Biomarkers, methods, kits and arrays for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer

Abstract

The present invention addresses the problem of providing a means for predicting the therapeutic effects of BCG intravesical infusion therapy in treating bladder cancer, more specifically, for predicting resistance to the BCG intravesical infusion therapy in treating bladder cancer, and / or possibility of relapse after the BCG intravesical infusion therapy in treating bladder cancer. The method according to the present invention for predicting the therapeutic effects of BCG intravesical infusion therapy in treating bladder cancer involves a step for detecting the presence or absence of two or more single nucleotide polymorphisms. Thus, the method of the invention comprises predicting resistance to the BCG intravesical infusion therapy in treating bladder cancer, and / or predicting possibility of relapse after the BCG intravesical infusion therapy in treating bladder cancer.

Description

[Technical Field]

[0001] The present invention relates to a biomarker for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, specifically, resistance to intravesical BCG therapy in the treatment of bladder cancer and / or the likelihood of recurrence after intravesical BCG therapy in the treatment of bladder cancer. The present invention also relates to an array and kit for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, specifically, resistance to intravesical BCG therapy in the treatment of bladder cancer and / or the likelihood of recurrence after intravesical BCG therapy in the treatment of bladder cancer, and a method for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, specifically, resistance to intravesical BCG therapy in the treatment of bladder cancer and / or the likelihood of recurrence after intravesical BCG therapy in the treatment of bladder cancer. [Background technology]

[0002] Bladder cancer is a malignant tumor that develops in the urothelial mucosa of the bladder. Early detection and appropriate treatment can improve the prognosis of bladder cancer, so technical means for early detection and appropriate treatment are being investigated. For example, Patent Document 1 discloses a bladder cancer-specific biomarker that includes a specific nucleic acid sequence or the like. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2012-513584 [Non-patent literature]

[0004] [Non-Patent Document 1] Hinotsu S. et al., BJU Int 2011; 108; 187-195 Summary of the Invention [Problem to be solved by the invention]

[0005] Bladder cancer can be divided into two types: non-muscle-invasive bladder cancer, which does not penetrate the bladder muscle layer, and invasive bladder cancer, which penetrates the bladder muscle layer. Most bladder cancers are of the former type. Intravesical BCG therapy is an effective treatment for non-muscle-invasive bladder cancer. However, approximately 10% to 20% of patients with non-muscle-invasive bladder cancer exhibit resistance to intravesical BCG therapy. Treating these patients with intravesical BCG therapy and then undergoing other treatments, such as radical cystectomy, after resistance is determined can lead to treatment delays. To address this issue, a biomarker for predicting resistance to intravesical BCG therapy is needed.

[0006] Furthermore, it is known that non-muscle invasive bladder cancer has a high rate of recurrence (intravesical recurrence) even after intravesical BCG instillation therapy (e.g., Non-Patent Document 1). It is known that recurrence can be reduced by performing BCG maintenance instillation (maintenance therapy) after BCG induction therapy, but the high incidence of adverse events associated with maintenance therapy is a problem. Therefore, there is a need for a biomarker that can predict the possibility of intravesical recurrence after intravesical BCG instillation therapy.

[0007] Therefore, the object of the present invention is to provide a means for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, in particular, resistance to intravesical BCG therapy in the treatment of bladder cancer, and / or the possibility of recurrence after intravesical BCG therapy in the treatment of bladder cancer. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors have identified multiple single nucleotide polymorphisms that are correlated with the therapeutic effect of intravesical BCG therapy, particularly with resistance to intravesical BCG therapy in bladder cancer treatment and with recurrence after intravesical BCG therapy in bladder cancer treatment. The present invention is based on this novel finding.

[0009] That is, the present invention relates to, for example, the following inventions [1] to

[14] . [1] A method for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, comprising the step of detecting the presence or absence of two or more single nucleotide polymorphisms, the method comprising predicting resistance to intravesical BCG therapy in the treatment of bladder cancer and / or predicting the likelihood of recurrence after intravesical BCG therapy in the treatment of bladder cancer. [2] The method according to [1], wherein the two or more single nucleotide polymorphisms comprise two or more single nucleotide polymorphisms selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442, and rs13193250, and the method comprises predicting resistance to intravesical BCG therapy in the treatment of bladder cancer. [3] The method according to [1], wherein the two or more single nucleotide polymorphisms comprise two or more single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903, and the method comprises predicting the likelihood of recurrence after intravesical BCG therapy in the treatment of bladder cancer. [4] If two or more of the above single nucleotide polymorphisms are Two or more single nucleotide polymorphisms selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442, and rs13193250, and Two or more single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903 The method according to [1], wherein the method comprises predicting resistance to intravesical BCG therapy in the treatment of bladder cancer, and predicting the possibility of recurrence after intravesical BCG therapy in the treatment of bladder cancer. [5] A kit for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, comprising a reagent for detecting the presence or absence of two or more single nucleotide polymorphisms, wherein predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer comprises predicting resistance to intravesical BCG therapy in the treatment of bladder cancer, and / or predicting the possibility of recurrence after intravesical BCG therapy in the treatment of bladder cancer. [6] The kit according to [5], wherein the two or more single nucleotide polymorphisms comprise two or more single nucleotide polymorphisms selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442, and rs13193250, and wherein predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer comprises predicting resistance to intravesical BCG therapy in the treatment of bladder cancer. [7] The kit described in [5], wherein the two or more single nucleotide polymorphisms include two or more selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903, and predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer includes predicting the likelihood of recurrence after intravesical BCG therapy in the treatment of bladder cancer. [8] If two or more of the above single nucleotide polymorphisms are Two or more single nucleotide polymorphisms selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442, and rs13193250, and Two or more single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903 The kit according to [5], comprising: a) a method for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer; and b) a method for predicting resistance to intravesical BCG therapy in the treatment of bladder cancer; and c) a method for predicting the possibility of recurrence after intravesical BCG therapy in the treatment of bladder cancer. [9] An array for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, the array comprising probes for detecting the presence or absence of two or more single nucleotide polymorphisms, wherein predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer includes predicting resistance to intravesical BCG therapy in the treatment of bladder cancer, and / or predicting the possibility of recurrence after intravesical BCG therapy in the treatment of bladder cancer.

[10] The array according to [9], wherein the two or more single nucleotide polymorphisms comprise two or more single nucleotide polymorphisms selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442, and rs13193250; and predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer comprises predicting resistance to intravesical BCG therapy in the treatment of bladder cancer.

[11] The array described in [9], wherein the two or more single nucleotide polymorphisms comprise two or more selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903, and predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer comprises predicting the likelihood of recurrence after intravesical BCG therapy in the treatment of bladder cancer.

[12] If two or more of the above single nucleotide polymorphisms are Two or more single nucleotide polymorphisms selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442, and rs13193250, and Two or more single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903 The array according to [9], comprising:

[13] A biomarker for predicting resistance to intravesical BCG therapy in the treatment of bladder cancer, comprising a single nucleotide polymorphism selected from the group consisting of rs3738088, rs4250, rs11894207, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs73520681, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs61094339, rs1607282, rs7825442, and rs13193250.

[14] A method for predicting resistance to intravesical BCG therapy in the treatment of bladder cancer, comprising the step of detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs3738088, rs4250, rs11894207, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs73520681, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs61094339, rs1607282, rs7825442, and rs13193250.

[15] A kit for predicting resistance to intravesical BCG therapy in the treatment of bladder cancer, comprising a reagent for detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs3738088, rs4250, rs11894207, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs73520681, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs61094339, rs1607282, rs7825442, and rs13193250.

[16] An array for predicting resistance to intravesical BCG therapy in the treatment of bladder cancer, comprising probes for detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs3738088, rs4250, rs11894207, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs73520681, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs61094339, rs1607282, rs7825442, and rs13193250.

[17] A method for predicting the likelihood of recurrence after BCG therapy in the treatment of bladder cancer, comprising a single nucleotide polymorphism selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903.

[18] A kit for predicting the possibility of recurrence after BCG therapy in the treatment of bladder cancer, comprising a step of detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903.

[19] A reagent for predicting the possibility of recurrence after BCG therapy in the treatment of bladder cancer, comprising a reagent for detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903.

[20] An array for predicting the possibility of recurrence after BCG therapy in the treatment of bladder cancer, comprising probes for detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903. [Effects of the Invention]

[0010] The present invention can provide biomarkers for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, particularly resistance to intravesical BCG therapy in the treatment of bladder cancer, and / or the likelihood of recurrence after intravesical BCG therapy in the treatment of bladder cancer. The present invention also can provide arrays and kits for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, particularly resistance to intravesical BCG therapy in the treatment of bladder cancer, and / or the likelihood of recurrence after intravesical BCG therapy in the treatment of bladder cancer, as well as methods for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, particularly resistance to intravesical BCG therapy in the treatment of bladder cancer, and / or the likelihood of recurrence after intravesical BCG therapy in the treatment of bladder cancer.

[0011] Therefore, according to the present invention, it is possible to predict a patient's resistance to intravesical BCG instillation therapy before the patient undergoes intravesical BCG instillation therapy.Furthermore, according to the present invention, it is possible to predict the possibility of recurrence after intravesical BCG instillation therapy in a patient's bladder cancer treatment before the patient undergoes intravesical BCG instillation therapy, while the patient is receiving intravesical BCG instillation therapy, or after the patient has undergone intravesical BCG instillation therapy.As a result, it is expected that medical professionals will be able to select a more appropriate treatment for each individual bladder cancer patient, which will lead to a better prognosis for bladder cancer. [Brief explanation of the drawings]

[0012] [Figure 1]FIG. 1 shows the results of genome-wide association studies in Manhattan plots. [Figure 2] Graphs showing the survival rate for maintaining treatment response in the two-gene model (A) and the four-gene model (B). [Figure 3] FIG. 1 shows the results of genome-wide association studies in Manhattan plots. [Figure 4] Graph showing time to recurrence for the validation cohort (A) and exploratory cohort (B) in the two-gene model. DETAILED DESCRIPTION OF THE INVENTION

[0013] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following describes in detail the embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0014] [Biomarkers] In the present invention, biomarkers that can be used to predict the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer include biomarkers for predicting resistance to intravesical BCG therapy in the treatment of bladder cancer (BCG therapy resistance) and biomarkers for predicting the possibility of recurrence after BCG therapy in the treatment of bladder cancer (likelihood of recurrence). The biomarkers in the present invention consist of single nucleotide polymorphisms.

[0015] A single nucleotide polymorphism (SNP) is a type of genetic polymorphism. It refers to a single-nucleotide difference between individuals in the genomic DNA of a given species. It is a genetic polymorphism caused by a single-nucleotide substitution due to a point mutation in the base sequence. The frequency of SNPs is said to be greater than 1% of the population. Depending on their function, SNPs are classified as follows: rSNPs (regulatory SNPs) that cause mutations in promoter regions, cSNPs (coding SNPs) that cause mutations in exon regions, sSNPs (silent SNPs) that do not cause mutations in exon regions, iSNPs (intron SNPs) that cause mutations in intron regions, and gSNPs (genomic SNPs) that cause mutations in junk regions. Medical treatments that aim to avoid side effects and achieve efficient therapeutic effects by testing patients for SNPs before medication and determining the appropriate drug dosage based on their genotype are known as "personalized medicine."

[0016] The rs number is a number that refers to the SNP and indicates the accession number in the dbSNP database of the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / projects / SNP / ).

[0017] The sample used for analyzing single nucleotide polymorphisms is not particularly limited as long as it contains genomic DNA, and may be, for example, blood, tissue cells such as oral mucosal cells, body hair such as hair, urine, etc., and preferably whole blood, plasma, or serum. Analysis of single nucleotide polymorphisms can be performed, for example, by sequence analysis, TaqMan PCR, Invader PCR, etc. Devices used include, for example, a sequencer, a real-time PCR device, a fluorescence spectrophotometer, etc.

[0018] (Biomarkers associated with BCG therapy resistance) Biomarkers for predicting resistance to intravesical BCG therapy (BCG therapy resistance) in bladder cancer treatment according to this embodiment are rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442 and rs13193250. In this specification, these biomarkers are sometimes collectively referred to as "the 19 biomarkers according to this embodiment."

[0019] Intravesical BCG therapy is a treatment for bladder cancer, particularly for non-muscle-invasive bladder cancer. Intravesical BCG therapy involves instilling saline-based BCG (attenuated bovine tuberculosis) into the bladder through a catheter inserted into the bladder via the urethra, allowing the drug to come into contact with the bladder without urination for a set period of time. Resistance to intravesical BCG therapy refers to the ineffectiveness of intravesical BCG therapy. Resistance can be determined, for example, by assessing the presence or absence of residual bladder cancer 6 months after intravesical BCG therapy, the presence or absence of muscle-invasiveness after intravesical BCG therapy, the progression to metastatic bladder cancer after intravesical BCG therapy, and the need for radical cystectomy to control the underlying disease after intravesical BCG therapy.

[0020] The 19 biomarkers according to this embodiment are single nucleotide polymorphisms that are significantly higher in patients resistant to BCG intravesical instillation therapy than in patients not resistant to BCG intravesical instillation therapy in genome-wide SNP analysis. Because these single nucleotide polymorphisms are highly correlated with resistance to BCG intravesical instillation therapy, they can be used as biomarkers for predicting resistance to BCG intravesical instillation therapy in bladder cancer treatment. Detection of SNPs that are the 19 biomarkers according to this embodiment in genomic DNA analysis can determine whether a patient has or may have resistance to BCG intravesical instillation therapy in bladder cancer treatment. The 19 biomarkers according to this embodiment may be used alone, or multiple markers may be used in combination to further improve prediction accuracy.

[0021] The 19 biomarkers of this embodiment can assist in the diagnosis of bladder cancer, for example, by determining the therapeutic effect of intravesical BCG instillation therapy in the treatment of bladder cancer, predicting the therapeutic effect of intravesical BCG instillation therapy in the treatment of bladder cancer, and predicting the prognosis of bladder cancer patients after intravesical BCG instillation therapy in the treatment of bladder cancer.

[0022] rs3738088 is located in the intron region of the IGSF21 gene. The IGSF21 gene is located on human chromosome 1. The IGSF21 gene is a member of the immunoglobulin superfamily and is known to function as a receptor in immune responses.

[0023] The rs4250 gene is located near the TNFSF4 gene, 22,803 bp downstream of the gene. The TNFSF4 gene is located on human chromosome 1. The TNFSF4 gene binds to OX40 in T lymphocytes, contributing to the suppression of T lymphocyte cell death and cytokine release. Furthermore, rs4250 is located between TNFSF4 and TNFSF18 and is thought to affect the expression of these genes. Genes in the TNFSF family are thought to be involved in various immune responses. For example, TNFSF4 and TNFSF18 encode OX40L and glucocorticoid-inducible tumor necrosis factor (TNF) receptor-related protein ligand (GITRL), which activate T cells via their recognition receptors (OX40) and GITR, respectively. Signal transduction involving these molecules has been shown to be associated with the development of various immune-related diseases.

[0024] The rs11894207 mutation is located in the MPP4 gene. The MPP4 gene is located on human chromosome 2. The MPP4 gene is involved in photoreceptor polarity and cell-cell adhesion formation in the retina; however, its other functions remain largely unknown. rs11894207 causes a synonymous amino acid substitution in the MPP4 gene, suggesting that it may affect MPP4 gene expression. The MPP4 gene is a member of the MAGUK p55 subfamily and acts as a molecular scaffold to regulate membrane-associated cytoskeleton, ion channel and receptor clustering, signal transduction pathways, and cell junction formation.

[0025] The roles of MPP4, TMEM38B, and CXorf28 in the bladder and immune system remain to be explored. While the mechanism of BCG's antitumor activity remains unclear, infection of urothelial cells or bladder cancer cells with BCG, induction of an immune response, and enhanced antitumor activity are key steps in the antitumor efficacy of intravesical BCG therapy. Because TNFSF4 and TNFSF18 (TNFSFs) encode factors important for T cell activation and survival, they may be involved in the induction of an immune response during these steps. Furthermore, MPP4's involvement in the membrane-associated cytoskeleton and formation of cell junctions most likely contributes to the attachment and internalization of BCG in urothelial and bladder cancer cells. This suggests that rs4250 and rs11894207 influence the oncological outcome of BCG therapy.

[0026] rs161448 is located near the LMCD1-AS1 gene, 165,887 bp downstream of the LMCD1-AS1 gene. The LMCD1-AS1 gene is located on human chromosome 3. The LMCD1-AS1 gene is classified as a non-coding RNA, and its function is largely unknown.

[0027] The rs2764326 and rs2814707 locate near the MOB3B / IFNK gene. rs2764326 is located 5563 bp upstream of the MOB3B / IFNK gene, and rs2814707 is located 6618 bp upstream of the MOB3B / IFNK gene. The MOB3B / IFNK gene is located on human chromosome 9. The function of the MOB3B gene remains largely unknown. IFNK is located on the opposite strand of the MOB3B gene and encodes interferon-κ, a type 1 interferon. Type 1 interferons are cytokines produced by immune cells in response to the invasion of pathogens such as viruses.

[0028] The rs3787194 and rs58081719 loci are located in the intron region of the NFATC2 gene. The NFATC2 gene is located on human chromosome 20. The NFATC2 gene is activated by T cell receptor stimulation, translocates to the nucleus, and exhibits transcriptional activity. Therefore, it is known to play an important role in the immune response of T cells.

[0029] rs3095966 is located near the LINC01098 gene, 150,455 bp downstream of the LINC01098 gene. The LINC01098 gene is located on human chromosome 4. The LINC01098 gene is classified as a non-coding RNA, and its function is largely unknown.

[0030] rs73520681 is located near the TMEM38B gene, 622921 bp downstream of the gene, and may affect TMEM38B gene expression. The TMEM38B gene is located on human chromosome 9. The TMEM38B gene is involved in regulating intracellular calcium concentration, and abnormalities in this gene are known to cause congenital osteogenesis imperfecta.

[0031] rs16877113 is located near the DTNBP1 gene, 94101 bp upstream of the DTNBP1 gene. The DTNBP1 gene is located on human chromosome 6. The DTNBP1 gene is involved in the formation of intracellular organelles such as melanosomes, platelet dense granules, and lysosomes, and abnormalities in this gene are known to cause Hermansky-Pudlak syndrome.

[0032] rs16887173 is located in the intron region of the BMP5 gene. The BMP5 gene is located on human chromosome 6. The BMP5 gene is a member of the TGF-β superfamily and is thought to be involved in the development of osteoarthritis and various cancers.

[0033] rs10269584 is located in an intron region of a gene located on human chromosome 7.

[0034] rs11772249 is located near the SHH gene, 15,944 bp downstream of the gene. The SHH gene is located on human chromosome 7. The SHH gene plays an important role in regulating organogenesis in vertebrates. Even in adults, it is thought to regulate stem cell division and be involved in cancer development.

[0035] rs118137814 is located in the intron region of the ATRNL1 gene. The ATRNL1 gene is located on human chromosome 10. The ATRNL1 gene encodes attractin-like protein 1, and its function remains largely unknown.

[0036] rs61094339 is located near the CXorf28 gene, 82081 bp upstream of the CXorf28 gene. The CXorf28 gene is located on the human X chromosome. The CXorf28 gene is classified as a non-coding RNA, and its function is largely unknown.

[0037] rs1607282 is located near the SERTM1 gene, 138,889 bp upstream of the SERTM1 gene. The SERTM1 gene is located on human chromosome 13. The SERTM1 gene encodes serine-rich and transmembrane domain-containing protein 1, and its function remains largely unknown.

[0038] rs7825442 is located in an intron region of the DLGAP2 gene, which is located on human chromosome 8. The DLGAP2 gene is known to function in neuronal synapses.

[0039] rs13193250 is located near the SLC22A23 gene, 13,357 bp upstream of the gene. The SLC22A23 gene is located on human chromosome 6. The SLC22A23 gene is a member of the SLC transporter superfamily and is thought to be involved in material transport.

[0040] (Biomarkers associated with the likelihood of recurrence) The biomarkers for predicting the likelihood of recurrence after BCG therapy in bladder cancer treatment according to this embodiment are single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903. In this specification, these biomarkers are sometimes collectively referred to as the "12 biomarkers according to this embodiment."

[0041] Recurrence after intravesical BCG therapy refers to the recurrence of non-muscle-invasive bladder cancer (also referred to as intravesical recurrence) after intravesical BCG therapy. Generally, if recurrence does not occur within five years from the initial onset, the probability of recurrence is extremely low. Therefore, recurrence after intravesical BCG therapy in this specification refers to no recurrence within five years, preferably no recurrence within ten years, from the initial onset of bladder cancer. Furthermore, to determine recurrence, for example, regular urine cytology or cystoscopy is performed after intravesical BCG therapy, and if recurrence is suspected, a bladder biopsy or bladder tumor resection is performed, and intravesical recurrence can be determined by pathological diagnosis of the biopsy obtained from the bladder biopsy or bladder tumor resection.

[0042] The 12 biomarkers according to this embodiment are single nucleotide polymorphisms that are significantly detected in patients who relapsed after intravesical BCG therapy compared to patients who did not relapse after intravesical BCG therapy in genome-wide SNP analysis. Because these single nucleotide polymorphisms are highly correlated with intravesical recurrence after intravesical BCG therapy, they can be used as biomarkers for predicting the likelihood of recurrence after intravesical BCG therapy in bladder cancer treatment. Detection of SNPs that are the 12 biomarkers according to this embodiment in genomic DNA analysis can determine whether there is a high likelihood or possibility of recurrence after intravesical BCG therapy in bladder cancer treatment. The 12 biomarkers according to this embodiment may be used alone, or multiple markers may be used in combination to further improve prediction accuracy.

[0043] The 12 biomarkers according to this embodiment can assist in the diagnosis of bladder cancer, for example, by determining the therapeutic effect of intravesical BCG instillation therapy in the treatment of bladder cancer, predicting the therapeutic effect of intravesical BCG instillation therapy in the treatment of bladder cancer, predicting the prognosis of bladder cancer patients after intravesical BCG instillation therapy in the treatment of bladder cancer, etc. Furthermore, the combination of the 19 biomarkers and 12 biomarkers according to this embodiment can assist in the diagnosis of bladder cancer, for example, by predicting the therapeutic effect of intravesical BCG instillation therapy in the treatment of bladder cancer, predicting the prognosis of bladder cancer patients after intravesical BCG instillation therapy in the treatment of bladder cancer, etc.

[0044] The rs363765 mutation is located in an intron region of the POF1B gene. The MAN1A1 gene is located on the X chromosome in humans. The POF1B gene is expressed in the epidermis, oropharynx, and gastrointestinal tract and is known to bind to non-muscle actin filaments, but its function remains largely unknown.

[0045] rs6986852 is located near the GDF6 gene, 143,784 bp downstream of the GDF6 gene. The GDF6 gene is located on human chromosome 8. The GDF6 gene is a member of the TGF-β (transforming growth factor-β) superfamily, and mutations in this gene are known to cause congenital malformations such as Klippel-Feil syndrome and microphthalmia.

[0046] rs9374832 is located near the MAN1A1 gene, 452,066 bp upstream of the MAN1A1 gene. The MAN1A1 gene is located on human chromosome 6. The MAN1A1 gene encodes a type II transmembrane protein, class I mammalian Golgi 1,2-mannosidase, and is known to be involved in the progression of various cancers.

[0047] rs35176001 is located in the intron region of the TACC2 gene. The TACC2 gene is located on human chromosome 10. The TACC2 gene is involved in cell division through microtubule regulation in centrosome localization, and is known to be involved in the progression of various cancers.

[0048] rs2127120 is located in the intron region of the LOC101928135 gene, which is located on human chromosome 3. The LOC101928135 gene is classified as a non-coding RNA, and its function is largely unknown.

[0049] rs4277759 is located near the SFRP2 gene, 154,169 bp upstream of the SFRP2 gene. The SFRP2 gene is located on human chromosome 4. The SFRP2 gene is known to function as a regulator of Wnt signaling, which controls cell polarity and malignant transformation, and is involved in the progression of various cancers.

[0050] rs73664140 is located in the intron region of the PDCL gene, which is located on human chromosome 9. Although the function of the PDCL gene remains largely unknown, its association with allergic diseases has been reported.

[0051] rs1607282 is located near the SERTM1 gene, 138,889 bp upstream of the SERTM1 gene. The SERTM1 gene is located on human chromosome 13. The SERTM1 gene encodes serine-rich and transmembrane domain-containing protein 1, and its function remains largely unknown.

[0052] rs12141654 is located near the LOC101927412 gene, 289311 bp downstream of the LOC101927412 gene. The LOC101927412 gene is classified as a non-coding RNA, and its function is largely unknown.

[0053] rs4541358 is located in the intron region of the LOC101928135 gene, which is located on human chromosome 3. The LOC101928135 gene is classified as a non-coding RNA, and its function is largely unknown.

[0054] rs12373386 is located in the 3´-untranslated region of the GNAL gene. The GNAL gene is located on human chromosome 18. The GNAL gene is expressed in the central nervous system and has been reported to be associated with neurological and psychiatric disorders.

[0055] rs17637903 is located near the C16orf82 gene, 182,906 bp upstream of the C16orf82 gene. The C16orf82 gene is located on human chromosome 16. The C16orf82 gene is thought to encode a protein, but its function remains largely unknown.

[0056] [Prediction method] The prediction method of the present invention is a method for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, and includes a step of detecting the presence or absence of two or more single nucleotide polymorphisms. Here, predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer includes predicting resistance to intravesical BCG therapy in the treatment of bladder cancer and / or predicting the possibility of recurrence after intravesical BCG therapy in the treatment of bladder cancer.

[0057] In the prediction method of the present invention, the sample used for prediction is not particularly limited as long as it is a sample containing nucleic acid derived from a subject patient, for example, genomic DNA (which may be nuclear genomic DNA and may further include mitochondrial genomic DNA), or a sample containing chromosomal DNA in which a single nucleotide polymorphism to be detected exists. Examples of such a sample include blood, tissue cells such as oral mucosal cells, body hair such as hair, and urine, and are preferably whole blood, plasma, or serum. Prior to the step of detecting a single nucleotide polymorphism, genomic DNA may be extracted and purified as necessary. The method for extracting and purifying genomic DNA is not particularly limited, and can be carried out using, for example, a commercially available genome purification kit.

[0058] (Method for predicting BCG therapy resistance) The method for predicting resistance to intravesical BCG therapy in bladder cancer treatment according to this embodiment (sometimes referred to herein simply as the "method for predicting resistance to BCG therapy") comprises the step of detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442, and rs13193250.

[0059] The single nucleotide polymorphisms to be detected are two or more single nucleotide polymorphisms selected from the 19 biomarkers according to this embodiment. As described above, the 19 biomarkers according to this embodiment are single nucleotide polymorphisms that are significantly detected in a patient group resistant to intravesical BCG therapy compared to a patient group not resistant to intravesical BCG therapy. Because these single nucleotide polymorphisms are highly correlated with resistance to intravesical BCG therapy, the method for predicting resistance to BCG therapy according to this embodiment can predict resistance to intravesical BCG therapy in bladder cancer treatment for an individual patient.

[0060] The single nucleotide polymorphisms to be detected are two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, or all nineteen types of single nucleotide polymorphisms selected from the group consisting of the 19 biomarkers according to this embodiment. Preferred combinations of single nucleotide polymorphisms include two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, and rs118137814; The number of types is 13 or more, 14 or more, 15 or more, or all 16 types, and the number of types is two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 11 or more, 12 or more, 13 or more, or all 14 types selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs3787194, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, and rs118137814.

[0061] Particularly preferred combinations of single nucleotide polymorphisms include a combination of single nucleotide polymorphisms comprising rs73520681 and rs61094339, a combination of single nucleotide polymorphisms comprising rs4250, rs11894207, rs73520681 and rs61094339, and a combination of single nucleotide polymorphisms comprising rs73520681, rs61094339, rs4250, rs11894207, rs73520681 and rs61094339.

[0062] The step of detecting the presence or absence of a single nucleotide polymorphism is not particularly limited as long as it includes a step that can detect the presence or absence of single nucleotide polymorphisms (SNPs), which are the 19 biomarkers according to this embodiment. The procedures and devices and reagents used in the detection step vary depending on the detection method, but can be appropriately selected by those skilled in the art. The method of detecting SNPs is not particularly limited, and examples include conventional SNP analysis methods such as the Restriction Fragment Length Polymorphism (RFLP) method, in which a genomic region containing an SNP is amplified by PCR, treated with a restriction enzyme, and the DNA fragments are separated by agarose electrophoresis, and the resulting fragments are detected based on differences in fragment length, and the Single Strand Conformation Polymorphism (SSCP) method, in which RFLP PCR is performed on single-stranded DNA, electrophoresis is performed while maintaining the three-dimensional structure, and differences in three-dimensional structure caused by SNPs are detected based on differences in mobility. Other examples include SNP analysis methods such as TaqMan PCR, SNaP shot, Invander, mass spectrometry, and DNA chip methods, as well as DNA microarray methods.

[0063] In addition to the detection step, the method for predicting BCG therapy resistance according to this embodiment may further include a step of determining the presence or absence of resistance to intravesical BCG therapy in bladder cancer treatment based on the detection results of the presence or absence of the two or more single nucleotide polymorphisms. If even one of the two or more single nucleotide polymorphisms to be detected is detected, it can be determined that the subject patient is resistant to intravesical BCG therapy in bladder cancer treatment, or is likely to be resistant. The more single nucleotide polymorphisms detected, the higher the accuracy of the prediction is considered to be, but if even one single nucleotide polymorphism is detected, there is a sufficient possibility that the subject is resistant to intravesical BCG therapy in bladder cancer treatment. Preferably, a subject in which two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or even more preferably all, of the two or more single nucleotide polymorphisms to be detected is detected can be determined to be resistant to intravesical BCG therapy in bladder cancer treatment, or is likely to be resistant. Furthermore, this prediction method is intended to indicate the possibility of resistance and does not involve diagnosis or treatment (medical intervention) by a doctor.

[0064] The method for predicting resistance to BCG therapy according to this embodiment is preferably performed before or during intravesical BCG therapy, and more preferably before intravesical BCG therapy. Because SNPs in humans are thought to remain constant throughout their lives, the method for predicting resistance to BCG therapy according to this embodiment only needs to be performed once in a lifetime, but may be performed two or more times to further improve prediction accuracy.

[0065] (Method for predicting the likelihood of recurrence) The method for predicting the possibility of recurrence after intravesical BCG instillation therapy in bladder cancer treatment according to this embodiment (sometimes referred to herein simply as the "method for predicting the possibility of recurrence") comprises the step of detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903.

[0066] The single nucleotide polymorphisms to be detected are two or more single nucleotide polymorphisms selected from the 12 biomarkers according to this embodiment. As described above, the 12 biomarkers according to this embodiment are single nucleotide polymorphisms that are significantly detected in patients who relapsed after intravesical BCG therapy compared to patients who did not relapse after intravesical BCG therapy. Because these single nucleotide polymorphisms are highly correlated with intravesical recurrence after intravesical BCG therapy, the method for predicting the likelihood of recurrence according to this embodiment can predict the likelihood of recurrence after intravesical BCG therapy in bladder cancer treatment for each patient.

[0067] The single nucleotide polymorphisms to be detected are two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve of the single nucleotide polymorphisms selected from the group consisting of the 12 biomarkers according to this embodiment. A preferred combination of single nucleotide polymorphisms is two or more, three or more, or all of the single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs73664140, and rs17637903.

[0068] Particularly preferred combinations of single nucleotide polymorphisms include those containing rs363765 and rs6986852.

[0069] The step of detecting the presence or absence of a single nucleotide polymorphism is not particularly limited as long as it includes a step that can detect the presence or absence of single nucleotide polymorphisms (SNPs) that are the 12 biomarkers according to this embodiment. Specific detection methods are as described above.

[0070] In addition to the detection step, the method for predicting the likelihood of recurrence according to this embodiment may further include a step of determining the likelihood of recurrence after intravesical BCG instillation therapy for bladder cancer treatment based on the detection results of the presence or absence of the two or more single nucleotide polymorphisms. If even one of the two or more single nucleotide polymorphisms to be detected is detected, it can be determined that the target patient has a high or possible recurrence after intravesical BCG instillation therapy for bladder cancer treatment. The more single nucleotide polymorphisms detected, the higher the accuracy of the prediction is considered to be, but even if even one single nucleotide polymorphism is detected, there is a sufficient likelihood of recurrence after intravesical BCG instillation therapy for bladder cancer treatment. Preferably, a target patient in which two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or even more preferably all, of the two or more single nucleotide polymorphisms to be detected are detected can be determined to have a high or possible recurrence after intravesical BCG instillation therapy for bladder cancer treatment. Furthermore, this prediction method is intended to indicate the possibility of recurrence after intravesical BCG therapy, and does not involve diagnosis or treatment (medical procedures) by a doctor.

[0071] The method for predicting the likelihood of recurrence according to this embodiment may be performed before, during, or after intravesical BCG instillation therapy, but is preferably performed before intravesical BCG instillation therapy. Simultaneous performance of the method for predicting resistance to BCG therapy and the method for predicting the likelihood of recurrence is preferred, since it allows prediction of resistance to BCG therapy and the likelihood of recurrence with a single measurement, thereby reducing the burden on the patient. Because SNPs in humans are thought to remain constant throughout their lifetime, the method for predicting the likelihood of recurrence according to this embodiment only needs to be performed once in a lifetime, but may be performed more than once to further improve prediction accuracy.

[0072] [Prediction Kit] The kit for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer of the present invention (sometimes simply referred to as a "prediction kit" in this specification) comprises a reagent for detecting the presence or absence of two or more single nucleotide polymorphisms. Here, predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer includes predicting resistance to intravesical BCG therapy in the treatment of bladder cancer and / or predicting the possibility of recurrence after intravesical BCG therapy in the treatment of bladder cancer.

[0073] (BCG therapy resistance prediction kit) The kit for predicting resistance to intravesical BCG therapy in bladder cancer treatment according to this embodiment (sometimes referred to herein simply as a "BCG therapy resistance prediction kit") comprises reagents for detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442, and rs13193250.

[0074] The single nucleotide polymorphisms to be detected are two or more single nucleotide polymorphisms selected from the 19 biomarkers according to this embodiment. As described above, the 19 biomarkers according to this embodiment are single nucleotide polymorphisms that are significantly detected in a patient group resistant to intravesical BCG therapy compared to a patient group not resistant to intravesical BCG therapy. Because these single nucleotide polymorphisms are highly correlated with resistance to intravesical BCG therapy, the use of the kit for predicting resistance to BCG therapy according to this embodiment makes it possible to predict resistance to intravesical BCG therapy in bladder cancer treatment for individual patients.

[0075] The single nucleotide polymorphisms to be detected are two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, or all nineteen types of single nucleotide polymorphisms selected from the group consisting of the 19 biomarkers according to this embodiment. Preferred combinations of single nucleotide polymorphisms include two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, and rs118137814; The number of types is 13 or more, 14 or more, 15 or more, or all 16 types, and the number of types is two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 11 or more, 12 or more, 13 or more, or all 14 types selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs3787194, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, and rs118137814.

[0076] Particularly preferred combinations of single nucleotide polymorphisms include a combination of single nucleotide polymorphisms comprising rs73520681 and rs61094339, a combination of single nucleotide polymorphisms comprising rs4250, rs11894207, rs73520681 and rs61094339, and a combination of single nucleotide polymorphisms comprising rs73520681, rs61094339, rs4250, rs11894207, rs73520681 and rs61094339.

[0077] The BCG therapy resistance prediction kit according to this embodiment may include two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all 12 of the 19 biomarkers according to this embodiment for detecting the presence or absence of single nucleotide polymorphisms. For example, the kit may include primers or probes corresponding to the single nucleotide polymorphisms. The kit may further include restriction enzymes, polymerases, dNTPs, ddNTPs, etc., and may further include buffers, instructions, etc. The primers are preferably designed to amplify regions containing single nucleotide polymorphisms and to have restriction enzyme sites at appropriate locations. The probes are preferably designed to hybridize with single nucleotide polymorphisms. The primers, probes, and ddNTPs may be labeled, such as with fluorescent labels, for detection. The reagents included in the kit may vary depending on the SNP analysis method. For example, in the case of conventional SNP analysis methods such as RFLP and SSCP, the kit may contain a primer corresponding to the single nucleotide polymorphism, and may further contain reagents necessary for the PCR reaction, such as polymerase, polynucleotides, and restriction enzymes. In the case of the SNaP shot method, the kit may contain a primer and fluorescently labeled ddNTP, which are used to perform a single-base extension reaction and are then analyzed by capillary electrophoresis. In the case of comprehensive SNP analysis methods using probes, the kit may contain a fluorescently labeled probe, and may also contain a reagent for labeling the genomic DNA of the subject.

[0078] (Recurrence probability prediction kit) The kit for predicting the likelihood of recurrence after intravesical BCG instillation therapy in bladder cancer treatment according to this embodiment (sometimes simply referred to herein as a "kit for predicting the likelihood of recurrence") comprises reagents for detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903.

[0079] The single nucleotide polymorphisms to be detected are two or more single nucleotide polymorphisms selected from the 12 biomarkers according to this embodiment. As described above, the 12 biomarkers according to this embodiment are single nucleotide polymorphisms that are significantly detected in patients who have relapsed after intravesical BCG therapy compared to patients who have not relapsed after intravesical BCG therapy. Because these single nucleotide polymorphisms are highly correlated with intravesical recurrence after intravesical BCG therapy, the use of the kit for predicting the likelihood of recurrence according to this embodiment makes it possible to predict the likelihood of recurrence after intravesical BCG therapy in bladder cancer treatment for individual patients.

[0080] The single nucleotide polymorphisms to be detected are two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve single nucleotide polymorphisms selected from the group consisting of the 12 biomarkers according to this embodiment. A preferred combination of single nucleotide polymorphisms is two or more, three or more, or all selected from the group consisting of rs363765, rs6986852, rs73664140, and rs17637903.

[0081] Particularly preferred combinations of single nucleotide polymorphisms include those containing rs363765 and rs6986852.

[0082] The recurrence probability prediction kit according to this embodiment may include two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve of the 12 biomarkers according to this embodiment for detecting the presence or absence of single nucleotide polymorphisms. For example, the kit may include primers or probes corresponding to the single nucleotide polymorphisms. The kit may further include restriction enzymes, polymerases, dNTPs, ddNTPs, etc., and may further include buffers, instructions, etc. The primers are preferably designed to amplify the region containing the single nucleotide polymorphism and to have a restriction enzyme site at an appropriate location. The probes are preferably designed to hybridize with the single nucleotide polymorphism. The primers, probes, and ddNTPs may be labeled, such as with a fluorescent label, for detection. The reagents included in the kit may vary depending on the SNP analysis method. For example, in the case of conventional SNP analysis methods such as RFLP and SSCP, the kit may contain a primer corresponding to the single nucleotide polymorphism, and may further contain reagents necessary for the PCR reaction, such as polymerase, polynucleotides, and restriction enzymes. In the case of the SNaP shot method, the kit may contain a primer and fluorescently labeled ddNTP, which are used to perform a single-base extension reaction and are then analyzed by capillary electrophoresis. In the case of comprehensive SNP analysis methods using probes, the kit may contain a fluorescently labeled probe, and may also contain a reagent for labeling the genomic DNA of the subject.

[0083] The prediction kit of the present invention may include a BCG therapy resistance prediction kit and / or a recurrence probability prediction kit, and if it includes both a BCG therapy resistance prediction kit and a recurrence probability prediction kit, it is preferable because one kit can simultaneously predict both BCG therapy resistance and recurrence probability, thereby reducing the burden on patients.

[0084] [Prediction Array] The array for predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer of the present invention comprises probes for detecting the presence or absence of two or more single nucleotide polymorphisms, wherein predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer includes predicting resistance to intravesical BCG therapy in the treatment of bladder cancer and / or predicting the possibility of recurrence after intravesical BCG therapy in the treatment of bladder cancer. (BCG therapy resistance prediction array) The array for predicting resistance to intravesical BCG therapy in bladder cancer treatment according to this embodiment (sometimes referred to herein simply as the "BCG therapy resistance prediction array") comprises probes for detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442, and rs13193250.

[0085] The BCG therapy resistance prediction array according to this embodiment is an array primarily used in a DNA chip method (microarray method), and may be a microarray. The array or microarray may be a device in which probes for detecting the presence or absence of two or more types of single nucleotide polymorphisms to be detected, such as DNA probes, are aligned and preferably immobilized on a substrate such as a glass slide by spots or the like. The DNA probe may be any probe that hybridizes with the single nucleotide polymorphism to be detected. The array comprises two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, six or more, seventeen or more, seventeen or more, eighteen or more, or all nineteen types of single nucleotide polymorphisms selected from the group consisting of the 19 biomarkers of this embodiment.Preferred combinations of single nucleotide polymorphisms include two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, and rs118137814; The number of types is 13 or more, 14 or more, 15 or more, or all 16 types, and the number of types is two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 11 or more, 12 or more, 13 or more, or all 14 types selected from the group consisting of rs73520681, rs61094339, rs4250, rs11894207, rs3738088, rs161448, rs2764326, rs3787194, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, and rs118137814.

[0086] Particularly preferred combinations of single nucleotide polymorphisms include a combination of single nucleotide polymorphisms comprising rs73520681 and rs61094339, a combination of single nucleotide polymorphisms comprising rs4250, rs11894207, rs73520681 and rs61094339, and a combination of single nucleotide polymorphisms comprising rs73520681, rs61094339, rs4250, rs11894207, rs73520681 and rs61094339.

[0087] The single nucleotide polymorphisms to be detected are two or more single nucleotide polymorphisms selected from the 19 biomarkers according to this embodiment. As described above, the 19 biomarkers according to this embodiment are single nucleotide polymorphisms that are significantly detected in a patient group resistant to intravesical BCG therapy compared to a patient group not resistant to intravesical BCG therapy. Because these single nucleotide polymorphisms are highly correlated with resistance to intravesical BCG therapy, resistance to intravesical BCG therapy in bladder cancer treatment can be predicted by using the prediction array according to this embodiment.

[0088] When using the BCG therapy resistance prediction array according to this embodiment, genomic DNA or chromosomal DNA is extracted and purified from a sample derived from a patient, fluorescently labeled for detection, and then attached to the array. The presence or absence of hybridization between the DNA probes provided on the array and the patient's DNA is detected using a fluorescent substance.

[0089] (Recurrence probability prediction array) The array for predicting the likelihood of recurrence after intravesical BCG instillation therapy in bladder cancer treatment according to this embodiment (sometimes simply referred to herein as the "recurrence likelihood prediction array") comprises probes for detecting the presence or absence of two or more single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903.

[0090] The recurrence probability prediction array according to this embodiment is an array primarily used in a DNA chip method (microarray method) and may be a microarray. The array or microarray may be a device in which probes for detecting the presence or absence of two or more single nucleotide polymorphisms to be detected, such as DNA probes, are aligned and preferably immobilized on a substrate such as a glass slide by spotting or the like. The DNA probes may be any probes that hybridize with the single nucleotide polymorphisms to be detected. The array includes two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve probes for detecting the presence or absence of two or more, preferably three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve single nucleotide polymorphisms selected from the group consisting of the 12 biomarkers according to this embodiment. A preferred combination of single nucleotide polymorphisms is two or more, three or more, or all of the single nucleotide polymorphisms selected from the group consisting of rs363765, rs6986852, rs73664140, and rs17637903.

[0091] Particularly preferred combinations of single nucleotide polymorphisms include those containing rs363765 and rs6986852.

[0092] The single nucleotide polymorphisms to be detected are two or more single nucleotide polymorphisms selected from the 12 biomarkers according to this embodiment. As described above, the 12 biomarkers according to this embodiment are single nucleotide polymorphisms that are significantly detected in patients who have relapsed after intravesical BCG therapy compared to patients who have not relapsed after intravesical BCG therapy. Because these single nucleotide polymorphisms have a high correlation with intravesical recurrence after intravesical BCG therapy, the use of an array for predicting the likelihood of recurrence according to this embodiment can predict the likelihood of recurrence after intravesical BCG therapy in bladder cancer treatment.

[0093] When using the recurrence probability prediction array according to this embodiment, genomic DNA or chromosomal DNA is extracted and purified from a sample derived from a patient, fluorescently labeled for detection, and then attached to the array. The presence or absence of hybridization between the DNA probes provided on the array and the patient's DNA is detected using a fluorescent substance.

[0094] The predictive array of the present invention may include a predictive array for resistance to BCG therapy and / or a predictive array for the likelihood of recurrence, and if it includes both a predictive array for resistance to BCG therapy and a predictive array for the likelihood of recurrence, this is preferred because it allows a single array to simultaneously predict both resistance to BCG therapy and the likelihood of recurrence, thereby reducing the burden on patients. [Example]

[0095] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0096] <Test Example 1: Genome-wide SNP analysis of BCG therapy resistance> Genomic DNA was extracted from blood samples of 44 patients with non-muscle-invasive bladder cancer who had received intravesical BCG therapy (exploratory cohort, Table 1). SNP analysis of the extracted genomic DNA was performed using the Japonica Array Version 2 (Toshiba Corporation), which consists of 659,253 SNPs. SNP analysis was performed according to the protocol described in the instruction manual (Thermo Fisher Scientific). The Japonica Array Version 2 is an array equipped with the Axiom platform (Thermo Fisher Scientific), developed by the Tohoku Medical Megabank Organization, Tohoku University, National University Corporation, for the analysis of Japanese genomic information.

[0097] In Table 1 and Table 3 below, the grade indicates the histological malignancy of bladder cancer, with higher numbers indicating higher malignancy. The T category indicates the extent of bladder cancer. Ta, T1, and Tis indicate non-invasive papillary carcinoma, carcinoma in which the tumor has invaded the subepithelial connective tissue, and carcinoma in situ, respectively. Resistance to intravesical BCG therapy refers to the presence of any of the following: residual bladder cancer 6 months after intravesical BCG therapy, muscle invasiveness after intravesical BCG therapy, progression to metastatic bladder cancer after intravesical BCG therapy, or radical cystectomy for the purpose of controlling the primary disease after intravesical BCG therapy. On the other hand, non-resistant to intravesical BCG therapy means that there was no residual bladder cancer 6 months after intravesical BCG therapy, no muscle invasion after intravesical BCG therapy, no progression to metastatic bladder cancer after intravesical BCG therapy, and no radical cystectomy for the purpose of controlling the primary disease after intravesical BCG therapy.

[0098] [Table 1]

[0099] Genotyping was performed using Thermo Fisher Scientific Genotyping Console software version 4.2, which generated PED-format files containing individual genotype information, which were used for statistical analysis.

[0100] After SNP analysis, a genome-wide association study (GWAS) was performed to examine the correlation between BCG therapy resistance and SNPs. GWAS was performed using PLINK software version 1.07 and evaluated by chi-square test (p value < 1.0 × 10 -5 ) During the calculation process, markers that deviated from the Hardy-Weinberg rule were excluded. The results of the GWAS are shown in a Manhattan plot (Figure 1). The GWAS identified 19 SNPs that were significantly detected in the BCG intravesical therapy resistant group compared to the BCG non-resistant group. The results are shown in Table 2.

[0101] [Table 2]

[0102] <Test Example 2: Targeted SNP Analysis> Genomic DNA was extracted from blood samples of 47 patients with non-muscle-invasive bladder cancer who had received intravesical BCG therapy (validation study cohort, Table 3). SNP analysis was performed on the extracted genomic DNA for the 14 SNPs listed in Table 2 (only the "adopted" SNPs out of 19 SNPs). SNP analysis was performed using pre-designed TaqMan SNP Genotyping Assays (Life Technologies) and TaqMan Gene Expression Master Mix (Life Technologies) according to the protocol described in the instruction manual.

[0103] [Table 3]

[0104] After SNP analysis, odds ratios (OR) were estimated using logistic regression to examine the correlation between BCG therapy resistance and SNPs. The results are shown in Table 4. Hazard ratios (HR) were estimated using a Cox proportional hazards model. The results are shown in Table 5. From the results in Tables 4 and 5, the SNPs that were associated with BCG resistance in both the exploratory and validation cohorts were rs4250, rs11894207, rs73520681, and rs61094339.

[0105] [Table 4] [Table 5]

[0106] <Test Example 3: Prediction of BCG therapy resistance using an SNP panel> Using four SNPs with odds ratios of 2 or more in the validation test cohort of Test Example 2, a two-gene model (two-gene SNP panel) combining rs73520681 and rs61094339, and a four-gene model (four-gene SNP panel) combining rs4250, rs11894207, rs73520681, and rs61094339 were constructed.

[0107] Using both models, odds ratios and hazard ratios were calculated for the exploratory cohort and the validation cohort. The results are shown in Tables 6 and 7. The survival rate of the validation cohort was also measured. The results are shown in Figure 2. Figures 2(A) and (B) show the survival rate of the two-gene model and the four-gene model, respectively.

[0108] [Table 6] [Table 7]

[0109] The results in Figure 2 show the time it took for resistance to intravesical BCG therapy to develop in the validation cohort. Approximately 40% of high-risk patients (with one or two risk alleles) in the two-gene model showed resistance to intravesical BCG therapy two years after intravesical BCG therapy. These patients can be considered to have moderate resistance to intravesical BCG therapy. Furthermore, approximately 70% of high-risk patients (with two to four risk alleles) in the four-gene model showed resistance to intravesical BCG therapy two years after intravesical BCG therapy. These patients can be considered to have severe resistance to intravesical BCG therapy.

[0110] <Test Example 4: Genome-wide SNP analysis for recurrence probability> As in Test Example 1, genomic DNA was extracted from blood samples of 44 patients with non-muscle-invasive bladder cancer who had received intravesical BCG therapy (exploratory cohort, Table 8). SNP analysis of the extracted genomic DNA was performed using the Japonica Array Version 2 (Toshiba Corporation), which consists of 659,253 SNPs. SNP analysis was performed according to the protocol described in the instruction manual (Thermo Fisher Scientific). The Japonica Array Version 2 is an array equipped with the Axiom platform (Thermo Fisher Scientific), which was developed by the Tohoku Medical Megabank Organization, Tohoku University, a national university corporation, for the analysis of Japanese genomic information.

[0111] In Table 8 and Table 10 below, the grade and T category have the same meanings as in Table 1. Recurrence after intravesical BCG therapy means that cancer was histologically confirmed in the bladder after treatment. On the other hand, no recurrence after intravesical BCG therapy means that cancer was not histologically confirmed in the bladder after treatment.

[0112] [Table 8]

[0113] Genotyping was performed using Thermo Fisher Scientific Genotyping Console software version 4.2, which generated PED-format files containing individual genotype information, which were used for statistical analysis.

[0114] After SNP analysis, a genome-wide association study (GWAS) was performed to examine the correlation between recurrence and SNPs. GWAS was performed using PLINK software version 1.07 and evaluated by chi-square test (p value < 1.0 × 10 -5) During the calculation process, markers that deviated from the Hardy-Weinberg rule were excluded. The results of the GWAS were shown in a Manhattan plot (Figure 3). The GWAS identified 12 SNPs that were significantly detected in the group of patients with recurrence after intravesical BCG therapy compared to the group of patients without recurrence after intravesical BCG therapy. The results are shown in Table 9.

[0115] [Table 9]

[0116] <Test Example 5: Targeted SNP Analysis> Genomic DNA was extracted from blood samples of 47 patients with non-muscle invasive bladder cancer (validation test cohort, Table 10) who underwent intravesical BCG therapy as in Test Example 2. The extracted genomic DNA was subjected to SNP analysis for the 12 types of SNPs shown in Table 9. SNP analysis was performed using pre-designed TaqMan SNP Genotyping Assays (Life Technologies, Inc.) and TaqMan Gene Expression Master Mix (Life Technologies, Inc.) according to the protocol described in the instruction manual.

[0117] [Table 10]

[0118] After SNP analysis, hazard ratios were estimated using a Cox proportional hazards model to examine the correlation between recurrence and SNPs. The results are shown in Table 11. In the exploratory cohort, the 12 SNPs shown in Table 11 were associated with recurrence. In both the validation cohort and exploratory cohort, the SNPs associated with recurrence were rs363765 and rs6986852.

[0119] [Table 11]

[0120] <Test Example 6: Prediction of recurrence probability using SNP panel> Based on the results of Test Example 5, a two-gene model (two-gene SNP panel) combining rs363765 and rs6986852 was constructed.

[0121] Using the two-gene model, hazard ratios were calculated for the exploratory cohort and the validation cohort. The results are shown in Table 12. The survival rate for the validation cohort was also measured. The results are shown in Figure 4. Figures 4(A) and (B) show the recurrence time for the validation cohort and the exploratory cohort, respectively.

[0122] [Table 12]

[0123] Figure 4 shows the time to recurrence after intravesical BCG therapy in the validation cohort (A) and exploratory cohort (B). It was found that patients in the validation cohort, who were in the high-risk group, had a higher risk of recurrence after intravesical BCG therapy. On the other hand, it was also found that patients in the exploratory cohort, who were in the high-risk group, had a higher risk of recurrence after intravesical BCG therapy. These findings demonstrate that the two-gene model combining rs363765 and rs6986852 can effectively predict recurrence after BCG therapy.

Claims

1. A kit for predicting a therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, the kit comprising a reagent for detecting the presence or absence of two or more single nucleotide polymorphisms, wherein predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer comprises predicting resistance to intravesical BCG therapy in the treatment of bladder cancer, and predicting the possibility of recurrence after intravesical BCG therapy in the treatment of bladder cancer, The two or more single nucleotide polymorphisms are Two or more single nucleotide polymorphisms selected from the group consisting of rs73520681, rs61094339, rs4250 and rs11894207, and A kit comprising rs363765, and rs6986852.

2. The two or more single nucleotide polymorphisms further include one or more single nucleotide polymorphisms selected from the group consisting of rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442, and rs13193250; and one or more single nucleotide polymorphisms selected from the group consisting of rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903 The kit of claim 1 , comprising:

3. An array for predicting a therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer, the array comprising probes for detecting the presence or absence of two or more single nucleotide polymorphisms, wherein predicting the therapeutic effect of intravesical BCG therapy in the treatment of bladder cancer includes predicting resistance to intravesical BCG therapy in the treatment of bladder cancer, and predicting the possibility of recurrence after intravesical BCG therapy in the treatment of bladder cancer; The two or more single nucleotide polymorphisms are Two or more single nucleotide polymorphisms selected from the group consisting of rs73520681, rs61094339, rs4250 and rs11894207, and rs363765, and rs6986852.

4. The two or more single nucleotide polymorphisms further include one or more single nucleotide polymorphisms selected from the group consisting of rs3738088, rs161448, rs2764326, rs2814707, rs3787194, rs58081719, rs3095966, rs16877113, rs16887173, rs10269584, rs11772249, rs118137814, rs1607282, rs7825442, and rs13193250; and one or more single nucleotide polymorphisms selected from the group consisting of rs9374832, rs35176001, rs2127120, rs4277759, rs73664140, rs1607282, rs12141654, rs4541358, rs12373386, and rs17637903 4. The array of claim 3, comprising:

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