived from normal tissue.

[0026] The determination method of this embodiment can also be said to be a method for collecting data for determining whether or not a biological sample is derived from a cancer patient. The method for collecting data does not include medical treatment by a doctor.

[0027] That is, the present invention can be said to provide a method for collecting data for determining whether a biological sample is derived from a cancer patient, the method comprising a step of measuring the expression level of the SPSB2 protein or gene in the biological sample, wherein the measured expression level of the protein or gene provides data for determining whether the biological sample is derived from a cancer patient. A higher expression level of the SPSB2 protein or gene in the biological sample compared to a control indicates that the biological sample is derived from a cancer patient.

[0028] Alternatively, the present invention can be said to provide a method for collecting data for determining whether a subject has cancer, the method comprising a step of measuring the expression level of the SPSB2 protein or gene in a biological sample derived from the subject, wherein the measured expression level of the protein or gene provides data for determining whether the subject has cancer. A higher expression level of the SPSB2 protein or gene in the biological sample derived from the subject compared to a control indicates that the subject has cancer. In the data collection method, examples of cancer include bladder cancer, pancreatic cancer, and hepatocellular carcinoma.

[0029] Furthermore, as described later in the Examples, the inventors analyzed bladder cancer patients and hepatocellular carcinoma patients and found that high expression of the SPSB2 protein or SPSB2 gene tends to be associated with a poor prognosis. Therefore, the determination method of this embodiment can also be considered a method for predicting prognosis.

[0030] In other words, the present invention can be said to provide a method for predicting the prognosis of a cancer patient, which comprises a step of measuring the expression level of the SPSB2 protein or SPSB2 gene in a biological sample derived from the cancer patient, and wherein a higher expression level of the measured protein or gene compared to a control indicates a poor prognosis for the cancer patient. In the method for predicting the prognosis of a cancer patient, examples of cancer include bladder cancer, pancreatic cancer, and hepatocellular carcinoma.

[0031] [Anti-cancer drug screening method] In one embodiment, the present invention provides a method for screening anticancer agents, which includes a step of measuring the expression level of SPSB2 protein or SPSB2 gene in cancer cells cultured in the presence of a test substance, and a significant decrease in the expression level compared to the expression level of SPSB2 protein or SPSB2 gene in the absence of the test substance indicates that the test substance is an anticancer agent.

[0032] As will be described later in the Examples, the more malignant a cancer cell is, the higher the expression level of the SPSB2 protein or SPSB2 gene tends to be. Therefore, a test substance that reduces the expression level of the SPSB2 protein or SPSB2 gene can be said to be a candidate for an anticancer drug.

[0033] 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.

[0034] In the screening method of this embodiment, the cancer cells may be cancer cells derived from bladder cancer, pancreatic cancer, hepatocellular carcinoma, or the like.

[0035] Alternatively, the cancer cells may be cisplatin-resistant cells obtained by culturing the cancer cells in the presence of gradually increasing concentrations of cisplatin. Cisplatin-resistant cells tend to be resistant to anticancer drugs other than cisplatin and tend to be highly malignant.

[0036] [Other embodiments] In one embodiment, the present invention provides a method for treating cancer, comprising the steps of measuring the expression level of SPSB2 protein or SPSB2 gene in a biological sample derived from a subject, wherein a higher expression level compared to a control indicates that the subject is suffering from cancer, and, if the subject is suffering from cancer, surgically removing cancerous tissue from the subject or administering anticancer drug treatment to the subject.

[0037] In the treatment method of this embodiment, examples of cancer include bladder cancer, pancreatic cancer, and hepatocellular carcinoma. Examples of biological samples include serum, plasma, urine, and tissue. In particular, when the cancer is bladder cancer, examples of biological samples include urine and tissue. Regarding urine, exosomes may be extracted from urine to prepare the biological sample.

[0038] As will be described later in the Examples, when urine is used as a biological sample, the expression level of the SPSB2 protein or SPSB2 gene may be high not only in cancer patients but also in patients with urinary tract infections. In this case, whether or not a subject has a urinary tract infection can be diagnosed by a urine culture test or the like. That is, a subject with a high expression level of the SPSB2 protein or SPSB2 gene but without a urinary tract infection can be diagnosed as having cancer.

[0039] In the treatment method of this embodiment, examples of anticancer drugs include cisplatin, M-VAC (a combination of methotrexate, vinblastine, adriamycin, and cisplatin), GC (a combination of cisplatin and Gemzar), and antibody-drug conjugates (ADCs) targeting Nectin-4, such as enfortumab vedotin, padsev, and Keytruda (pemprolizumab).

[0040] These anticancer agents are generally administered by intravenous infusion. The dosage of these anticancer agents varies depending on the patient's symptoms, weight, age, sex, etc., but a person skilled in the art can appropriately select an appropriate dosage. [Example]

[0041] The present invention will now be described in more detail with reference to experimental examples, but the present invention is not limited to these examples.

[0042] [Experimental Example 1] In previous research, the inventors have already produced numerous bladder cancer-specific antibodies. They have also established a technique for identifying target proteins using autoantibodies in serum using the dot blot method, and have successfully identified numerous autoantibodies against bladder cancer. They examined the reactivity of these bladder cancer-specific antibodies and autoantibodies against bladder cancer with the serum and tumor tissues of numerous bladder cancer patients they had collected. As a result, they identified the SPSB2 protein or SPSB2 gene as a new cancer marker candidate.

[0043] [Experimental Example 2] The expression level of the SPSB2 gene in bladder cancer tissue was examined using the public database, The Cancer Genome Atlas (TCGA). Figure 1 is a graph showing the results of the study. In Figure 1, the vertical axis indicates the expression level of the SPSB2 gene. Furthermore, "Normal" indicates the expression level of the SPSB2 gene in normal tissue, and "Primary tumor" indicates the expression level of the SPSB2 gene in bladder cancer tissue. The results revealed that the expression level of the SPSB2 gene was significantly increased in bladder cancer tissue.

[0044] [Experimental Example 3] Tissue sections prepared from radical cystectomy specimens were immunostained with anti-SPSB2 antibodies. The specimens were from 126 patients who underwent radical cystectomy at Kitasato University Hospital between 1990 and 2015. Immunostaining was performed using a Bond-MAX automated immunostainer (Leica).

[0045] The intensity of nuclear staining by anti-SPSB2 antibodies in tumor cells in tissue sections was compared with the intensity of nuclear staining by anti-SPSB2 antibodies in surrounding cells, and evaluated using three levels according to the following criteria: Criteria 0-1 were classified as the low SPSB2 protein expression group, and criteria 2 was classified as the high SPSB2 protein expression group, and the following analysis was performed.

[0046] (Evaluation criteria) 0: Low expression 1: Equivalent 2: High expression

[0047] Figures 2(a) to (c) are photographs showing representative immunostaining results. Figure 2(a) is a photograph showing the results of immunostaining of normal urothelial tissue. Figure 2(b) is a photograph showing the results of immunostaining of bladder cancer tissue classified as a group with low SPSB2 protein expression. Figure 2(c) is a photograph showing the results of immunostaining of bladder cancer tissue classified as a group with high SPSB2 protein expression.

[0048] [Experimental Example 4] Based on the results of Experimental Example 3, the correlation between SPSB2 protein expression and clinicopathological factors was analyzed. The analysis results are shown in Table 1 below. In Table 1, the "p value" indicates the p value calculated by Fisher's exact test. p<0.05 was considered to indicate a significant difference. Bold indicates a significant difference. As a result, it was revealed that SPSB2 protein expression is correlated with gender, depth of invasion (pT stage), grade of atypia, and vascular invasion.

[0049] [Table 1]

[0050] Table 2 below shows the results of examining the correlation between the expression of S100A8 protein, S100A9 protein, Uroplakin III protein, and HNRNPA3 protein, which have been reported to be bladder cancer markers, and the expression of SPSB2 protein. In Table 2, the "p value" indicates the p value calculated by Fisher's exact test. p<0.05 was considered to indicate a significant difference. Bold indicates a significant difference.

[0051] As a result, it was revealed that the expression of SPSB2 protein was correlated with the expression of S100A8 protein, S100A9 protein, Uroplakin III protein, and HNRNPA3 protein.

[0052] [Table 2]

[0053] [Experimental Example 5] Based on the results of Experimental Example 3, the relationship between SPSB2 protein expression and prognosis was analyzed. Figure 3 is a graph showing the results of an analysis of cancer-specific survival rates using the Kaplan-Meier method. In Figure 3, "SPSB2 low" indicates the results for the low SPSB2 protein expression group, and "SPSB2 high" indicates the results for the high SPSB2 protein expression group. Additionally, "Number at risk" indicates the number of survivors at each time point. The results revealed that the high SPSB2 protein expression group had a significantly higher risk of death from bladder cancer.

[0054] Figure 4 is a graph showing the results of an analysis of progression-free survival rates using the Kaplan-Meier method based on the results of Experimental Example 3. In Figure 4, "SPSB2 low" indicates the results for the low SPSB2 protein expression group, and "SPSB2 high" indicates the results for the high SPSB2 protein expression group. Additionally, "Number at risk" indicates the number of progression-free survivors at each time point. The results revealed that the high SPSB2 protein expression group had a significantly shorter time to bladder cancer recurrence.

[0055] Table 3 below shows the results of univariate and multivariate analyses based on the Cox proportional hazards model for cancer-specific survival. Table 4 below shows the results of univariate and multivariate analyses based on the Cox proportional hazards model for progression-free survival. In Tables 3 and 4, "HR" indicates hazard ratio, and "95% CI" indicates 95% confidence interval. p<0.05 was considered to indicate a significant difference. Bold indicates a significant difference.

[0056] As a result, it was revealed that SPSB2 protein expression is an independent factor for cancer-specific survival and progression-free survival, as well as lymph node metastasis.

[0057] [Table 3]

[0058] [Table 4]

[0059] [Experimental Example 6] We investigated the expression of SPSB2 protein in cancer types other than bladder cancer. Figures 5(a)-(c) are photographs showing representative results of immunostaining with anti-SPSB2 antibodies on tissue sections of pancreatic cancer, hepatocellular carcinoma, and ovarian cancer. Figure 5(a) shows the results for a pancreatic cancer tissue section, Figure 5(b) shows the results for a hepatocellular carcinoma tissue section, Figure 5(c) shows the results for an ovarian cancer tissue section, and Figure 5(d) shows the results for a normal pancreatic tissue section.

[0060] The results showed that SPSB2 protein was strongly expressed in pancreatic cancer, and moderately expressed in hepatocellular carcinoma and ovarian cancer, while it was weakly or non-expressed in renal, prostate, esophageal, gastric, colon, breast, and lung cancer.

[0061] [Experimental Example 7] The expression level of the SPSB2 gene in hepatocellular carcinoma tissue was examined using the public database, The Cancer Genome Atlas (TCGA). Figure 6 is a graph showing the results of the examination. In Figure 6, the vertical axis indicates the expression level of the SPSB2 gene. Furthermore, "Normal" indicates the expression level of the SPSB2 gene in normal tissue, and "Primary tumor" indicates the expression level of the SPSB2 gene in hepatocellular carcinoma tissue.

[0062] As a result, it was revealed that the expression level of the SPSB2 gene was significantly increased in hepatocellular carcinoma tissues.

[0063] Figure 7 is a graph showing the relationship between SPSB2 gene expression levels and prognosis in hepatocellular carcinoma in TCGA samples. In Figure 7, "High expression" indicates the results for the high SPSB2 protein expression group, and "Low / Medium expression" indicates the results for the low to medium SPSB2 protein expression group. The results revealed that the high SPSB2 gene expression group in hepatocellular carcinoma patients had a significantly poorer prognosis.

[0064] [Experimental Example 8] The abundance of SPSB2 protein in serum and urinary exosomes from healthy individuals and bladder cancer patients was examined by Western blotting.

[0065] First, exosomes were extracted from serum and urine samples using Total Exosome Isolation Reagent (Thermo Fisher Scientific).

[0066] Next, SPSB2 protein was detected using the extracted exosomes. Furthermore, Western blotting of CD9, an exosome marker, confirmed that exosomes were successfully extracted. The molecular weight of SPSB2 protein is approximately 26 kDa, while that of CD9 is approximately 24 kDa.

[0067] Figure 8 is a photograph showing the results of Western blotting of serum exosomes. In Figure 8, "C" indicates exosomes derived from healthy individuals, and "T" indicates exosomes derived from bladder cancer patients.

[0068] As a result, CD9 was detected, confirming that exosomes were extracted. Furthermore, it was revealed that the presence of SPSB2 protein was not detected in serum exosomes from either healthy individuals or bladder cancer patients.

[0069] Figure 9 is a photograph showing the results of Western blotting of urinary exosomes. In Figure 9, "C" indicates exosomes derived from healthy individuals, and "T" indicates exosomes derived from bladder cancer patients.

[0070] As a result, CD9 was detected, confirming that exosomes were extracted. urine The presence of SPSB2 protein was not detected in urinary exosomes, whereas the presence of SPSB2 protein was detected in urinary exosomes from bladder cancer patients.

[0071] [Experimental Example 9] The abundance of SPSB2 protein in urine from healthy individuals, patients with urinary tract stones, patients with urinary tract infection, and patients with bladder cancer was quantified by ELISA (enzyme-linked immunosorbent assay) and examined.

[0072] The subjects were 91 bladder cancer patients who underwent urine collection immediately before transurethral bladder tumor resection at Kitasato University Hospital between 2009 and 2015. Table 5 below shows the background of the bladder cancer patients. In Table 5, "NMIBC" indicates non-muscle-invasive bladder cancer, and "MIBC" indicates muscle-invasive bladder cancer. The specific gravity of all urine samples was corrected to 1.002, and the amount of SPSB2 protein present was measured by ELISA.

[0073] [Table 5]

[0074] Figure 10 is a graph showing the quantitative values ​​of SPSB2 protein in urine samples from each group. Figure 10 also shows the results of the Mann-Whitney U test. In Figure 10, "BC" indicates the results for bladder cancer patients, "Healthy" indicates the results for healthy individuals, "Stone" indicates the results for urinary stone patients, and "UTI" indicates the results for urinary tract infection patients.

[0075] As a result, the urinary S P S B The abundance of the two proteins was higher than that of healthy individuals and patients with urinary stones, but lower than that of patients with urinary tract infections, which can be diagnosed by urine culture tests.

[0076] Figure 11 is a graph showing the quantitative values ​​of SPSB2 protein in urine samples from patients with non-muscle invasive bladder cancer and patients with muscle invasive bladder cancer. Figure 11 also shows the results of analysis using the Mann-Whitney U test. In Figure 11, "NMIBC" indicates non-muscle invasive bladder cancer, and "MIBC" indicates muscle invasive bladder cancer.

[0077] As a result, the urinary S P S B The abundance of these two proteins was found to be higher in muscle-invasive bladder cancer.

[0078] Figure 12 is a graph showing the quantitative values ​​of SPSB2 protein in urine samples from patients with bladder cancer of pathological grades 1 and 2 and from patients with bladder cancer of pathological grade 3. Figure 12 also shows the results of analysis by the Mann-Whitney U test.

[0079] As a result, the urinary S P S B The abundance of these two proteins tended to be higher in patients with pathological grade 1 and 2 bladder cancer.

[0080] Figure 13 is a graph showing the quantitative values ​​of SPSB2 protein in urine samples from patients with muscle-invasive bladder cancer and patients with urinary tract infection. Figure 13 also shows the results of a Mann-Whitney U test. In Figure 13, "MIBC" indicates muscle-invasive bladder cancer, and "UTI" indicates the results from patients with urinary tract infection.

[0081] As a result, the urinary S levels in patients with muscle-invasive bladder cancer and urinary tract infection were P S B No significant difference was observed in the abundance of the two proteins. Urinary tract infections can be diagnosed by urine culture tests, etc.

[0082] [Experimental Example 10] The relationship between SPSB2 protein expression and prognosis was analyzed based on the results of Experimental Example 9. Figure 14 shows an ROC curve created based on the quantitative values ​​of SPSB2 protein in urine samples from bladder cancer patients and healthy individuals measured in Experimental Example 9. As a result, it was revealed that the area under the ROC curve (AUC) was 0.7791.

[0083] Figure 15 shows an ROC curve created based on the quantitative values ​​of SPSB2 protein in urine samples from muscle-invasive bladder cancer patients and healthy individuals measured in Experimental Example 9. As a result, it was revealed that the area under the ROC curve (AUC) was 0.8699.

[0084] Next, based on the ROC curve, the cutoff value was set at 162.8 ng / mL (sensitivity 58.2%, specificity 80.0%), and survival analysis was performed with urinary SPSB2 protein levels of less than 162.8 ng / mL representing the low SPSB2 protein expression group and 162.8 ng / mL or higher representing the high SPSB2 protein expression group.

[0085] Figure 16 is a graph showing the results of an analysis of cancer-specific survival rates using the Kaplan-Meier method. In Figure 16, "SPSB2 Low" indicates the results for the low SPSB2 protein expression group, and "SPSB2 High" indicates the results for the high SPSB2 protein expression group. Additionally, "Number at risk" indicates the number of survivors at each time point. The results revealed that the high SPSB2 protein expression group had a significantly higher risk of death from bladder cancer.

[0086] Figure 17 is a graph showing the results of an analysis of progression-free survival rates using the Kaplan-Meier method. In Figure 17, "SPSB2 Low" indicates the results for the low SPSB2 protein expression group, and "SPSB2 High" indicates the results for the high SPSB2 protein expression group. Additionally, "Number at risk" indicates the number of progression-free survivors at each time point. As a result, no significant difference was observed in the time to bladder cancer recurrence between the low SPSB2 protein expression group and the high SPSB2 protein expression group.

[0087] Table 6 below shows the results of univariate and multivariate analyses of cancer-specific survival rates based on the Cox proportional hazards model. In Table 6, "NMIBC" indicates non-muscle-invasive bladder cancer, "MIBC" indicates muscle-invasive bladder cancer, "G3" indicates grade 3, "G1,2" indicates grade 1 or 2, "HR" indicates hazard ratio, and "95% CI" indicates 95% confidence interval. p<0.05 was considered to indicate a significant difference. Bold indicates a significant difference.

[0088] As a result, it was revealed that SPSB2 protein expression, along with the depth of cancer invasion, is a prognostic factor for cancer-specific survival.

[0089] [Table 6]

[0090] [Experimental Example 11] Human bladder cancer cell lines T24 and 5637 were cultured in the presence of increasing concentrations of cisplatin to obtain cisplatin-resistant cell lines, T24CDDPR and 5637CDDPR, respectively. T24CDDPR and 5637CDDPR are considered to be more malignant than T24 and 5637.

[0091] Next, the expression levels of SPSB2 protein and β-actin protein were measured by Western blotting in the T24, T24CDDPR, 5637, and 5637CDDPR cell lines.

[0092] Figure 18(a) is a photograph showing the results of Western blotting. Figure 18(b) is a graph of the results of Figure 18(a). The vertical axis of Figure 18(b) shows the expression level of SPSB2 relative to the expression level of β-actin, with "*" indicating a significant difference at p<0.05 and "**" indicating a significant difference at p<0.01.

[0093] As a result, it was revealed that T24CDDPR and 5637CDDPR had significantly higher SPSB2 expression levels than T24 and 5637, respectively.

[0094] This result indicates that the higher the malignancy of the cancer, the higher the expression level of SPSB2 in the cancer cells. [Industrial Applicability]

[0095] The present invention can provide a new cancer diagnostic technique.

Claims

1. a substance that specifically binds to the SPRY domain-containing SOCS box protein 2 (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; It is used to measure the expression level of SPSB2 protein or SPSB2 gene in a biological sample, and a higher expression level of the measured protein or gene compared to a control indicates that the biological sample is derived from a cancer patient, the cancer is bladder cancer, pancreatic cancer, or hepatocellular carcinoma; The biological sample is a cancer tissue. Cancer diagnostic kit.

2. comprising a substance that specifically binds to the SPSB2 protein, It is used to measure the expression level of SPSB2 protein in a biological sample, and a higher expression level of the measured protein compared to a control indicates that the biological sample is derived from a cancer patient; the cancer is bladder cancer, The biological sample is urine from a subject who is not suffering from a urinary tract infection. Cancer diagnostic kit.

3. 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; It is used to measure the expression level of SPSB2 protein or SPSB2 gene in a biological sample derived from a cancer patient, and a higher expression level of the measured protein or gene compared to a control indicates a poor prognosis for the cancer patient, the cancer is bladder cancer or hepatocellular carcinoma, The biological sample is a cancer tissue. A kit for determining the prognosis of cancer patients.

4. comprising a substance that specifically binds to the SPSB2 protein, It is used to measure the expression level of SPSB2 protein in a biological sample derived from a cancer patient, and a higher expression level of the measured protein compared to a control indicates a poor prognosis for the cancer patient, the cancer is bladder cancer, The biological sample is urine from a bladder cancer patient who is not suffering from a urinary tract infection. A kit for determining the prognosis of cancer patients.

5. measuring the expression level of SPSB2 protein or SPSB2 gene in a biological sample, a higher expression level of the measured protein or gene compared to a control indicates that the biological sample is derived from a cancer patient; the cancer is bladder cancer, pancreatic cancer, or hepatocellular carcinoma; The biological sample is a cancer tissue. Methods for determining biological samples.

6. measuring the expression level of SPSB2 protein in a biological sample; a higher expression level of the measured protein compared to a control indicates that the biological sample is derived from a cancer patient; the cancer is bladder cancer, The biological sample is urine from a subject who is not suffering from a urinary tract infection. Methods for determining biological samples.

7. A method for collecting data for determining whether or not a subject is suffering from cancer, the method comprising: measuring an expression level of SPSB2 protein or SPSB2 gene in a biological sample derived from the subject; data on the measured expression level of the protein or gene is data for determining whether or not the subject is suffering from cancer; and a higher expression level of the measured protein or gene compared to a control indicates that the biological sample is derived from a cancer patient; the cancer is bladder cancer, pancreatic cancer, or hepatocellular carcinoma; The biological sample is a cancer tissue. Methods (excluding medical procedures performed by physicians).

8. A method for collecting data for determining whether or not a subject is suffering from cancer, the method comprising: measuring an expression level of SPSB2 protein in a biological sample derived from the subject; the measured data on the expression level of the protein is data for determining whether or not the subject is suffering from cancer; and a higher expression level of the measured protein compared to a control indicates that the biological sample is derived from a cancer patient; the cancer is bladder cancer, The biological sample is urine from a subject who is not suffering from a urinary tract infection. Methods (excluding medical procedures performed by physicians).

9. A method for predicting the prognosis of a cancer patient, comprising a step of measuring an expression level of SPSB2 protein or SPSB2 gene in a biological sample derived from the cancer patient, a higher expression level of the measured protein or gene compared to a control indicates a poor prognosis for the cancer patient; the cancer is bladder cancer or hepatocellular carcinoma, The biological sample is a cancer tissue. method.

10. A method for predicting the prognosis of a cancer patient, comprising a step of measuring an expression level of SPSB2 protein in a biological sample derived from the cancer patient, a higher expression level of the measured protein compared to a control indicates a poor prognosis for the cancer patient; the cancer is bladder cancer, The biological sample is urine from a bladder cancer patient who is not suffering from a urinary tract infection. method.

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