JP6695586B2 - Diagnostic agent and kit for use in predicting recurrence risk of pancreatic cancer, and prediction method - Google Patents

Description

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

  The only curative treatment for pancreatic cancer is surgical curative resection. However, since the symptoms are poor, about 80% of the cases are already unresectable at the time of initial diagnosis (Non-patent Documents 1 and 2). Therefore, although chemotherapy is important for prolonging the prognosis, pancreatic cancer is generally resistant to chemotherapy (Non-Patent Document 3). The recurrence lesion after the initial treatment is considered to be formed by selective growth of the chemoresistant strain, and it is expected that the chemoresistance is further enhanced as compared with that before the treatment. In recent years, the development of new chemotherapy regimens and the introduction of molecular-targeted therapy with erlotinib, which is an EGFR tyrosine kinase inhibitor, have achieved some results (Non-Patent Documents 4 and 5), but ultimately resistance to them has been achieved. A sex strain appears and the disease is out of control. Moreover, it is known that even if radical resection can be performed, recurrence occurs at a high rate. Since it is said that malignant tumors successively acquire new gene mutations suitable for their own survival (Non-patent Document 6), it is necessary to perform radical resection earlier in order to prolong the prognosis. It is important to diagnose recurrence / metastasis and start treatment earlier.

  As a predictor of recurrence of pancreatic cancer, preoperative serum CA19-9 value (Non-patent document 7), preoperative CRP value (Nonpatent document 8), preoperative platelet count (Nonpatent document 8), etc. are generally said. ing.

Donghui L, Keping X, Robert W, et al. Pancreatic cancer. Lancet 2004; 363: 1049-1057. Butturini G, Stocken DD, Wente MN, et al. Influence of resection margins and treatment on survival in patients with pancreatic cancer: meta-analysis of randomized controlled trials. Arch Surg 2008; 143: 75-83. Arumugam T, Ramachandran V, Fournier KF, et al. Epithelial to Mesenchymal Transition Contributes to Drug Resistance in Pancreatic Cancer. Cancer Res 2009; 69: 5820-5828. Conroy T, Desseigne F, Ychou M et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer.N Engl J Med 2011; 364: 1817-1825. Moore MJ, Goldstein D, Hamm J, et al. Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol. 2007; 25: 1960. -1966. Yachida S and Donahue-lacobuzio CA. Evolution and dynamics of pancreatic cancer progression. Oncogene 2013; 32: 5253-5260. Humphris JL, Chang DK, Johns AL, et al. The prognostic and predictive value of serum CA19.9 in pancreatic cancer. Ann Oncol. 2012; 23 (7): 1713-1722. Miura T, Hirano S, Nakamura T, et al. A new preoperative prognostic scoring system to predict prognosis in patients with locally advanced pancreatic body cancer who undergo distal pancreatectomy with en bloc celiac axis resection: a retrospective cohort study.Surgery. 2014; 155 (3): 457-467.

  Preoperative serum CA19-9 levels, preoperative CRP levels, and preoperative platelet counts, which are listed as predictive factors for recurrence of pancreatic cancer, vary from report to report. Moreover, the preoperative serum CA19-9 level, preoperative CRP level, and preoperative platelet count were not all pancreatic cancer-specific, and no certain opinion has been obtained regarding their efficacy.

  Therefore, it is required to elucidate clinically available recurrence predicting factors.

  Therefore, an object of the present invention is to provide a predictive diagnostic agent and kit for pancreatic cancer recurrence risk using a factor specific to recurrence of pancreatic cancer, which is highly useful as a means for predicting recurrence of pancreatic cancer, and a prediction method. It is in. A further object of the present invention is to provide a predictive diagnostic agent and kit using a factor capable of linking the predictive result of pancreatic cancer recurrence risk to treatment, and a predictive method.

As a result of intensive studies to solve the above problems, the present inventors have found that high expression of AMAP1 protein and EPB41L5 protein in locally advanced pancreatic cancer excised tissue contributes to significant early recurrence and shortened survival period.
The present invention has been completed based on this finding.

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

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

The micrograph of the immunohistochemical staining (The anti-EPB41L5 antibody is used as an example) acquired in the Example is shown. The result of having investigated the correlation between the translation product expression of each gene and survival time is shown. 2 shows the cds region nucleotide sequence of EPB41L5 (NCBI Reference Sequence: NM_020909.3). The amino acid sequence of EPB41L5 (NCBI Reference Sequence: NP_065960.2) is shown. A comparison of the primary structures of EPB41L5 and EPB41L4b is shown. Fig. 2 shows a comparison of the genomes of EPB41L4b isoform1 and isoform2. The comparison of the amino acid of EPB41L4b isoform1 and isoform2 is shown. The alignment analysis is shown for EPB41L5 (NP_065960.2) and EPB41L4b (NP_061987.3061987.3). The cds region base sequence of AMAP1 (NCBI Reference Sequence: NM_018482.3) is shown. The amino acid sequence of AMAP1 (NCBI Reference Sequence: NP_060952.2) is shown. The information of the gene (ArfGAP family) having homology with AMAP1 is shown. 1 shows the cds region nucleotide sequence (NCBI Reference Sequence: NM_003887.2) of the AMAP2 gene having homology with AMAP1. The amino acid sequence of AMAP2 having homology with AMAP1 (NCBI Reference Sequence: NP_003878.1) is shown. The data of alignment analysis of AMAP2 which has homology with AMAP1 are shown. The antigenic site (peptide (193 amino acids) including amino acids 541 to 733) of the pockclonal antibody against EPB41L5 is illustrated. The scheme of a series of operations for immunizing a rabbit with an antigen, collecting antisera, and purifying antibodies is shown. The result of affinity purification of EPB41L5 antibody is shown. The result of affinity purification of EPB41L5 antibody is shown. The information on the antigenic site of the antibody against AMAP1, the procedure for cloning the antigenic site, the information on the antibody purification, etc. are shown together. The information on the antigenic site of the antibody against GEP100, the cloning procedure of the antigenic site, and the information on the antibody purification and the like are shown together.

Diagnostic Agent According to one aspect of the present invention, a diagnostic agent for use in predicting a recurrence risk of pancreatic cancer, which specifically comprises a translation product of any one gene selected from the group consisting of AMAP1 and EPB41L5. Diagnostic agents are provided that contain a recognizable protein and / or polypeptide.

  In the present invention, the term “pancreatic cancer” refers to pancreatic ductal adenocarcinoma which accounts for the majority of malignant neoplasms originating in the pancreas.

  In the present invention, the “risk of recurrence” means the risk of recurrence of cancer in a patient who has been treated for pancreatic cancer after completion of the treatment. Recurrent cancers include locally recurrent pancreatic cancer and / or metastatic recurrent pancreatic cancer. The risk of recurrence mainly means the risk of early recurrence within 5 years after receiving the treatment for pancreatic cancer. However, the recurrence risk may include a medium-term recurrence risk of more than 5 years and less than 10 years after receiving the treatment for pancreatic cancer, or a late recurrence risk of more than 10 years. Furthermore, the treatment method for pancreatic cancer is not particularly limited. For example, chemotherapy including anticancer drug administration, administration of molecular target drugs that have recently been introduced into metastatic pancreatic cancer, or pancreaticoduodenectomy to remove the head side of the pancreas including the lesion site, or caudal to remove the tail side of the pancreatic body. A pancreatectomy method etc. are mentioned.

The method of “prediction of recurrence risk” will be described later in “Prediction method”.
The diagnostic agent of the present invention can be used to predict the risk of recurrence of pancreatic cancer. This can be understood from the following results in a group of patients who received treatment for pancreatic cancer and had recurrence of pancreatic cancer thereafter. Patients with high expression of the translation product of a specific gene in pancreatic tissue are statistically relatively early after treatment for pancreatic cancer, compared with patients without high expression of translation product of the specific gene in pancreatic tissue. Recurrence of pancreatic cancer (for example, within 5 years) is shown with a statistically significant difference. This point will be specifically shown in Examples described later. The specific gene is specifically at least one gene selected from the group consisting of AMAP1 and EPB41L5. Patients with high expression of the translation product of any one of these genes are at high risk of relapse with a statistically significant difference. Furthermore, patients with high expression of the translation products of any two of the above genes have a higher risk of relapse with a statistically significant difference. That is, when high expression of the translation product of the above gene is observed in pancreatic cancer tissue, it is predicted that the patient has a high risk of recurring cancer early after the treatment of pancreatic cancer. The details will be described in the method of predicting the risk of recurrence of pancreatic cancer, which will be described later. Thus, in the present invention, "predicting the risk of recurrence of pancreatic cancer" is premised on detecting high expression of a translation product of the above-mentioned specific gene in pancreatic tissue. Therefore, the diagnostic agent of the present invention for use in the prediction is specifically a reagent used for detecting those gene translation products, and is a reagent for any one gene selected from the group consisting of AMAP1 and EPB41L5. It contains a protein and / or polypeptide capable of specifically recognizing a translation product.

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

  2 Among the EPB41L5 family, EPB41L5 has the most homology to EPB41L5. EPB41L4b contains 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 comparison of the primary structures of EPB41L5 and EPB41L4b. FIG. 6 shows a comparison of EPB41L4b isoform1 and isoform2 genomes. FIG. 7 shows a comparison of the amino acid sequences of EPB41L4b isoform1 and isoform2. FIG. 8 shows the alignment analysis of 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 of EPB41L5 (NP_065960.2) (SEQ ID NO: 2) and EPB41L4b (NP_061987.3) (SEQ ID NO: 4), in the C-terminal side (the sequence from the 541st position on the EPB41L5 standard) Both sequences are very 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 antibody specific to EPB41L5 protein or a fragment thereof is an antibody or a fragment thereof having an amino acid sequence serving as an epitope in the amino acid sequence region from 541st position to 733th position 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 of the cds region of AMAP1 (NCBI Reference Sequence: NM_018482.3) (SEQ ID NO: 5). 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 nucleotide 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 of AMAP2 (NCBI Reference Sequence: NP_003878.1) (SEQ ID NO: 8) which has homology with AMAP1. FIG. 14 shows data of alignment analysis of AMAP2 having homology with AMAP1.

  As shown in FIG. 14, as a result of alignment analysis of 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 are significantly different. There is an area. This region of AMAP1 becomes an effective antigen in producing an antibody specific to the protein. In the present invention, an antibody specific to AMAP1 protein or a fragment thereof may be an antibody or a fragment thereof having an amino acid sequence serving as an epitope in the amino acid sequence region from the 935th position to the 1002nd position of AMAP1 (SEQ ID NO: 6). Appropriate.

  The protein and / or polypeptide capable of specifically recognizing the translation product of the above gene is not particularly limited as long as it can specifically bind to the translation product of one of the above genes. Examples include an antibody against the translation product and / or a fragment thereof. Examples of the antibodies and / or fragments thereof include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, humanized antibodies and the like. As for the method for preparing the protein and the polypeptide that can specifically recognize the translation product of the gene, any method can be adopted as long as it can retain the specific binding to the gene translation product. 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.

  In addition, the specific binding property to the gene translation product of the protein and polypeptide capable of specifically recognizing the translation product of the gene may be such that the translation product of the gene may coexist with the translation product of the gene. It only needs to be distinguishable from the translation product of the gene. Therefore, for example, even if a protein and a polypeptide capable of specifically recognizing a translation product of a gene in which some modification (substitution, deletion, addition, etc.) is added to the gene, the translation product of the gene is As long as it has a specific binding property to a gene translation product that can be distinguished from the translation products of other genes that may possibly coexist with the translation product of the gene, the “specifically recognizes the translation product of the gene of the present invention” Possible proteins and / or polypeptides ".

  Furthermore, in the diagnostic agent of the present invention, a single diagnostic agent may include only one protein or polypeptide capable of specifically recognizing a translation product of one of the above genes, Alternatively, two or more kinds of proteins and / or polypeptides capable of specifically recognizing each of the translation products of the above genes may be contained. In addition, the above protein and / or polypeptide may be labeled with a labeling substance for detecting a gene translation product. Examples of the labeling substance include fluorescent substances (including fluorescent proteins such as GFP and fluorescent low molecular weight compounds such as fluoroscein). However, it is not intended to be limited to those having a label 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 those proteins and / or polypeptides (primary antibody) can be used. There are commercially available detection kits for systems using secondary antibodies. In this system, for example, a secondary antibody against the primary antibody is labeled with peroxidase, and the secondary antibody is further specifically bound to the primary antibody that specifically binds to the translation product of the gene of interest in the sample. A gene translation product can be detected by adding a chromogenic substrate (for example, 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 that can specifically recognize the translation products of each of the above genes, for example, two or more of them can be used. The proteins and / or polypeptides of 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 buffering agents, preservatives, antifreeze agents and the like. Examples of the buffer include tris hydrochloride, potassium phosphate, sodium phosphate and the like. Examples of the preservatives include sodium azide and the like. Examples of the antifreezing agent include glycerol and the like.

  The diagnostic agent of the present invention can be used for detecting a translation product of a gene of interest in order to determine the expression level of a predetermined gene translation product in step (A) of the method of predicting a recurrence risk of pancreatic cancer described below.

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

  The kit of the present invention provides elements such as reagents and members that can be used in carrying out the method for predicting the recurrence risk of pancreatic cancer described below, and is characterized by containing the above-mentioned diagnostic agent.

  In the kit of the present invention, the diagnostic agent of the present invention comprises (1) only one protein or polypeptide capable of specifically recognizing a translation product of one gene of AMAP1 and EPB41L5 in a single diagnostic agent By being included, it can be a diagnostic agent capable of detecting the translation product of one gene. Alternatively, the diagnostic agent of the present invention comprises (2) the single diagnostic agent containing two or more kinds of proteins and / or polypeptides capable of specifically recognizing translation products of these genes, respectively. The above-mentioned diagnostic agent may be a single diagnostic agent capable of detecting a plurality of gene translation products. Furthermore, in the kit of the present invention, a diagnostic agent capable of detecting the translation product of one gene of the above (1), and / or a single agent capable of detecting translation products of a plurality of genes of the above (2). Diagnostic agents may be included.

  The kit of the present invention comprises, in addition to the above-mentioned diagnostic agent, a blocking reagent, a labeled secondary antibody for use in detecting a protein and / or a polypeptide (antibody) contained in the diagnostic agent, a coloring reagent, and a risk of recurrence of pancreatic cancer. An instruction manual or the like which describes an experimental protocol regarding a method of predicting a can be optionally included. Examples of the blocking reagent include bovine serum albumin (BSA) and the like.

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

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

  In the method of the present invention, the “biological sample” is any biological sample collected from a patient who has been treated for pancreatic cancer. The biological sample is preferably pancreatic tissue from the viewpoint that the risk of recurrence can be accurately predicted. Further, as the pancreatic tissue, for example, the tissue of the pancreatic lesion site removed from the patient at the time of tumor removal in the treatment of pancreatic cancer, or the nearby pancreatic tissue where the lesion site was present after the tumor removal procedure. The pancreatic tissue collected separately from the enucleation can be mentioned. However, from the viewpoint of reducing the burden on the patient as much as possible and accurately predicting the risk of recurrence, the pancreatic tissue is preferably a tissue of a pancreatic cancer lesion site removed from the patient at the time of tumor resection. In the present invention, since the above-mentioned predictive factors can be detected by using paraffin-embedded tissues prepared from surgically resected specimens of pancreatic primary lesion and metastatic lesion for pathological diagnosis, all pathological diagnosed cases Can be searched for.

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

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

  The method of determining the translation product expression level of the above-mentioned predetermined gene in a 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) capable of specifically recognizing 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 expression level of the translation product of the gene by immunohistochemistry using the protein and / or polypeptide (diagnostic agent) 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 immunohistochemistry method. In the present invention, those known methods may be used, or a newly developed method may be used. The method for determining the expression level of the translation product such as immunohistochemistry is not particularly limited as long as it can determine the expression level of the target gene translation product.

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

  In the step (A) of the method of the present invention, the expression level of the translation product of the 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 reference is used. You can As an example of the biological sample used as a reference, a non-cancerous tissue (particularly a non-cancerous pancreatic tissue) collected from the same patient can be mentioned, 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 gene translation product expression signal intensity of the present invention in a non-cancerous tissue (particularly non-cancerous pancreatic tissue) collected from the same patient, and the gene translation product expression signal intensity of the present invention in a target biological sample, Detected by the same protein detection method and compared, whereby the translation product expression level of at least one gene defined in the present invention can be determined. Furthermore, in the "determination of the translation product expression level", the signal intensity of the translation product expression of at least one gene of the present invention in the target biological sample is, for example, "high" or "equivalent" in comparison with the above criteria. Alternatively, a ranking such as “low” can be used. Alternatively, for example, the signal intensity of the translation product expression of at least one gene of the present invention in the target biological sample can be scored based on the signal intensity used as the above criterion. The signal intensity is scored, for example, by setting the signal intensity of the gene translation product in the sample used as a reference to 0 and comparing the signal intensity with the signal intensity of the gene translation product in the target biological sample, for example, -1 to 3 Can be scored with integers such as. The ranking or scoring of the signal intensity of gene translation products can be appropriately determined.

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

  In order to predict the risk of pancreatic cancer recurrence 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 treatment of pancreatic cancer can be predicted based on the rank or the like or the scored value. More specifically, when the translation product expression level of the at least one gene in the target biological sample is ranked as "high", the patient has a relatively high risk of recurrence of pancreatic cancer. Can be understood. Furthermore, if a translation product expression level of any two of the above genes in a target biological sample is ranked as “high”, it is understood that the risk of recurrence of pancreatic cancer is relatively higher in the patient. be able to.

  On the other hand, if the translation product expression levels of the above two genes in the target biological sample are ranked as “equivalent” or “low”, the risk of recurrence of pancreatic cancer in the patient is increased. Can be understood to be relatively low. When the translation product expression level is scored, the recurrence risk of pancreatic cancer can be understood from the score itself. Alternatively, a score such as “high”, “low” or “moderate” is assigned to the score in advance, and the risk of recurrence of pancreatic cancer is “high”, “low” or “moderate” from the obtained scores. It can also be understood as "etc." Predicting the risk of recurrence of pancreatic cancer not only results in “high,” “low,” or “moderate,” but the risk of developing pancreatic cancer after treatment of pancreatic cancer is 50-90% (eg, 50%. , 60%, 70%, 80%, 90%, 95%, etc.) can be included.

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

  Specifically, the method includes means capable of treating or preventing recurrence of any pancreatic cancer, such as chemotherapy using an anticancer agent, hormone therapy, radiation therapy, surgical treatment, diet therapy, life guidance, and the like. The translation product (biomarker) of the gene used in the present invention is composed of a group of proteins forming a signal pathway involved in invasion and metastasis of cancer and treatment resistance. The present inventor has also found that inhibition of this signaling pathway significantly reduces cancer invasion and metastasis. Therefore, the prediction method of the present invention can be used to treat or prevent the recurrence of pancreatic cancer by applying a therapeutic method including inhibiting the above signaling pathway to a patient in which the recurrence of pancreatic cancer is predicted.

  This method, based on the prediction results obtained by the method of predicting the recurrence risk of pancreatic cancer, is not particularly limited as long as it treats or prevents the recurrence of pancreatic cancer, the method of predicting the recurrence risk of pancreatic cancer The method steps 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 to the following examples.

<Material and method>
(1) Patient Group 70 patients who had undergone pancreatic cancer resection surgery at Hokkaido University Hospital (Sapporo, Japan) in the past were analyzed.

(2) Immunohistochemistry (IHC)
Details of the method for producing the EPB41L5 antibody, AMAP1 antibody, and GEP100 antibody are described in paragraphs 0049 to 0053 of JP-A-2015-21800.

  The EPB41L5 antibody was prepared by immunizing rabbits with a peptide containing amino acids 558 to 733 of human protein as an antigen, collecting antiserum by a standard method, and affinity-purifying with the antigen. FIG. 15 shows the antigenic site (a peptide containing amino acids 541 to 733 (193 amino acids)) of a pockclonal antibody against EPB41L5. FIG. 16 shows a scheme of a series of operations for immunizing a rabbit with an antigen, collecting an antiserum, and purifying an antibody. The results of affinity purification of the obtained EPB41L5 antibody are shown in FIGS.

  Regarding the antibody against AMAP1, the information on the antigenic site of the antibody against AMAP1, the procedure for cloning the antigenic site, and the information on the antibody purification and the like are shown together in FIG.

  For reference, information on the antigenic site of the antibody against GEP100, the procedure for cloning the antigenic site, and information on the antibody purification and the like are shown together in FIG.

  It should be noted that there are reports of antibodies against AMAP1 and GEP100 before the publication of the above publication (Onodera et al., 2006; Morishige et al., 2008).

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

Then, the antigen activation treatment of the slice was performed for each antigen under the following conditions.
(I) EPB41L5 (EBL5): Incubated with citrate buffer (pH 6.0) at 95 ° C for 40 minutes;
(Ii) AMAP1 or GEP100: Incubated with antigen activation solution (EDTA solution, pH 9, purchased from Nichirei) at 95 ° C. for 40 minutes;
Endogenous peroxidase was then blocked by incubation in 0.3% H ₂ O ₂ / methanol at room temperature. After washing the sections with TBS, with the primary antibody against AMAP1 (1: 750), GEP100 (1: 750), or EPB41L5 (EBL5; 1: 1000) (the ratio in parentheses is the dilution ratio), 30 minutes at room temperature, Incubated.
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. Each section was counterstained with hematoxylin, washed with water, and then subjected to a clearing treatment / encapsulation treatment according to a stationary method.

(3) Evaluation All samples were blindly evaluated by a researcher who was familiar with the Arf6 pathway study to determine the degree of staining. 2 points for each staining intensity of tumor cells (when staining is very dark compared to the staining degree of non-cancerous pancreatic tissue), 1 point (when higher than the staining degree of non-cancerous pancreatic tissue) or 0 Points were scored (if comparable or low compared to the degree of staining of non-cancerous pancreatic tissue). In addition, the ratio of those tumor cells in the whole tissue is expressed as 0.0 to 1.0 (0% is 0, 10 to 90% is 0.1 to 0.9, 100% is 1.0), and is 0 × (0 point ratio) + 1 × The IHC score was defined as (percentage of 1 point) + 2 x (percentage of 2 points). The IHC score was calculated for each patient, and the patients with higher than the median value were defined as the high expression group, and the patients with low values were defined as the low expression group.

(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. Cox proportional hazards regression was used for multivariate analysis, and the factors with significant difference in univariate analysis were adopted as independent variables. A p-value less than 0.05 was considered to have a statistically significant correlation. These analyzes were performed using EZR (Bone Marrow Transplant 2014; 48: 452-458).

<Results>
(1) Relationship between AMAP1, EPB41L5, and GEP100 translation product expression in patients and clinicopathologic features Table 1 shows the clinicopathologic features of the patients targeted this time.

A representative stained image is shown in FIG.
The median IHC scores of 70 patients were AMAP1 (0.13), EPB41L5 (0.07) and GEP100 (0.14).

(Analysis result)
2A and 2B are Kaplan-Meier curves showing the overall survival rate and recurrence-free survival rate for patients with high expression and low expression of translation products of each gene of AMAP1, EPB41L5, and GEP100. Patients with high expression of AMAP1 and EPB41L5 relapse significantly earlier than those with low expression (P = 0.0014, P = 0.00768, respectively) and have a poor prognosis (P = 0.0012, P = 0.000127, respectively). I understood it.

  Further, as shown in FIGS. 2C to D, when any two of AMAP1, EPB41L5, and GEP100 are highly expressed, or when all three are highly expressed (indicated as “All-high” in the figure, out of three Compared with the case where at least one of them had low expression (denoted as "Others" in the figure), the tendency of early recurrence and poor prognosis was observed.

  When these results were combined with clinicopathological features, univariate analysis and multivariate analysis were performed, and it was found that a tumor diameter larger than 2 cm and high expression of AMAP1 / EPB41L5 were independent poor prognostic factors (Table. 2).

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

SEQ ID NO: 1: nucleotide sequence of EPB41L5 gene SEQ ID NO: 2: amino acid sequence of EPB41L5 SEQ ID NO: 3: nucleotide sequence of band 4.1-like protein 4B isoform 1 SEQ ID NO: 4: nucleotide sequence of band 4.1-like protein 4B isoform 2 SEQ ID NO: 5 : AMAP1 gene cds region nucleotide sequence SEQ ID NO: 6: AMAP1 amino acid sequence SEQ ID NO: 7: AMAP2 gene cds region nucleotide sequence SEQ ID NO: 8 AMAP2 amino acid sequence

Claims (10)

A diagnostic agent for use in predicting the risk of pancreatic cancer recurrence,
A diagnostic agent containing a protein and / or a polypeptide capable of specifically recognizing a translation product of any one gene selected from the group consisting of AMAP1 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 predicting the risk of recurrence of pancreatic cancer, comprising the diagnostic agent according to claim 1 or 2. (A) in a biological sample taken from a patient treated for pancreatic cancer, and at least one gene child of determining the translation product expression levels selected from the group consisting of AMAP1 and EPB41L5
And a method of predicting the risk of recurrence of pancreatic cancer. In step (A), that determine the translation product expression levels of all genes selected from the group consisting of AMAP1 and EPB41L5, The method of claim 4. The method according to claim 4 or 5, wherein the biological sample is pancreatic tissue. In the step (A), the translation product expression level of the at least one gene is determined by an immunohistochemistry method using a protein and / or a polypeptide that can specifically recognize the translation product of the gene. 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 step (A), to determine the translation product expression levels of said at least one gene in the biological sample as a control expression level of the respective translation product of said at least one gene in non-cancerous pancreatic tissue of the patient, and step and said at least one gene translation product expression levels determined (a), the further comprising the contrasting and corresponding control level of expression, method towards according to any one of claims 4 8. In step (A), to determine the translation product expression levels of all of the above genes in the biological sample translation products each of the expression levels of all of the above genes in non-cancerous pancreatic tissue of the patient as a control, and the step (A It said a translation product expression levels of all genes determined in), comprising further to comparing the corresponding control level of expression, method towards according to any one of claims 4 8.