JP2015021800A - Diagnostic drug and kit for use in prediction of recurrence risk of renal cancer and prediction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a prediction diagnostic drug and kit for a recurrence risk of a renal cancer and a prediction method therefor that are high in availability as means for predicting recurrence of the renal cancer, using a factor specific to the recurrence of the renal cancer and capable of a prediction result of the recurrence risk of the renal cancer to a medial treatment.SOLUTION: A diagnostic drug for predicting recurrence risk of a renal cancer includes a protein and/or polypeptide capable of specifically recognizing a translation product of any one gene selected from a group composed of AMAP1, GEP100 and EPB41L5, and a kit for predicting the recurrence risk of a renal cancer includes the diagnostic drug. A method for predicting the recurrence risk of the renal cancer is configured to: (A) determine an expression level of a translation product of at least one gene selected from the group composed of the AMAP1, the GEP100 and the EPB41L5 in a biological specimen collected from a patient having undergone a medical treatment of the renal cancer; and (B) predict a recurrence risk of the renal cancer of the patient on the basis of the expression level of the translation product determined in the step of (A).

Description

  The present invention relates to a diagnostic agent and kit for use in predicting the recurrence risk of renal cancer, and a method for predicting the recurrence risk of renal cancer.

  The main treatments for renal cancer confined to the kidney (without metastasis) include renal-conserving surgery (partial nephrectomy) and radical nephrectomy. However, even in cases where radical nephrectomy has been performed, metastasis is observed during follow-up in about 30% of cases. In metastatic renal cell carcinoma, anti-cancer chemotherapy and radiotherapy are considered to be ineffective, and although immunotherapy such as interferon or interleukin 2 has been performed, the response rate is as low as about 10%. . In recent years, molecular targeted therapy has been introduced with vascular endothelial growth factor: VEGF and its receptors targeting sorafenib, sunitinib, axitinib and mammalian target of rapamycin (mTOR) inhibitors everolimus and temsirolimus. However, it is known that it will eventually become resistant to treatment. After a definitive diagnosis of renal cancer, the presence or absence of distant metastases, stage, and the size of the tumor are examined, and based on the results, an appropriate one of these treatment methods is selected and implemented. After treatment of renal cancer based on these treatments, follow-up of the prognosis by image examination such as CT examination is indispensable in order to cope with recurrence and metastasis. Later, prognostic prediction including recurrence of kidney cancer and metastasis is important.

  As factors for predicting the recurrence of renal cancer, factors relating to inflammatory reactions such as erythrocyte, CRP (C-reactive protein) and IAP (Immunosuppressive Acidic Protein) have attracted attention. It has been shown that erythema and CRP values have a significant correlation with the renal cancer-specific survival rate (Non-Patent Documents 1 to 7), and these factors are recommended as subjects for pre-treatment testing. On the other hand, for IAP, although usefulness as a predictive factor for recurrence of renal cancer has been suggested from a study based on clinical cases (Non-patent Documents 8 to 11), the number of cases used in the survey is not necessarily sufficient. It cannot be said that its usefulness as a predictive factor for renal cancer recurrence is unknown.

  Furthermore, in recent years, it has been suggested that blood pyruvate kinase M2 and thymidine kinase 1 may be predictive of recurrence of renal cell carcinoma after nephrectomy as predictors of recurrence of renal cancer (Non-patent literature). 12).

Sengupta S, Lohse CM, Cheville JC, Leibovich BC, Thompson RH, Webster WS, Frank I, Zincke H, Blute ML, Kwon ED.The preoperative erythrocyte sedimentation rate is an independent prognostic factor in renal cell carcinoma.Cancer. 2006; 106 (2): 304-12. Ljungberg B, Grankvist K, Rasmuson T. Serum acute phase reactants and prognosis in renal cell carcinoma. Cancer. 1995; 76 (8): 1435-9. Lamb GW, McMillan DC, Ramsey S, Aitchison M. The relationship between the preoperative systemic inflammatory response and cancer-specific survival in patients undergoing potentially curative resection for renal clear cell cancer.Br J Cancer. 2006; 94 (6): 781- Four. Masuda H, Kurita Y, Fukuta K, Mugiya S, Suzuki K, Fujita K. Significant prognostic factors for 5-year survival after curative resection of renal cell carcinoma.Int J Urol. 1998; 5 (5): 418-22. Fujikawa K, Matsui Y, Oka H, Fukuzawa S, Takeuchi H. Serum C-reactive protein level and the impact of cytoreductive surgery in patients with metastatic renal cell carcinoma.J Urol. 1999; 162 (6): 1934-7. Atzpodien J, Royston P, Wandert T, Reitz M; DGCIN-German Cooperative Renal Carcinoma Chemo-Immunotherapy Trials Group.Metastatic renal carcinoma comprehensive prognostic system.Br J Cancer. 2003; 88 (3): 348-53. Michela de Martino, Tobias Klatte, Christoph Seemann, Matthias Waldert, Andrea Haitel, Georg Schatzl, Mesut Remzi † and Peter Weibl. Validation of serum C-reactive protein (CRP) as an independent prognostic factor for disease-free survival in patients with localized renal cell carcinoma (RCC). BJU International Early View, Article first published online: 15 MAR 2013 Masuda H, Kurita Y, Suzuki K, Fujita K, Aso Y. Predictive value of serum immunosuppressive acidic protein for staging renal cell carcinoma: comparison with other tumour markers. Br J Urol. 1997; 80 (1): 25-9. Igarashi T, Tobe T, Kuramochi H, Akakura K, Ichikawa T, Hamano S, Suzuki N, Furuya Y, Ito H. Serum immunosuppressive acidic protein as a potent prognostic factor for patients with metastatic renal cell carcinoma.Jpn J Clin Oncol. 2001 ; 31 (1): 13-7. Matsumoto K, Iwamura M, Muramoto M, Suyama K, Tabata K, Minei S, Hirai S, Baba S. [Prognostic value of serum immunosuppressive acidic protein in renal cell carcinoma] Nippon Hinyokika Gakkai Zasshi. 2002; 93 (4): 548 -54. Kawata N, Yamaguchi K, Hirakata H, Hachiya T, Yoshida T, Takimoto Y. Immunosuppressive acidic protein detects high nuclear grade localized renal cell carcinoma. Urology. 2005; 66 (4): 736-40. Benjamin Nisman, Vladimir Yutkin, Hovav Nechushtan, Ofer N. Gofrit, Tamar Peretz, Simon Gronowitz, and Dov PodeOBJECTIVES Pyruvate kinase type M2 Circulating Tumor M2 Pyruvate Kinase and Thymidine Kinase 1 Are Potential Predictors for Disease Recurrence in Renal Cell Carcinoma After Nrectomy 2010 Aug; 76 (2): 513.e1-6.

  As noted above, erythema and CRP levels have been shown to have a significant correlation with renal cancer-specific survival, but these factors remain recommended for pretreatment testing and are It is a general marker indicating the degree, and its usefulness as a specific factor that can predict the recurrence of renal cancer is not critical. Regarding IAP, as described above, the usefulness of IAP as a predictive factor for renal cancer recurrence remains unclear.

  Furthermore, although the usefulness of blood pyruvate kinase M2 and thymidine kinase 1 as predictors of recurrence of renal cell carcinoma after nephrectomy has been suggested, they are also used as predictors of recurrence of renal cancer in actual clinical settings. Applicable technology has not been reached.

  In addition, the biomarker (CRP) does not clearly show the molecular mechanism of how the protein is related to cancer malignancy or recurrence. Therefore, there is a lack of knowledge on how to treat these biomarker positive patients, and even if the biomarker is found to be positive, both patients and doctors are confused.

  Therefore, an object of the present invention is to provide a diagnostic agent and kit for predicting a recurrence risk of a renal cancer, a method useful for predicting the recurrence of the renal cancer, and a prediction method that are highly useful as a means for predicting the recurrence of the renal cancer using a factor specific to the recurrence of the renal cancer. It is to provide. A further object of the present invention is to provide a predictive diagnostic agent and kit, and a prediction method using a factor that can link the prediction result of the recurrence risk of renal cancer to treatment.

  As a result of intensive studies to solve the above problems, the present inventors have found that the expression of at least one gene in a specific group of genes belonging to the Arf6 pathway is significantly correlated with the recurrence of renal cancer. The present invention has been completed based on such knowledge.

That is, the present invention relates to the following (1) to (3).
(1) A diagnostic agent for use in predicting the recurrence risk of renal cancer,
A diagnostic agent comprising a protein and / or polypeptide capable of specifically recognizing a translation product of any one gene selected from the group consisting of AMAP1, GEP100, and EPB41L5.
(2) A kit for use in predicting recurrence risk of renal cancer, comprising the diagnostic agent of the present invention.
(3) (A) determining a translation product expression level of at least one gene selected from the group consisting of AMAP1, GEP100, and EPB41L5 in a biological sample collected from a patient treated for renal cancer; B) A method for predicting the recurrence risk of renal cancer, comprising predicting the recurrence risk of renal cancer of the patient based on the translation product expression level determined in step (A).

  According to the present invention, there are provided a diagnostic agent and kit for predicting a recurrence risk of a renal cancer and a prediction method, which are highly useful as a means for predicting the recurrence of the renal cancer using a factor specific to the recurrence of the renal cancer. be able to. The biomarker that is a factor used in the present invention is composed of a group of proteins that form a signal pathway involved in cancer invasion and metastasis and treatment resistance. The present inventor has also found that inhibition of this signal pathway significantly reduces cancer invasion and metastasis. Therefore, it is also possible to provide a therapeutic policy for a patient who is predicted to have a recurrence of renal cancer using the predictive diagnostic agent and kit and the prediction method of the present invention.

The micrograph of the immunohistochemical dyeing | staining acquired in the Example (using anti-EPB41L5 antibody as an example) is shown. The result of investigating the correlation with the translation product expression of each gene, the period (FIGS. 2A-2D) from the completion of treatment of renal cancer to recurrence, and the survival period (FIG. 2E) is shown. This shows the cds region base sequence (NCBI Reference Sequence: NM_020909.3) of EPB41L5. This shows the amino acid sequence of EPB41L5 (NCBI Reference Sequence: NP_065960.2). The primary structure comparison of EPB41L5 and EPB41L4b is shown. EPB41L4b shows a comparison of isoform1 and isoform2 genomes. A comparison of the amino acids of EPB41L4b isoform1 and isoform2 is shown. Alignment analysis is shown for EPB41L5 (NP_065960.2) and EPB41L4b (NP_061987.3061987.3). This shows the base sequence of cds region of AMAP1 (NCBI Reference Sequence: NM_018482.3). The amino acid sequence of AMAP1 (NCBI Reference Sequence: NP_060952.2) is shown. Information on genes (ArfGAP family) homologous to AMAP1. 2 shows the cds region base sequence (NCBI Reference Sequence: NM_003887.2) of the AMAP2 gene having homology with AMAP1. Amino acid sequence (NCBI Reference Sequence: NP_003878.1) of AMAP2 having homology with AMAP1 is shown. Data of alignment analysis of AMAP2 having homology with AMAP1 is shown. This shows the nucleotide sequence (NCBI Reference Sequence: NM_014869.3) of cds region of GEP100. This shows the amino acid sequence of GEP100 (NCBI Reference Sequence: NP_055684.3). A protein group similar to GEP100 in the human genome is shown. A gene group having homology with GEP100 is shown. This shows the cds region base sequence of the BRAG1 gene having homology with GEP100 (NCBI Reference Sequence: NM_015075.1). The amino acid sequence of BRAG1 having homology with GEP100 (NCBI Reference Sequence: NP_055890.1) is shown. The data of the alignment analysis of BRAG1 which has homology with GEP100 are shown. This shows the cds region base sequence of the BRAG3 gene having homology with GEP100 (NCBI Reference Sequence: NM_015232.1). The amino acid sequence of BRAG3 (NCBI Reference Sequence: NP_056047.1) having homology with GEP100 is shown. The data of alignment analysis of BRAG3 which has homology with GEP100 is shown. The antigenic site of a polyclonal antibody against EPB41L5 (a peptide containing amino acids 541 to 733 (193 amino acids)) is illustrated. A scheme of a series of procedures for immunizing a rabbit with an antigen, collecting antiserum, and purifying the antibody is shown. The result of affinity purification of EPB41L5 antibody is shown. The result of affinity purification of EPB41L5 antibody is shown. Information on the antigen site of the antibody against AMAP1, cloning procedure of the antigen site, preparation of the antigen, immunization to rabbit, collection of antiserum, and purification of the antibody are shown together. Information on the antigen site of the antibody against GEP100, cloning procedure of the antigen site, preparation of the antigen, immunization to rabbit, collection of antiserum, and purification of the antibody are shown together.

Diagnostic Agent According to one aspect of the present invention, a diagnostic agent for use in predicting the recurrence risk of renal cancer,
Provided is a diagnostic agent containing a protein and / or polypeptide capable of specifically recognizing the translation product of any one gene selected from the group consisting of AMAP1, GEP100, and EPB41L5.

  In the present invention, “renal cancer” refers to renal cell carcinoma that occupies the largest number of primary malignant neoplasms of the kidney. Most of renal cell carcinoma is clear cell renal cell carcinoma.

  In the present invention, “risk of recurrence” means the risk that a patient who has been treated for renal cancer will relapse cancer after the treatment is completed. Cancer that recurs includes one or both of renal cancer and metastatic renal cell carcinoma. The risk of recurrence mainly means the risk of early recurrence within 5 years after receiving treatment for renal cancer. However, the risk of recurrence may include a medium-term recurrence risk of more than 5 years after receiving treatment for renal cancer and within 10 years, or a late recurrence risk of more than 10 years. Furthermore, the method for treating renal cancer is not particularly limited. For example, chemotherapy including administration of anticancer drugs, administration of interferon and / or interleukin 2 and molecular targeted drugs recently introduced to metastatic renal cancer, or partial excision of the kidney including the lesion to preserve the kidney Such as partial nephrectomy.

The “prediction risk prediction” method will be described later in “Prediction Method”.
The diagnostic agent of the present invention can be used to predict the risk of recurrence of renal cancer. This can be understood from the following results in a group of patients who have been treated for renal cancer and who later relapsed. Patients with high expression of a translation product of a specific gene in renal tissue are relatively The recurrence of renal cancer at an early stage (eg, within 5 years) is shown with a statistically significant difference. This point is concretely shown in the Example mentioned later. Specifically, the specific gene is at least one gene selected from the group consisting of AMAP1, GEP100, and EPB41L5. Patients with high expression of translation products of any one of these genes have a high risk of recurrence with statistical significance. Furthermore, patients with high expression of any two translation products of the above genes have a higher risk of recurrence with statistical significance, and patients with high expression of the three translation products of the above genes The risk of recurrence with the statistically significant difference is the highest. That is, when high expression of the translation product of the above gene is observed in renal cancer tissue, the patient is predicted to be at high risk of recurrence of cancer early after treatment of renal cancer. Details will be described in a method for predicting the recurrence risk of kidney cancer, which will be described later. Thus, in the present invention, “prediction of recurrence risk of renal cancer” is based on the premise of detecting high expression of the translation product of the specific gene in renal tissue. Therefore, the diagnostic agent of the present invention for use in the prediction is specifically a reagent for use in detecting these gene translation products, and any one selected from the group consisting of AMAP1, GEP100, and EPB41L5 It contains a protein and / or polypeptide that can specifically recognize the translation product of one gene.

<Gene EPB41L5>
The gene EPB41L5 (abbreviation EBL5) (GeneID: 57669) is a binding partner of AMAP1 in cancer invasion and is essential for invasion pseudopod formation.

  2 EPB41L4b is most homologous to EPB41L5 in the EPB41L5 family. EPB41L4b has band 4.1-like protein 4B isoform 1 [Homo sapiens] (NP_060894.2) (SEQ ID NO: 3) and band 4.1-like protein 4B isoform 2 [Homo sapiens] (NP_061987.3) (SEQ ID NO: 4). is there. isoform1 is a short form. FIG. 5 shows a primary structure comparison of EPB41L5 and EPB41L4b. FIG. 6 shows a comparison of EPB41L4b isoform1 and isoform2 genomes. FIG. 7 shows a comparison between the amino acid sequence of EPB41L4b isoform1 and the amino acid sequence of isoform2. FIG. 8 shows alignment analysis for EPB41L5 (NP_065960.2) (SEQ ID NO: 2) and EPB41L4b (NP_061987.3) (SEQ ID NO: 4).

  As shown in FIG. 8, as a result of alignment analysis for EPB41L5 (NP_065960.2) (SEQ ID NO: 2) and EPB41L4b (NP_061987.3) (SEQ ID NO: 4), on the C-terminal side (sequence after 541th on the basis of EPB41L5) Both sequences are greatly different, and the C-terminal sequence of EPB41L5 is an effective antigen for producing a specific antibody against the protein. In the present invention, the specific antibody against EPB41L5 protein or a fragment thereof is an antibody or fragment thereof having an amino acid sequence that serves as an epitope in the amino acid sequence region from the 541st to 733th positions of EPB41L5 (SEQ ID NO: 2). Is appropriate.

<Gene AMAP1>
The gene AMAP1 (GeneID: 50807) is a downstream effector of Arf6 in cancer invasion. FIG. 9 shows the nucleotide sequence (NCBI Reference Sequence: NM — 018482.3) (SEQ ID NO: 5) of cds region of AMAP1. FIG. 10 shows the amino acid sequence of AMAP1 (NCBI Reference Sequence: NP — 060952.2) (SEQ ID NO: 6). FIG. 11 shows information on genes (ArfGAP family) having homology with AMAP1. FIG. 12 shows the cds region base sequence (NCBI Reference Sequence: NM — 003887.2) (SEQ ID NO: 7) of the AMAP2 gene having homology with AMAP1. FIG. 13 shows the amino acid sequence (NCBI Reference Sequence: NP_003878.1) (SEQ ID NO: 8) of AMAP2 having homology with AMAP1. FIG. 14 shows alignment analysis data of AMAP2 having homology with AMAP1.

  As shown in FIG. 14, as a result of alignment analysis for AMAP1 (NCBI Reference Sequence: NP_060952.2) (SEQ ID NO: 6) and AMAP2 (NCBI Reference Sequence: NP_003878.1) (SEQ ID NO: 8), the two sequences differ greatly. There is an area. This region of AMAP1 becomes an effective antigen for producing a specific antibody against the protein. In the present invention, the specific antibody against AMAP1 protein or a fragment thereof is an antibody or fragment thereof having an amino acid sequence serving as an epitope in the amino acid sequence region from the 935th to the 1002nd vicinity of AMAP1 (SEQ ID NO: 6). Is appropriate.

<Gene GEP100>
Gene GEP100 (GeneID: 9922) is an Arf6 activator in cancer invasion.
FIG. 15 shows the base sequence of the GEP100 cds region (NCBI Reference Sequence: NM — 014869.3) (SEQ ID NO: 9). FIG. 16 shows the amino acid sequence of GEP100 (NCBI Reference Sequence: NP — 055684.3) (SEQ ID NO: 10). FIG. 17 shows a protein group having homology with GEP100 in the human genome. FIG. 18 shows a gene group having homology with GEP100. It can be seen that GEP100 is highly homologous to the BRAG family. FIG. 19 shows the nucleotide sequence (NCBI Reference Sequence: NM — 015075.1) (SEQ ID NO: 11) of the BRAG1 gene having high homology with GEP100. FIG. 20 shows the amino acid sequence of BRAG1 (NCBI Reference Sequence: NP — 055890.1) (SEQ ID NO: 12) having high homology with GEP100. FIG. 21 shows alignment analysis data of GEP100 and BRAG1. FIG. 22 shows the nucleotide sequence (NCBI Reference Sequence: NM — 015232.1) (SEQ ID NO: 13) of the BRAG3 gene having high homology with GEP100. FIG. 23 shows the amino acid sequence of BRAG3 (NCBI Reference Sequence: NP — 056047.1) (SEQ ID NO: 14) having high homology with GEP100. FIG. 24 shows alignment analysis data of GEP100 and BRAG3.

  As a result of alignment analysis for GEP100 (NCBI Reference Sequence: NP_055684.3) (SEQ ID NO: 10) and BRAG1 (NCBI Reference Sequence: NP_055890.1) (SEQ ID NO: 12) shown in FIG. 21, GEP100 (NCBI Reference Sequence shown in FIG. 24) Sequence: NP_055684.3) (SEQ ID NO: 10) and BRAG3 amino acid sequence (NCBI Reference Sequence: NP_056047.1) (SEQ ID NO: 14). Therefore, it becomes an effective antigen for producing specific antibodies against proteins in these sequence regions. In the present invention, the specific antibody against GEP100 protein or a fragment thereof is an antibody or fragment thereof having an amino acid sequence that serves as an epitope in the amino acid sequence region from the 139th to 248th of GEP100 (SEQ ID NO: 10). Is appropriate.

  The protein and / or polypeptide capable of specifically recognizing the translation product of the gene is not particularly limited as long as it can specifically bind to the translation product of one of the genes. For example, an antibody against the translation product and / or a fragment thereof can be mentioned. Examples of antibodies and / or fragments thereof include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, humanized antibodies, and the like. As a method for preparing proteins and polypeptides capable of specifically recognizing the translation product of the gene, any method can be adopted as long as specific binding to the gene translation product can be maintained. For example, a desired polyclonal antibody, monoclonal antibody, single chain antibody, humanized antibody and the like can be prepared by a known method using the protein and / or fragment thereof as the gene translation product as an antigen.

  The specific binding property to the gene translation product possessed by the protein and polypeptide that can specifically recognize the translation product of the above-mentioned gene is that the translation product of the gene can coexist with the translation product of the gene. It is sufficient if it can be distinguished from the translation product of the gene. Therefore, for example, even in the case of proteins and polypeptides that can specifically recognize a translation product of a gene in which a partial modification (substitution, deletion, addition, etc.) is added to the gene, As long as it has a specific binding property to a gene translation product that can be distinguished from the translation product of another gene that may coexist with the translation product of the gene, the “translation product of the gene is specifically recognized” of the present invention. Possible proteins and / or polypeptides ”.

  Furthermore, in the diagnostic agent of the present invention, the single diagnostic agent may contain only one protein or polypeptide that can specifically recognize the translation product of one of the above genes, Alternatively, two or more kinds of proteins and / or polypeptides capable of specifically recognizing each translation product of the gene may be included. In addition, the protein and / or polypeptide may be labeled with a labeling substance for detection of gene translation products. Examples of the labeling substance include fluorescent substances (for example, fluorescent proteins such as GFP, and fluorescent low molecular compounds such as fluorescein). However, it is not intended to be limited to those having labels with these labeling substances. When the protein and / or polypeptide is not labeled in the diagnostic agent of the present invention, the translation product of the gene can be detected by a known method. For such detection, for example, a system using a secondary antibody that binds to the protein and / or polypeptide (primary antibody) can be used. There are commercially available detection kits for systems using secondary antibodies. In this system, for example, the secondary antibody against the primary antibody is labeled with peroxidase, and the secondary antibody is further specifically bound to the primary antibody specifically bound to the target gene translation product in the sample. A gene translation product can be detected by adding a chromogenic substrate (eg, 3,3′-diaminobenzidine tetrahydrochloride) to the sample and observing the color development. Furthermore, in the present invention, when one diagnostic agent contains two or more proteins and / or polypeptides capable of specifically recognizing each of the translation products of the above genes, for example, two or more of them may be used. These proteins and / or polypeptides can be labeled with different fluorescent substances that emit fluorescence of different wavelengths. Thereby, each target gene translation product can be detected in the same sample.

  Diagnostic agents can optionally include buffers, preservatives, cryoprotectants, and the like. Examples of the buffer include tris hydrochloride, potassium phosphate, sodium phosphate and the like. Examples of the preservative include sodium azide. Examples of the antifreezing agent include glycerol.

  The diagnostic agent of the present invention can be used for detection of a translation product of a given gene in order to determine the expression level of the gene translation product in step (A) of the method for predicting the risk of recurrence of renal cancer, which will be described later.

Kit According to another aspect of the present invention, there is provided a kit for use in predicting the risk of recurrence of renal cancer, comprising the above-described diagnostic agent.

  The kit of the present invention provides components such as reagents and members that can be used in carrying out a method for predicting the recurrence risk of renal cancer, which will be described later, and includes the above-described diagnostic agent.

  In the kit of the present invention, the diagnostic agent of the present invention is (1) a protein or polypeptide 1 that can specifically recognize a translation product of one gene of AMAP1, GEP100, and EPB41L5 in a single diagnostic agent. By including only the species, the diagnostic product can detect the translation product of one gene. Alternatively, the diagnostic agent of the present invention comprises (2) two or more kinds of proteins and / or polypeptides each capable of specifically recognizing each of the translation products of these genes in a single diagnostic agent. The diagnostic agent may be a single diagnostic agent capable of detecting a plurality of the gene translation products. Furthermore, in the kit of the present invention, a diagnostic agent capable of detecting the translation product of one gene of (1) above and / or a single agent capable of detecting a plurality of gene translation products of (2) above. Diagnostic agents may be included.

  In addition to the above diagnostic agents, the kit of the present invention includes a blocking reagent, a labeled secondary antibody for use in detecting a protein and / or polypeptide (antibody) contained in the diagnostic agent, a coloring reagent, and recurrence of renal cancer. Instructions describing the experimental protocol regarding the method of predicting risk can optionally be included. Examples of the blocking reagent include bovine serum albumin (BSA).

Prediction Method According to another aspect of the present invention, there is provided a method for predicting the risk of recurrence of renal cancer, comprising the following steps (A) and (B).
(A) determining a translation product expression level of at least one gene selected from the group consisting of AMAP1, GEP100, and EPB41L5 in a biological sample collected from a patient treated for renal cancer;
(B) predicting the recurrence risk of renal cancer in the patient based on the translation product expression level determined in step (A),

  In the method of the present invention, “kidney cancer” and “treatment of kidney cancer” are as described above. In the method of the present invention, the patient is a patient who has been treated for renal cancer.

  In the method of the present invention, the “biological sample” is any biological sample collected from a patient who has been treated for renal cancer. The biological sample is preferably kidney tissue from the viewpoint that the risk of recurrence can be accurately predicted. Furthermore, as the renal tissue, for example, a tissue of a kidney lesion site removed from a patient at the time of tumor removal in the treatment of renal cancer, or a nearby kidney tissue where the lesion site was present, Later, kidney tissue collected separately from the excision can be mentioned. However, from the viewpoint of reducing the burden on the patient as much as possible and predicting the risk of recurrence with high accuracy, the renal tissue is preferably a tissue of a renal cancer lesion site removed from the patient at the time of tumor removal. In the present invention, since the above-mentioned predictive factor can be detected using a paraffin-embedded tissue prepared from a surgically excised specimen of a primary kidney and metastasis for pathological diagnosis, all cases in which pathological diagnosis has been performed You can search for.

  In step (A) of the method of the present invention, a translation product expression level of at least one gene selected from the group consisting of AMAP1, GEP100, and EPB41L5 is determined in the biological sample. These genes are genes belonging to the Arf6 pathway. As shown in the Examples, when the gene belonging to the Arf6 pathway is expressed at the tissue level in the renal tissue of a renal cancer patient and the operation of the Arf6 pathway is estimated, the patient is relatively early after treatment of renal cancer. High risk of recurrence. Therefore, when the high expression of at least one of the genes belonging to the Arf6 pathway is confirmed, the operation of the Arf pathway, which is considered to promote recurrence after the treatment of renal cancer, is estimated, and the patient is promptly It can be judged that the probability of recurrence of renal cancer is high.

  In the step (A) of the method of the present invention, as long as the translation product expression level of at least one gene selected from the group consisting of AMAP1, GEP100, and EPB41L5 is determined for the biological sample, Is not particularly limited. The determination of the translation product expression level of the at least one gene may be performed, for example, on the entire biological sample, or may be performed on a specific type of cell in the biological sample, or The cytoplasm and the cell nucleus may be separately performed in the cells present in the sample. When the determination of the translation product expression level of the at least one gene is carried out separately in the cytoplasm and the cell nucleus in the cells present in the biological sample, the result of the determined translation product expression level is the result for the cytoplasm and the cell nucleus. Two types of results are obtained. These two types of results can be used for the later-described step (B).

  The method for determining the translation product expression level of the predetermined gene in the biological sample used in step (A) of the method of the present invention is not particularly limited. Specifically, as described above for the diagnostic agent of the present invention, the expression level of the translation product of the gene is determined using a protein or polypeptide (diagnostic agent) that can specifically recognize the translation product of the gene to be detected. A method of determining is available. Specifically, it is preferable to determine the expression level by accurately grasping the expression intensity and expression distribution of the gene translation product in a tissue section that is a biological sample. In particular, determining the translation product expression level of the gene by immunohistochemistry using these proteins and / or polypeptides (diagnostics) can more accurately grasp the expression intensity and distribution of the gene translation product. It is preferable from the viewpoint. Various known methods can be used for the immunohistochemical method. In the present invention, these known methods may be used, or a newly developed method may be used. The translation product expression level determination method such as immunohistochemistry is not particularly limited as long as the expression level of the target gene translation product can be determined.

  Furthermore, in the present invention, in order to ensure specific detection of a gene translation product, the protein and / or polypeptide is preferably an antibody against a gene translation product to be detected and / or a fragment thereof. . This point is as described in the description of the diagnostic agent of the present invention.

  In step (A) of the method of the present invention, the expression level of the translation product of a predetermined gene of the present invention is determined based on the signal intensity detected by an arbitrary protein detection method in a biological sample, and comparison with this standard is used. I can do it. Examples of biological samples used as a reference include non-cancerous tissues (particularly non-cancerous renal tissues) collected from the same patient, and the expression level of the gene translation product of the present invention in this tissue is used as a control. be able to. In this case, the non-cancerous tissue (especially non-cancerous kidney tissue) collected from the same patient and the gene translation product expression signal intensity according to the present invention in the target biological sample, By detecting and comparing with the same protein detection method, the translation product expression level of at least one gene of the present invention can be determined. Furthermore, in “determination of translation product expression level”, the signal intensity of translation product expression of at least one gene of the present invention in the subject biological sample in the target biological sample is, for example, “high”, “equivalent” Alternatively, a ranking such as “low” can be given. Alternatively, for example, the signal intensity of the translation product expression of at least one gene of the present invention in the subject biological sample in the target biological sample can be scored based on the signal intensity used as the reference. For example, the signal intensity of the gene translation product in the sample used as a reference is set to 0, and the signal intensity is compared with the signal intensity of the gene translation product in the target biological sample. It can be scored with an integer such as. Ranking and scoring of signal intensity of gene translation products can be determined as appropriate.

  In step (B) of the method of the present invention, the recurrence risk of renal cancer of the patient is predicted based on the translation product expression level determined in step (A).

In order to predict the risk of recurrence of renal cancer in step (B), specifically, as described above with respect to step (A), “high”, “equivalent” or “low” determined by comparison with the above criteria The risk of recurrence after renal cancer treatment can be predicted based on a rank such as “” or a scored value. More specifically, when the expression level of the translation product of the at least one gene in the target biological sample is “high”, the risk of recurrence of renal cancer is relatively high for the patient. Can be understood as expensive. Furthermore, if the expression level of the translation product of any two of the above-mentioned genes in the target biological sample is ranked “high”, the patient is understood to have a relatively higher risk of recurrence of kidney cancer. If the expression level of the translation products of the three genes is ranked “high”, it can be understood that the patient has the highest risk of recurrence of kidney cancer. it can.
On the other hand, if the expression level of translation products of all three genes in the target biological sample is ranked as “equivalent” or “low”, the risk of recurrence of renal cancer is determined for the patient. Can be understood to be relatively low. When the translation product expression level is scored, the risk of recurrence of renal cancer can be understood from the score itself. Alternatively, a score such as “high”, “low”, or “medium” that has a risk of recurrence is assigned to the score in advance, and the recurrence risk of renal cancer is “high”, “low”, or “medium” from the obtained score. Or the like. Predicting the risk of recurrence of kidney cancer not only obtains a result such as “high”, “low” or “moderate”, but also has a risk of developing renal cancer after treatment of renal cancer of 50-90% (eg, , 50%, 60%, 70%, 80%, 90%, 95%, etc.) can also be included.

Treatment or Prevention Method Furthermore, according to another aspect of the present invention, a method for treating or preventing recurrence of renal cancer is provided. Specifically, the method for treating or preventing the recurrence of renal cancer is a method for treating or preventing the recurrence of renal cancer based on the prediction result obtained by the method for predicting the recurrence risk of the renal cancer. .

  Specifically, this method includes means capable of treating or preventing recurrence of any renal cancer, such as chemotherapy using an anticancer agent, hormonal therapy, radiation therapy, surgical treatment, diet therapy, lifestyle guidance, and the like. The translation product (biomarker) of the gene used in the present invention consists of a group of proteins that form a signal pathway involved in cancer invasion metastasis and treatment resistance. The present inventor has also found that inhibition of this signal pathway significantly reduces cancer invasion and metastasis. Therefore, by using the prediction method of the present invention, a patient whose renal cancer recurrence is predicted can be treated or prevented by applying a therapeutic method including inhibiting the signal pathway.

  This method is not particularly limited as long as it treats or prevents the recurrence of renal cancer based on the prediction result obtained by the method of predicting the recurrence risk of the renal cancer, and predicts the recurrence risk of the renal cancer. The method steps of the method to be performed may or may not be included.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not particularly limited by the following examples.

<Materials and methods>
(Materials and methods)
(1) Patient population This study was conducted as part of a study conducted with the permission of the Keio University Ethics Committee as a research project to investigate the resistance mechanism of molecular target therapy for renal cell carcinoma. Specifically, analysis was performed on 112 patients who had undergone surgery to remove kidney cancer in the past at Keio University Hospital (Tokyo, Japan).

(2) Immunohistochemistry (IHC)
EPB41L5 antibody immunizes rabbits with a peptide containing amino acids 558 to 733 of human protein as an antigen, and then collects antiserum by standard techniques and affinity-purifies with antigen as described in detail below. What was done was used.

FIG. 25 illustrates the antigenic site of a polyclonal antibody against EPB41L5 (a peptide containing amino acids 541 to 733 (193 amino acids)).
EPB41L5 antigen site cloning procedure
The HUVEC cDNA was used as a template, the antigen site was cloned using primers prepared at both ends of the antigen region, and inserted into the GST fusion protein expression vector pGEX-4T.
Preparation of EPB41L5 antigen
Escherichia coli BL21 is transformed using a plasmid in which the gene for the EPB41L5 antigenic site is incorporated into the GST fusion protein expression vector pGEX-4T-2. The transformed E. coli is cultured with shaking in LB medium at 37 ° C, and when OD ₆₀₀ reaches 0.7, IPTG is added to a final concentration of 0.2 mM to induce expression. Bacteria were collected after culturing at 25 ° C. for 2 hours.

Escherichia coli in which a large amount of antigen protein is expressed is suspended in a PBS buffer solution, crushed on ice by an ultrasonic crusher, and after removing complicated substances by centrifugation, the supernatant is collected. Since the antigen protein is expressed as a GST fusion protein, Glutathione Sepharose 4B that specifically recognizes and adsorbs the GST tag is packed in the column, and after equilibrating the column, a sample solution is added. After washing the column to remove complicated proteins, the GST fusion protein was eluted with a buffer containing glutathione.
The obtained antigen was immunized to a rabbit by a conventional method, antiserum was collected, and the antibody was purified. A series of operations was performed based on the scheme shown in FIG. The results of affinity purification of EPB41L5 antibody are shown in FIGS.

<Antibodies to AMAP1>
FIG. 29 collectively shows information on the antigenic site of the antibody against AMAP1, cloning procedure of the antigenic site, preparation of the antigen, immunization to rabbits, collection of antiserum, and purification of the antibody.

<Antibodies to GEP100>
FIG. 30 shows information on the antigen site of the antibody against GEP100, information on the antigen site cloning procedure, antigen preparation, rabbit immunization, antiserum collection, and antibody purification.

  In addition, antibodies against AMAP1 and GEP100 have been reported in the past (Onodera et al., 2006; Morishige et al., 2008).

  Immunohistochemical staining was performed using formalin-fixed, paraffin-embedded serial sections with a thickness of 4 μm as follows. All slides were first deparaffinized in xylene, dehydrated in graded alcohol, then sections were tris-buffered saline (TBS: 25 mM Tris-HCl (pH 7.4), 137 mM NaCl) , 2.7 mM KCl).

Then, the antigen activation process of the section | slice was performed about each antigen on the following conditions.
(I) EPB41L5 (EBL5): incubation with citrate buffer (pH 6.0) at 95 ° C. for 40 minutes;
(Ii) AMAP1 or GEP100: incubation with an antigen activation solution (EDTA solution, pH 9, purchased from Nichirei) at 95 ° C. for 40 minutes;

The endogenous peroxidase was then blocked by incubation in 0.3% H ₂ O ₂ / methanol at room temperature. Sections were washed with TBS and then incubated with primary antibody against AMAP1 (1: 500), GEP100 (1: 100), or EPB41L5 (EBL5; 1: 1000, ratio in parentheses is dilution factor) for 30 minutes at room temperature After further washing with TBS, each primary antibody was detected using EnVision (trademark) (DAKO, Japan) as a secondary antibody, and color was developed using diaminobenzidine. After counterstaining and washing with water, a clearing process and an encapsulating process were performed according to a steady method.

(3) Evaluation All samples were independently “blind” and “high” according to the degree of staining by a pathologist-certified physician specializing in renal tumors and researchers familiar with the Arf6 pathway. A two-stage evaluation was made: when the staining was dark compared to the staining degree of cancerous kidney tissue) or “low” (when the staining was comparable or light compared to the staining degree of non-cancerous kidney tissue).

(4) Statistical analysis In this analysis, the survival rate was calculated by the Kaplan-Meier method, and the significant difference was tested by the Log-rank method. Predictor variables that were candidates for analysis were AMAP1, GEP100, EPB4IL5 (EBL5), and p values less than 0.05 were judged to have a statistically significant correlation. These analyzes were performed using Stat View ver.5 for WINDOWS.

<Result>
(1) Relationship between expression of translation products of AMAP1, EPB41L5 and GEP100 in patients and clinicopathological features Table 1 below shows the relationship between the expression of translation products of each gene and clinicopathological features in patients.

(Analysis result)
FIGS. 2A to 2C are Kaplan-Meier curves showing the period of time and the rate at which recurrence was avoided for patients with high expression and low expression of translation products of each gene of AMAP1, EPB41L5, and GEP100. In any of AMAP1, EPB41L5, and GEP100, patients with high expression have significant early recurrence compared with those with low expression (P <0.0001, <0.0001, and 0.0003, respectively). I understood.

  As shown in FIG. 2D, when all three of AMAP1, EPB41L5, and GEP100 are highly expressed, all three are lowly expressed or one to two of the three are lowly expressed (indicated as “Others” in the figure). The tendency of early recurrence was more noticeable than in the case.

  Furthermore, when all three of AMAP1, EPB41L5, and GEP100 were highly expressed, a significant (P = 0.0002) tendency to shorten the survival period was also observed (FIG. 2E).

  The present invention is useful in fields related to cancer diagnosis and treatment.

Claims (10)

A diagnostic agent for use in predicting the risk of recurrence of renal cancer,
A diagnostic agent comprising a protein and / or polypeptide capable of specifically recognizing a translation product of any one gene selected from the group consisting of AMAP1, GEP100, and EPB41L5. The diagnostic agent according to claim 1, wherein the protein and / or polypeptide is an antibody and / or a fragment thereof. A kit for use in prediction of recurrence risk of renal cancer, comprising the diagnostic agent according to claim 1 or 2. (A) determining a translation product expression level of at least one gene selected from the group consisting of AMAP1, GEP100, and EPB41L5 in a biological sample collected from a patient treated for renal cancer; and (B) Predicting the risk of recurrence of renal cancer in the patient based on the translation product expression level determined in (A),
A method for predicting the risk of recurrence of kidney cancer. 5. The method according to claim 4, wherein in step (A), the translation product expression levels of all genes selected from the group consisting of AMAP1, GEP100, and EPB41L5 are determined. The method according to claim 4 or 5, wherein the biological sample is kidney tissue. In step (A), the translation product expression level of the at least one gene is determined by an immunohistochemical method using a protein and / or polypeptide capable of specifically recognizing the translation product of each of the genes. Item 7. The method according to any one of Items 4 to 6. The method according to claim 7, wherein the protein and / or polypeptide is an antibody and / or a fragment thereof. In the step (A), the expression level of the translation product of the at least one gene in the biological sample is determined with reference to the expression level of each translation product of the at least one gene in the non-cancerous kidney tissue of the patient, and the step The method according to any one of claims 4 to 8, wherein the risk of recurrence of renal cancer in the patient is predicted according to the following criteria in (B):
(I) if the translation product expression level of the at least one gene determined in step (A) is higher than the expression level of the corresponding control, the patient predicts that the risk of recurrence of renal cancer is high; or (Ii) if the translation product expression level of the at least one gene determined in step (A) is lower than or equivalent to the expression level of the corresponding control, the patient is at risk for recurrence of renal cancer; Expect low. In the step (A), the expression levels of the translation products of all the genes in the biological sample are determined with reference to the expression levels of the translation products of all the genes in the non-cancerous kidney tissue of the patient, and the step (B 9) The method according to any one of claims 4 to 8, wherein the risk of recurrence of renal cancer in the patient is predicted according to the following criteria:
(I) if the translation product expression levels of all the genes determined in step (A) are higher than the expression levels of the corresponding controls, the patient is predicted to be at increased risk of renal cancer recurrence; or ( ii) If the translation product expression levels of all the genes determined in step (A) are lower than or equivalent to the expression levels of the corresponding controls, the patient has a low risk of recurrence of renal cancer Predict.