JP6695586B2 - Diagnostic agent and kit for use in predicting recurrence risk of pancreatic cancer, and prediction method - Google Patents
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Description
本発明は、膵癌の再発リスクの予測に用いるための診断薬及びキット、並びに膵癌の再発リスクを予測する方法に関する。 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.
膵癌の唯一の根治的治療は外科的根治切除である。しかし、症状に乏しいため、約80％の症例は初診断時すでに切除不能である（非特許文献１、２）。したがって、予後延長のためには化学療法が重要となるが、膵癌は概して化学療法抵抗性を有する（非特許文献３）。初回治療後の再発巣は、化学療法抵抗性株が選択的に増殖して形成されると考えられ、治療前に比べてさらに化学療法抵抗性が増強していると予想される。近年、新規化学療法レジメンの開発やEGFRチロシンキナーゼ阻害薬であるエルロチニブによる分子標的治療が導入され、ある程度の成果が得られているものの（非特許文献４、５）、最終的にはこれらに対する抵抗性株が出現し、病勢コントロール不能となる。また、根治切除できたとしても高率で再発することが知られている。悪性腫瘍は自らの生存に適した新たな遺伝子変異を次々に獲得していく（非特許文献６）と言われているため、予後延長のためにはより早期に根治切除することは元より、より早期に再発・転移を診断し、治療を開始することが重要である。 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.
膵癌の再発予測因子としては、術前血清CA19-9値（非特許文献７）、術前CRP値（非特許文献８）、術前血小板数（非特許文献８）などが一般的に言われている。 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.
膵癌の再発予測因子として挙げられている術前血清CA19-9値、術前CRP値、術前血小板数は、報告により様々である。また、術前血清CA19-9値、術前CRP値、術前血小板数はいずれも膵癌特異的とは言えず、その有効性に関しては一定の見解が得られていない。 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.
そのため、臨床的に利用可能な再発予測因子の解明が求められている。 Therefore, it is required to elucidate clinically available recurrence predicting factors.
そこで、本発明の目的は、膵癌の再発に特異的な因子を用いた、膵癌の再発予測手段としての有用性が高い、膵癌の再発リスクの予測診断薬及びキット、並びに予測方法を提供することにある。さらに本発明の目的は、膵癌の再発リスクの予測結果を治療に結びつけることが可能な因子を用いた予測診断薬及びキット、並びに予測方法を提供することにある。 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.
本発明者らは、上記課題を解決するため鋭意検討した結果、局所進行膵癌切除組織におけるＡＭＡＰ１蛋白およびＥＰＢ４１Ｌ５蛋白の高発現が有意な早期再発と生存期間短縮に寄与していることを見出した。
本発明は、この知見により完成されたものである。
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.
即ち、本発明は以下の（１）〜（３）に関する。
（１）膵癌の再発リスクの予測に用いるための診断薬であって、
ＡＭＡＰ１及びＥＰＢ４１Ｌ５からなる群から選択されるいずれか1つの遺伝子の翻訳産物を特異的に認識可能なタンパク質及び／又はポリペプチドを含有する、診断薬。
（２）膵癌の再発リスクの予測に用いるためのキットであって、上記本発明の診断薬を含む、キット。
（３）（Ａ）膵癌の治療を受けた患者から採取した生体試料において、ＡＭＡＰ１及びＥＰＢ４１Ｌ５から成る群から選択される少なくとも１つの遺伝子の翻訳産物発現レベルを決定すること；並びに
（Ｂ）工程（Ａ）において決定した翻訳産物発現レベルに基づいて、上記患者の膵癌の再発リスクを予測すること、を含む、膵癌の再発リスクを予測する方法。
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).
本発明によれば、膵癌の再発に特異的な因子を用いた、膵癌の再発予測手段としての有用性が高い、膵癌の再発リスクの予測診断薬及びキット、並びに予測方法を提供することができる。本発明で用いる因子であるバイオマーカーは、癌の浸潤転移や治療抵抗性に関与するシグナル経路を形成する一群の蛋白質からなるものである。このシグナル経路を阻害すると、癌の浸潤転移などを著しく減じる事も本発明者は見いだしている。従って、本発明の予測診断薬及びキット、並びに予測方法を用いて、膵癌の再発が予測される患者について治療方針を提供することも可能である。 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.
診断薬
本発明の一の態様によれば、膵癌の再発リスクの予測に用いるための診断薬であって、ＡＭＡＰ１及びＥＰＢ４１Ｌ５からなる群から選択されるいずれか1つの遺伝子の翻訳産物を特異的に認識可能なタンパク質及び／又はポリペプチドを含有する、診断薬が提供される。
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.
本発明において、「膵癌」とは、膵臓原発の悪性新生物の最も多くを占める膵管腺癌を指す。 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.
本発明において、「再発リスク」とは、膵癌の治療を受けた患者が、当該治療の完了後に癌を再発するリスクを意味する。再発する癌は、局所再発膵癌及び転移性再発膵癌のいずれか一方または両方を包含する。再発リスクは、膵癌の治療を受けた後、例えば、５年以内の早期再発リスクを主に意味する。但し、再発リスクは、膵癌の治療を受けた後５年を超え、１０年以内の中期再発リスク、あるいは１０年を超える後期再発リスクを含み得る。さらに、膵癌の治療法は、特に限定されるものでは無い。例えば、抗癌剤投与を含む化学療法、近年転移性膵癌に導入されている分子標的薬の投与、或いは病巣部を含めて膵頭部側を切除する膵頭十二指腸切除術や膵体尾部側を切除する尾側膵切除術等が挙げられる。 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.
「再発リスクの予測」の方法については、「予測方法」において後述する。
本発明の診断薬は、膵癌の再発リスクの予測に用いることができる。このことは、膵癌の治療を受け、かつその後膵癌が再発した患者群における以下の結果から理解できる。膵組織で特定遺伝子の翻訳産物の高発現が見られる患者は、膵組織で当該特定遺伝子の翻訳産物の高発現が見られない患者と比較して、統計的に膵癌の治療後、比較的早期（例えば、５年以内）に膵癌を再発する事が統計的有意差を持って示される。この点は、後述する実施例で具体的に示される。特定遺伝子とは、具体的には、ＡＭＡＰ１及びＥＰＢ４１Ｌ５から成る群から選択される少なくとも１つの遺伝子である。これら遺伝子の何れか１つの翻訳産物の高発現が見られる患者は統計的有意差を持って再発するリスクが高い。さらに、上記遺伝子の何れか２つの翻訳産物の高発現が見られる患者は、統計的有意差を持って再発するリスクがさらに高くなる。即ち、膵癌組織において上記遺伝子の翻訳産物の高発現が認められる場合、当該患者は膵癌の治療後に早期に癌を再発するリスクが高いと予測される。詳細は、後述する、膵癌の再発リスクを予測する方法において説明する。このように、本発明において「膵癌の再発リスクの予測」とは、膵組織における上記特定遺伝子の翻訳産物の高発現を検出することを前提としている。そのため、当該予測に用いるための本発明の診断薬は、具体的にはそれら遺伝子翻訳産物の検出に用いるための試薬であって、ＡＭＡＰ１及びＥＰＢ４１Ｌ５からなる群から選択されるいずれか１つの遺伝子の翻訳産物を特異的に認識可能なタンパク質及び／又はポリペプチドを含有するものである。
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.
＜遺伝子ＥＰＢ４１Ｌ５＞
遺伝子ＥＰＢ４１Ｌ５（略称ＥＢＬ５）（ＧｅｎｅＩＤ：５７６６９）は、癌浸潤におけるＡＭＡＰ１の結合パートナーであり、浸潤仮足形成に必須である。
<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 EPB41L5ファミリーの中でもEPB41L5に最も相同性があるのはEPB41L4bである。EPB41L4bには、band 4.1-like protein 4B isoform 1 [Homo sapiens](NP_060894.2)（配列番号3）とband 4.1-like protein 4B isoform 2 [Homo sapiens](NP_061987.3)（配列番号4）がある。isoform1は、short formである。図５にEPB41L5とEPB41L4bの一次構造比較を示す。図６にEPB41L4b isoform1とisoform2のゲノムの比較を示す。図７にEPB41L4b isoform1のアミノ酸配列とisoform2のアミノ酸配列の比較を示す。図８にEPB41L5(NP_065960.2)（配列番号2）とEPB41L4b(NP_061987.3)（配列番号4）についてアラインメント解析を示す。 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).
図８に示すように、EPB41L5(NP_065960.2)（配列番号2）とEPB41L4b(NP_061987.3)（配列番号4）についてアラインメント解析の結果、C末端側(EPB41L5 基準で541番目以降の配列)において両者配列は大きく異なっており、EPB41L5のC末端配列は当該タンパク 質に対する特異的な抗体を作製する上で有効な抗原になる。本発明においては、EPB41L5タンパク 質に対する特異的な抗体またはその断片は、EPB41L5（配列番号2）の541番目から733番目付近のアミノ酸配列領域にエピトープとなるアミノ酸配列を有する抗体またはその断片であることが適当である。 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.
＜遺伝子ＡＭＡＰ１＞
遺伝子ＡＭＡＰ１（ＧｅｎｅＩＤ：５０８０７）は、癌浸潤におけるＡｒｆ６の下流エフェクターである。図９にAMAP1のcds領域塩基配列（NCBI Reference Sequence: NM_018482.3）（配列番号5）を示す。図１０にAMAP1のアミノ酸配列（NCBI Reference Sequence: NP_060952.2)（配列番号6）を示す。図１１にAMAP1と相同性がある遺伝子（ArfGAP family）の情報を示す。図１２にAMAP1と相同性があるAMAP2遺伝子のcds領域塩基配列(NCBI Reference Sequence: NM_003887.2)（配列番号7）を示す。図１３にAMAP1と相同性があるAMAP2のアミノ酸配列(NCBI Reference Sequence: NP_003878.1)（配列番号8）を示す。図１４にAMAP1と相同性があるAMAP2のアライメント解析のデータを示す。
<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.
図１４に示すように、AMAP1（NCBI Reference Sequence: NP_060952.2)（配列番号6）とAMAP2 (NCBI Reference Sequence: NP_003878.1)（配列番号8）についてのアラインメント解析の結果、両者配列に大きく異なる領域がある。AMAP1のこの領域は、当該タンパク質に対する特異的な抗体を作製する上で有効な抗原になる。本発明においては、AMAP1タンパク質に対する特異的な抗体またはその断片は、AMAP1（配列番号6）の935番目から1002番目付近のアミノ酸配列領域にエピトープとなるアミノ酸配列を有する抗体またはその断片であることが適当である。 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.
上記遺伝子の翻訳産物を特異的に認識可能なタンパク質及び／又はポリペプチドとは、上記遺伝子のうち一の遺伝子の翻訳産物に特異的に結合し得るものであれば特に限定されるものでは無い。例えば、当該翻訳産物に対する抗体及び／又はその断片が挙げられる。抗体及び／又はその断片としては、ポリクローナル抗体、モノクローナル抗体、一本鎖抗体、ヒト化抗体等が挙げられるが、これらに限定されるものではない。上記遺伝子の翻訳産物を特異的に認識可能なタンパク質及びポリペプチドの調製方法については、遺伝子翻訳産物に対する特異的結合を保持し得る限り、どのような方法も採用し得る。例えば、当該遺伝子翻訳産物となるタンパク質及び／又はその断片を抗原として、公知の手法により所望のポリクローナル抗体、モノクローナル抗体、一本鎖抗体、ヒト化抗体等を調製することが出来る。 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.
尚、上記遺伝子の翻訳産物を特異的に認識可能なタンパク質及びポリペプチドが有する遺伝子翻訳産物に対する特異的結合性は、当該遺伝子の翻訳産物を、当該遺伝子の翻訳産物と共存可能性がある他の遺伝子の翻訳産物と識別できる程度であれば良い。従って、例えば、上記遺伝子に一部改変（置換、欠失、付加等）が加わっている遺伝子の翻訳産物を特異的に認識可能なタンパク質及びポリペプチドであっても、上記遺伝子の翻訳産物を、当該遺伝子の翻訳産物と共存可能性がある他の遺伝子の翻訳産物と識別できる程度の遺伝子翻訳産物に対する特異的結合性を有するものであれば、本発明の「遺伝子の翻訳産物を特異的に認識可能なタンパク質及び／又はポリペプチド」に含めることができる。 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 ".
さらに、本発明の診断薬においては、単一の診断薬に、上記遺伝子のうち１つの遺伝子の翻訳産物を特異的に認識可能なタンパク質又はポリペプチド１種のみが含まれていてもよいし、或いは上記遺伝子の翻訳産物それぞれをそれぞれ特異的に認識可能な２種以上のタンパク質及び／又はポリペプチドが含まれていてもよい。加えて、上記タンパク質及び／又はポリペプチドは、遺伝子翻訳産物の検出のために、標識物質で標識されていてもよい。標識物質としては、蛍光物質（例えば、ＧＦＰ等の蛍光タンパク質、並びにフルオロセイン等の蛍光低分子化合物を含む）等が挙げられる。但し、これら標識物質による標識を有する物に限定される意図ではない。本発明の診断薬において上記タンパク質及び／又はポリペプチドが標識されていない場合には、当該遺伝子の翻訳産物を公知の方法により検出することができる。そのような検出には、例えば、それらタンパク質及び／又はポリペプチド（一次抗体）に結合する二次抗体を用いたシステムを用いることができる。二次抗体を用いたシステムには市販の検出キットがある。このシステムでは、例えば、一次抗体に対する二次抗体がペルオキシダーゼで標識されており、試料中で目的遺伝子翻訳産物に特異的に結合した一次抗体にさらに上記二次抗体を特異的に結合させ、ペルオキシダーゼの発色基質（例えば、3,3'-ジアミノベンジジンテトラヒドロクロライド）を当該試料に添加して発色を観察することにより遺伝子翻訳産物の検出することが出来る。さらに、本発明において、１つの診断薬に、上記遺伝子の翻訳産物それぞれをそれぞれ特異的に認識可能な２以上のタンパク質及び／又はポリペプチドが含まれていている場合には、例えば、それら２以上のタンパク質及び／又はポリペプチドをそれぞれ異なる波長の蛍光を発する異種の蛍光物質で標識することができる。これにより、同一試料において目的の遺伝子翻訳産物それぞれを検出することが出来る。 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.
診断薬は、任意に、緩衝剤、防腐剤、凍結防止剤等を含み得る。緩衝剤としては、例えば、トリス塩酸塩、リン酸カリウム、リン酸ナトリウム等を挙げることかできる。防腐剤としては、例えば、アジ化ナトリウム等を挙げることができる。凍結防止剤としては、例えば、グリセロール等を挙げることができる。 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.
本発明の診断薬は、後述する膵癌の再発リスクを予測する方法の工程（Ａ）において、所定の遺伝子翻訳産物の発現レベルを決定するために当該遺伝子の翻訳産物検出に用いることができる。 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.
キット
本発明の別の態様によれば、膵癌の再発リスクの予測に用いるためのキットであって、上述の診断薬を含む、キットが提供される。
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.
本発明のキットは、後述する膵癌の再発リスクを予測する方法を実施する上で用い得る試薬及び部材等の要素をキットとして提供するものであり、上記診断薬を含むことを特徴とする。 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.
本発明のキットにおいては、本発明の診断薬は、（１）単一の診断薬において、ＡＭＡＰ１及びＥＰＢ４１Ｌ５のうち１つの遺伝子の翻訳産物を特異的に認識可能なタンパク質又はポリペプチド１種のみが含まれることにより１つの遺伝子の翻訳産物の検出が可能である診断薬であることができる。あるいは、本発明の診断薬は、（２）単一の診断薬においてそれら遺伝子の翻訳産物それぞれをそれぞれ特異的に認識可能な２種以上のタンパク質及び／又はポリペプチドが含まれることにより当該単一の診断薬が複数の上記遺伝子翻訳産物を検出可能である単一の診断薬であってもよい。さらに、本発明のキットにおいては、上記（１）の１つの遺伝子の翻訳産物の検出が可能である診断薬、及び／又は上記（２）の複数の遺伝子翻訳産物の検出が可能である単一の診断薬が含まれ得る。 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.
本発明のキットには、上記診断薬に加えて、ブロッキング試薬、診断薬に含まれるタンパク質及び／又はポリペプチド（抗体）の検出に用いるための標識化二次抗体、発色試薬、膵癌の再発リスクを予測する方法に関する実験プロトコルを記述した指示書等が任意に含まれ得る。ブロッキング試薬としては、例えば、牛血清アルブミン（ＢＳＡ）等を挙げることができる。 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.
予測方法
本発明の別の態様によれば、以下工程（Ａ）及び（Ｂ）を含む、膵癌の再発リスクを予測する方法が提供される。
（Ａ）膵癌の治療を受けた患者から採取した生体試料において、ＡＭＡＰ１及びＥＰＢ４１Ｌ５から成る群から選択される少なくとも１つの遺伝子の翻訳産物発現レベルを決定する工程、
（Ｂ）工程（Ａ）において決定した翻訳産物発現レベルに基づいて、上記患者の膵癌の再発リスクを予測する工程、
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),
本発明の方法において、「膵癌」、並びに「膵癌の治療」については、前記の通りである。本発明の方法において、患者とは、膵癌の治療を受けた患者である。 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.
本発明の方法において、「生体試料」とは、膵癌の治療を受けた患者から採取された任意の生体試料である。生体試料は、膵組織であることが再発リスクを精度良く予測し得るというという観点で好ましい。さらに、膵組織としては、例えば、膵癌の治療において腫瘍摘出術の際に患者から摘出した膵臓病変部位の組織、又は、病変部位が存在していた近傍の膵組織であって腫瘍摘出術の後に、摘出術とは別に採取した膵組織を挙げることができる。但し、患者への負担を極力軽減し、かつ再発リスクを精度良く予測し得るという観点から、膵組織は、腫瘍摘出術の際に患者から摘出した膵癌病変部位の組織であることが好ましい。尚、本発明では病理診断のために膵臓原発巣および転移巣の外科切除検体から作製されたパラフィン包埋組織を用いて上述の予測因子を検出可能であるため、病理診断が行われたすべて症例について検索が可能である。 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.
本発明の方法の工程（Ａ）においては、前記生体試料において、ＡＭＡＰ１及びＥＰＢ４１Ｌ５から成る群から選択される少なくとも１つの遺伝子の翻訳産物発現レベルを決定する。これら遺伝子は、Ａｒｆ６経路に属する遺伝子である。実施例で示すように、膵癌患者の膵組織においてＡｒｆ６経路に属する遺伝子が組織レベルで発現し、Ａｒｆ６経路の稼働が推定される場合には、当該患者は相対的に膵癌の治療後に早期再発するリスクが高い。従って、Ａｒｆ６経路に属する遺伝子のうちの少なくとも１つの遺伝子の高発現が確認された場合、膵癌の治療後の再発を促進するものと考えられるＡｒｆ経路の稼働が推定され、当該患者は早期に膵癌を再発する蓋然性が高いと判断し得る。 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.
本発明方法の工程（Ａ）において、ＡＭＡＰ１及びＥＰＢ４１Ｌ５から成る群から選択される少なくとも１つの遺伝子の翻訳産物発現レベルの決定は、上記生体試料に対して実施される限り、その方法については特に限定されるものでは無い。上記少なくとも１つの遺伝子の翻訳産物発現レベルの決定は、例えば、上記生体試料全体に対して実施してもよいし、或いは生体試料において特定種の細胞に対して実施してもよいし、或いは生体試料に存在する細胞において細胞質及び細胞核それぞれ別々に実施してもよい。上記少なくとも１つの遺伝子の翻訳産物発現レベルの決定が、生体試料に存在する細胞において細胞質及び細胞核それぞれ別々に実施された場合、決定した翻訳産物発現レベルの結果は、細胞質についての結果と細胞核についての結果の２種類が得られる。それら２種類の結果は、後述の工程（Ｂ）にそれぞれ用いることが出来る。 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.
本発明方法の工程（Ａ）において使用される、生体試料における上記所定の遺伝子の翻訳産物発現レベルを決定する方法は、特に限定されない。具体的には、本発明の診断薬について上記した如く、検出の対象となる遺伝子の翻訳産物を特異的に認識可能なタンパク質又はポリペプチド（診断薬）を用いて当該遺伝子の翻訳産物発現レベルを決定する方法を利用可能である。具体的には、生体試料である組織切片において遺伝子翻訳産物の発現強度及び発現分布を正確に把握して発現レベルを決定することが好ましい。特に、それらタンパク質及び／又はポリペプチド（診断薬）を用いた免疫組織化学法により当該遺伝子の翻訳産物発現レベルを決定することが、遺伝子翻訳産物の発現強度及び発現分布をより正確に把握できるという観点から好ましい。なお、免疫組織化学法については、各種公知の手法を用いることができる。本発明においては、それら公知の手法を用いても良いし、或いは新たに開発された手法を用いてもよい。免疫組織化学法等の翻訳産物発現レベル決定方法は、対象となる遺伝子翻訳産物の発現レベルを決定できるものである限り、特に限定されるものではない。 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.
さらに、本発明においては、遺伝子翻訳産物の特異的な検出を確保するために、上記タンパク質及び／又はポリペプチドは、検出の対象となる遺伝子翻訳産物に対する抗体及び／又はその断片であることが好ましい。この点は、本発明の診断薬についての説明で既述の通りである。 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.
本発明方法の工程（Ａ）において、本発明所定の遺伝子の翻訳産物の発現レベル決定には、生体試料において任意のタンパク質検出法で検出されたシグナル強度を基準とし、この基準との比較を用いることが出来る。基準に用いる生体試料の例としては、同一患者から採取した非癌性組織（特に、非癌性膵組織）を挙げることができ、この組織における本発明所定の遺伝子翻訳産物発現レベルを対照とすることができる。この場合、同一患者から採取した非癌性組織（特に、非癌性膵組織）における本発明所定の遺伝子翻訳産物発現シグナル強度と、対象生体試料における本発明所定の遺伝子翻訳産物発現シグナル強度を、同一のタンパク質検出法で検出し、比較するそれにより、本発明所定の少なくとも１つの遺伝子の翻訳産物発現レベルを決定することができる。さらに、「翻訳産物発現レベルの決定」においては、上記基準との比較において、対象生体試料における本発明所定の少なくとも１つの遺伝子の翻訳産物発現のシグナル強度が、例えば、「高い」、「同等」又は「低い」等のランク付けをすることもできる。或いは、例えば上記基準として用いるシグナル強度を基準として対象生体試料における本発明所定の少なくとも１つの遺伝子の翻訳産物発現のシグナル強度をスコア化することもできる。シグナル強度のスコア化は、例えば、基準として用いた試料における遺伝子翻訳産物のシグナル強度を０とし、そのシグナル強度と対象生体試料における遺伝子翻訳産物のシグナル強度を比較して、例えば、−１から３等の整数でスコア化することが出来る。遺伝子翻訳産物のシグナル強度のランク付けやスコア化は、適宜決定できる。 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.
本発明方法の工程（Ｂ）においては、工程（Ａ）において決定した翻訳産物発現レベルに基づいて、上記患者の膵癌の再発リスクを予測する。 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).
工程（Ｂ）において膵癌の再発リスクを予測するには、具体的には、工程（Ａ）に関し上記に説明した通り、上記基準との比較により決定した「高い」、「同等」又は「低い」等のランク、或いはスコア化した値に基づいて、膵癌治療後の再発リスクを予測することが出来る。より具体的には、対象生体試料における上記少なくとも１つの遺伝子の翻訳産物発現レベルが「高い」とのランク付けが得られた場合には、当該患者については、膵癌の再発リスクが相対的に高いと理解することができる。さらに、対象生体試料における上記いずれか２つの遺伝子の翻訳産物発現レベルが「高い」とのランク付けが得られた場合には、当該患者については、膵癌の再発リスクが相対的により高いと理解することができる。 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.
それに対して、対象生体試料における上記２つの遺伝子の翻訳産物発現レベルがいずれも「同等」又は「低い」とのランク付けが得られたされた場合には、当該患者については、膵癌の再発リスクが相対的に低いと理解することができる。翻訳産物発現レベルをスコア化した場合には、スコア自体から膵癌の再発リスクを理解することができる。或いは、予めスコアに再発リスクが「高い」、「低い」又は「中程度」等の判定を割り振っておき、得られたスコアから膵癌の再発リスクを「高い」、「低い」又は「中程度」等と理解することもできる。膵癌の再発リスクの予測は、「高い」、「低い」又は「中程度」のような結果を得るだけでなく、膵癌の治療後から膵癌を発症するリスクが５０〜９０％（例えば、５０％、６０％、７０％、８０％、９０％、９５％等）である期間がどの程度であるかを示す指標を含むこともできる。 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.
治療又は予防方法
さらに、本発明の別の態様によれば、膵癌の再発を治療又は予防する方法が提供される。具体的には、膵癌の再発を治療又は予防する方法とは、上記膵癌の再発リスクを予測する方法により得られた予測結果に基づいて、膵癌の再発を治療又は予防する方法である。
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.
具体的には、本方法は、抗癌剤を用いた化学療法、ホルモン療法、放射線療法、外科的処置、食事療法、生活指導等、任意の膵癌の再発を治療又は予防し得る手段を含む。本発明で用いる遺伝子の翻訳産物（バイオマーカー）は、癌の浸潤転移や治療抵抗性に関与するシグナル経路を形成する一群の蛋白質からなるものである。このシグナル経路を阻害すると、癌の浸潤転移などを著しく減じる事も本発明者は見いだしている。従って、本発明の予測方法を用いて、膵癌の再発が予測される患者について、上記シグナル経路を阻害することを含む治療方法を施して、膵癌の再発を治療又は予防し得る。 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.
本方法は、上記膵癌の再発リスクを予測する方法により得られた予測結果に基づいて、膵癌の再発を治療又は予防する限り特に限定されるものではなく、上記膵癌の再発リスクを予測する方法の方法工程を含んでもよいし、含まなくてもよい。 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.
以下の実施例により本発明をさらに具体的に説明するが、本発明は以下の実施例により特に限定されるものではない。 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.
＜材料及び方法＞
（１）患者集団
過去に北海道大学病院（日本国、札幌）にて膵癌摘出手術を受けた70人の患者を対象
にし、解析を行った。
<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）免疫組織化学（Immunohistochemistry；ＩＨＣ）
EPB41L5抗体、AMAP1抗体、GEP100抗体の作成方法は特開2015-21800号公報の段落0049〜0053にその詳細を記載している。
(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.
EPB41L5抗体は、ヒト蛋白質のアミノ酸558番目から733番目までを含むペプチドを抗原としウサギに免疫した後、標準的手法によって抗血清を採取、抗原にてアフィニティ精製したものを用いた。図１５にEPB41L5に対するポククローナル抗体の抗原部位（アミノ酸541番目から733番目までを含むペプチド(193アミノ酸)）を図示する。図１６に抗原をウサギに免疫し、抗血清を採取、抗体の精製を行う一連の操作のスキームを示す。得られたEPB41L5抗体のアフィニティー精製の結果を図１７及び１８に示す。 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.
AMAP1に対する抗体に関しては、AMAP1に対する抗体の抗原部位の情報、抗原部位のクローニング手順、及びに抗体の精製等に関する情報をまとめて図１９に示す。 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.
参考として、GEP100に対する抗体の抗原部位の情報、抗原部位のクローニング手順、及びに抗体の精製等に関する情報をまとめて図２０に示す。 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.
尚、AMAP1及びGEP100に対する抗体については上記公報発行以前にも報告がある（Onodera et al., 2006; Morishige et al., 2008）。 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).
免疫組織化学染色は、以下の通り、厚さ４μｍのホルマリン固定パラフィン包埋連続切
片を用いて実施した。全てのスライドは、最初にキシレンにおいて脱パラフィン処理を行
い、段階的なアルコールにおいて脱水処理を実施し、次いで、切片をトリス緩衝食塩水(T
BS: 25 mM Tris-HCl (pH 7.4), 137 mM NaCl, 2.7 mM KCl)でリンスした。
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).
その後、以下の条件により、各抗原について切片の抗原賦活化処理を行った。
（ｉ）EPB41L5（EBL5）：クエン酸緩衝液（ｐＨ６．０）で９５℃、４０分インキュベート；
（ｉｉ）AMAP1又はGEP100：抗原賦活化液（EDTA solution、ｐＨ９、Nichireiから購入）で９５℃、４０分インキュベート；
次いで、室温で０．３％Ｈ2Ｏ2／メタノールにインキュベートすることにより、内因性ペルオキシダーゼをブロックした。切片をTBSで洗浄した後、AMAP1（１：750）、GEP100（１：750）、又はEPB41L5（ＥＢＬ５；１：１０００）に対する一次抗体（括弧内の比率は希釈倍率）と共に、室温で３０分間、インキュベートした。
さらにＴＢＳで洗浄した後、二次抗体としてEnVision（商標）(DAKO、日本国)を用いて各一次抗体の検出を行い、ジアミノベンジジンを用いて発色させた。各切片は、ヘマトキシリンを用いて対比染色し、水洗した後、定常法に従い透徹処理・封入処理を行った。
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）評価
全ての試料は、盲検下で、Arf6経路の研究に精通した研究者がその染色度合いを判定した。腫瘍細胞の染色強度毎に2点（非癌性膵組織の染色度合いと比較して染色が非常に濃い場合）又は1点（非癌性膵組織の染色度合いと比較して高い場合）又は0点（非癌性膵組織の染色度合いと比較して同程度か低い場合）と点数付けした。また、それらの腫瘍細胞の組織全体に占める割合を0.0〜1.0（0％なら0, 10〜90％なら0.1〜0.9, 100%なら1.0）で表し、0×（0点の占める割合）＋1×（1点の占める割合）＋2×（2点の占める割合）をIHC scoreと定義した。患者毎にIHC scoreを計算し、中央値より高い患者を高発現群、低い患者を低発現群とした。
(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）統計分析
今回の解析では、Kaplan-Meier法により生存率を算定し、Log-rank法により有意差の検定を行った。多変量解析にはコックス比例ハザード回帰を用い、単変量解析にて有意差を認めた因子を独立変数として採用した。０．０５未満のｐ値は、統計的に有意な相関があると判断した。これらの解析は、EZR(Bone Marrow Transplant 2014;48:452-458)を用いて実施した。
(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).
＜結果＞
（１）患者におけるAMAP1、EPB41L5、GEP100の翻訳産物発現と臨床病理学的特徴との関係
今回対象となった患者の臨床病理学的特徴を表1に示す。
<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.
代表的な染色像を図１に示す。
70名の患者のmedian IHC scoreは、AMAP1(0.13)、EPB41L5(0.07)、GEP100（0.14）であった。
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).
（解析結果）
図２Ａ〜ＢはAMAP1、EPB41L5、GEP100の各遺伝子の翻訳産物が高発現の患者と低発現の患者について、全生存率と無再発生存率を示すKaplan-Meier曲線である。ＡＭＡＰ１、ＥＰＢ４１Ｌ５において、それが高発現の患者は低発現の患者と比べて有意に早期に再発し（それぞれＰ=0.0014、P=0.00768）、予後不良（それぞれP=0.0012、P=0.000127）であることがわかった。
(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.
また図２Ｃ〜Ｄに示すように、AMAP1、EPB41L5、GEP100のいずれか２つが高発現の場合は、もしくは３つ全てが高発現の場合は（図中「All-high」と表記、３つのうち少なくとも１つが低発現（図中「Others」と表記）の場合と比べてより顕著に早期再発および予後不良の傾向が認められた。 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.
これらの結果を臨床病理学的特徴と併せて単変量解析および多変量解析を施行すると、腫瘍径2cmより大きい、およびAMAP1・EPB41L5の高発現が独立した予後不良因子であることがわかった（表２）。 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).
本発明は、癌の診断および治療に関連する分野に有用である。 The present invention is useful in the fields related to cancer diagnosis and treatment.
配列番号１：EPB41L5遺伝子の塩基配列
配列番号２：EPB41L5のアミノ酸配列
配列番号３：band 4.1-like protein 4B isoform 1の塩基配列
配列番号４：band 4.1-like protein 4B isoform 2の塩基配列
配列番号５：AMAP1遺伝子のcds領域塩基配列
配列番号６： AMAP1のアミノ酸配列
配列番号７：AMAP2遺伝子のcds領域塩基配列
配列番号８：AMAP2のアミノ酸配列
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)
ＡＭＡＰ１及びＥＰＢ４１Ｌ５からなる群から選択されるいずれか1つの遺伝子の翻訳産物を特異的に認識可能なタンパク質及び／又はポリペプチドを含有する、診断薬。 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.
を含む、膵癌の再発リスクを予測する方法。 (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.
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