WO2014185466A1 - Prognosis evaluation method for pancreatic cancer - Google Patents

Prognosis evaluation method for pancreatic cancer Download PDF

Info

Publication number
WO2014185466A1
WO2014185466A1 PCT/JP2014/062875 JP2014062875W WO2014185466A1 WO 2014185466 A1 WO2014185466 A1 WO 2014185466A1 JP 2014062875 W JP2014062875 W JP 2014062875W WO 2014185466 A1 WO2014185466 A1 WO 2014185466A1
Authority
WO
WIPO (PCT)
Prior art keywords
ebi3
pancreatic cancer
prognosis
expression level
patient
Prior art date
Application number
PCT/JP2014/062875
Other languages
French (fr)
Japanese (ja)
Inventor
伸介 平岡
義典 石川
理恵 山崎
Original Assignee
独立行政法人国立がん研究センター
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 独立行政法人国立がん研究センター filed Critical 独立行政法人国立がん研究センター
Publication of WO2014185466A1 publication Critical patent/WO2014185466A1/en

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a prognostic evaluation method for pancreatic cancer that predicts the prognosis of pancreatic cancer.
  • Pancreatic cancer is one of the intractable cancers whose number of onsets and deaths hardly change. That is, pancreatic cancer is difficult to diagnose and treat and has the poorest prognosis among digestive malignant tumors. Therefore, in order to improve the prognosis of pancreatic cancer, it is important to establish an accurate treatment policy after evaluating the effectiveness of not only early diagnosis but also surgery and chemotherapy. Many markers for predicting the prognosis of cancer have been reported so far, but few markers for specifically predicting the prognosis of pancreatic cancer have been reported yet. The current situation is to wait for a determination from. Therefore, the development of biomarkers that enable appropriate selection of treatments for individual pancreatic cancer patients is urgently desired along with markers for early diagnosis.
  • EBI3 Epstein-Barr virus induced gene 3
  • EBI3 is a gene that was first expressed and attracted attention in a B cell line transformed in vitro with Epstein Barr virus (EBV) (Non-patent Document 1).
  • EBI3 is a component of IL-27 formed by heterodimerization with p28, a p35-related subunit of IL-12 (Non-patent Document 2).
  • EBI3 is a component of IL-35 formed by heterodimerization with IL-12 p35 (Non-patent Document 3).
  • IL-27 is thought to play an important role in initiating the Th1 immune response required for the immune response induced by IFN- ⁇ , while suppressing the differentiation of CD4 + T cells into Th2, Th17, iTreg, and IL Induces Tr1 differentiation of suppressor T cells producing -10 (Non-patent Document 4).
  • Non-Patent Document 5 the level of EBI3 increases in the blood of lung cancer patients, and the level of EBI3 gene expression in blood samples obtained from subjects (Patent Document 1) and the like have been reported that the subject can diagnose lung cancer and determine the risk of developing lung cancer.
  • the present invention identifies a marker useful for evaluating the prognosis of pancreatic cancer, evaluates the prognosis of pancreatic cancer using the marker, and evaluates the prognosis of pancreatic cancer using the marker It is related with providing the inspection kit for.
  • the present inventors used three cohorts of surgically resected cases of pancreatic cancer, and analyzed the transcription level, IL-12 family and IL-12 receptor family genes expressed in pancreatic cancer tissue.
  • the expression at the protein level was measured and analyzed (see FIG. 1)
  • the case group with high EBI3 gene expression showed a significantly longer survival time than the case group with low EBI3 gene expression, and EBI3 infiltrating the cancer tissue
  • the case group with a large number of expressed cells showed significantly longer survival than the case group with a small number of expressed cells, and found that the EBI3 gene and EBI3 can be significant markers for predicting the prognosis of pancreatic cancer.
  • the present invention relates to the following (1) to (6).
  • a method for evaluating the prognosis of pancreatic cancer comprising measuring the expression level of EBI3 in a biological sample isolated from a patient with pancreatic cancer.
  • the method according to (1) comprising the following steps a and b. a) measuring the expression level of EBI3 in a biological sample isolated from a patient with pancreatic cancer b) comparing the expression level of EBI3 with a control level (3) the expression level of EBI3 increases above the threshold level
  • the prognosis can be predicted by evaluating the pancreatic cancer tissue excised by surgery, for example, by the method of the present invention. It is also possible to monitor the status and progression of pancreatic cancer.
  • numerator (marker).
  • the graph on the left shows the relationship with survival rate (OS), and the graph on the right shows the relationship with recurrence-free survival (DFS).
  • the graph on the left shows the relationship with survival rate (OS), and the graph on the right shows the relationship with recurrence-free survival (DFS).
  • pancreatic cancer means a malignant tumor arising from the pancreas.
  • prognosis means a decrease or decrease in tumor volume due to surgical treatment or the like, or a course or ending after pancreatic cancer is reduced (eg, recurrence / tumor growth, life / death, etc.). To do. For example, life and death at a point in years or more after surgically complete tumor resection.
  • “good prognosis” means that the condition after attenuation or reduction of tumor growth due to surgical treatment or the like of a patient is not lethal for a long period of time (for example, 2-5 years). And cases of survival, non-metastasis, non-regression, non-recurrence, etc. That is, a state with a good prognosis includes a state in which even if some kind of disease such as recurrence / metastasis has been confirmed, its malignancy is low and it can survive.
  • a gene is used to include not only double-stranded DNA but also each single-stranded DNA such as sense strand and antisense strand constituting the DNA.
  • the length is not particularly limited. Therefore, in this specification, unless otherwise specified, a gene (DNA) is a double-stranded DNA containing human genomic DNA and a single-stranded DNA containing DNA (positive strand) and a sequence having a sequence complementary to the positive strand. Both double-stranded DNA (complementary strand) and fragments thereof are included.
  • the method for evaluating the prognosis of pancreatic cancer measures the expression level of EBI3 in a biological sample isolated from a patient with pancreatic cancer.
  • EBI3 expression of EBI3 gene and expression of EBI3 protein.
  • the EBI3 gene (Epstein-Barr virus induced gene 3) is a known gene registered as GenBank accession number: NM_005755.
  • the “EBI3 gene” includes not only the gene represented by the nucleotide sequence (SEQ ID NO: 1) but also a protein encoded by these (referred to as “EBI3” (SEQ ID NO: 2)) and biological activity. Genes encoding proteins for which are equivalent.
  • a DNA comprising a base sequence that hybridizes with a complementary sequence of the base sequence represented by SEQ ID NO: 1 under stringent conditions is included.
  • the stringent conditions are usually, for example, “1 ⁇ SSC, 0.1% SDS, 37 ° C.”, “0.5 ⁇ SSC, 0.1% SDS, 42 ° C. as washing conditions after hybridization. And “0.1 ⁇ SSC, 0.1% SDS, 65 ° C.”.
  • a complementary strand a strand consisting of a base sequence that is completely complementary to the target positive strand base sequence, and 90% or more, preferably 95% or more identity with the strand A chain consisting of a base sequence having can be exemplified.
  • the identity of the base sequence is calculated by, for example, the Lippmann-Pearson method (Lipman-Pearson method; Science, 227, 1435, (1985)), and genetic information processing software Genetyx-Win (Ver. 5.1.1). It can be calculated by analyzing the unit size to compare (ktup) as 2 using the software development) homology analysis (Search homology) program.
  • the biological sample used in the present invention may be a biological specimen isolated from a pancreatic cancer patient to be evaluated for prognosis, and is a patient's pancreatic cancer tissue.
  • the biological sample may be the sample separated and collected from the patient as it is, or may be pretreated appropriately for various measurements.
  • a paraffin section prepared from a specimen obtained from a patient can be used.
  • a protein extract or mRNA extract prepared from a specimen obtained from a patient can be used.
  • the method for measuring the expression level of EBI3 is not particularly limited as long as it can confirm EBI3 in a biological sample. That is, any method capable of detecting or quantifying the expression level of EBI3 may be used, and the expression level of EBI3 may be determined relative or absolutely. EBI3 expression may be detected or quantified at the protein level or may be detected or quantified at the nucleic acid (mRNA) level.
  • mRNA nucleic acid
  • an immunological measurement method using an antibody that specifically binds to EBI3 is simple and preferable.
  • the immunological measurement method include enzyme immunoassay (ELISA), radioimmunoassay, immunohistochemical staining, Western blot, immunoprecipitation, immunofluorescence, flow cytometry, and the like.
  • detection or quantification at the nucleic acid level include RT-PCR (preferably real-time RT-PCR), Northern blotting method, Branched DNA assay, in situ hybridization method and the like.
  • the antibody that specifically binds to EBI3 may be of any structure, size, and immunoglobulin class as long as it binds to EBI3, and may be monoclonal or polyclonal, labeled antibody, chimeric antibody It may be a humanized antibody. Moreover, it is also possible to use a part of an antibody (partial fragment) or a peptide containing a part of an antibody, and an antibody fragment that retains the binding action of an antibody to an antigen (EBI3). Examples of such antibody fragments include F (ab ') 2, Fab', Fab, single chain Fv (scFv) and the like.
  • Anti-EBI3 antibodies can be obtained by methods well known to those skilled in the art. For example, an expression vector for mammalian cells into which a polypeptide having all or part of EBI3 or a polynucleotide encoding the same is prepared and used as an antigen. After immunization of the animal with this antigen, immune cells obtained from the immunized animal and myeloma cells are fused to obtain a hybridoma, and an antibody is collected from the hybridoma culture. A monoclonal antibody against EBI3 can be obtained by subjecting the last collected antibody to antigen-specific purification using a polypeptide corresponding to EBI3 used as an antigen or a part thereof.
  • an animal is immunized with the same antigen as described above, blood is collected from the immunized animal, serum is separated from the blood, and antigen-specific purification using the antigen is performed on the serum. Can be obtained.
  • Antibody fragments can be obtained by enzymatic treatment of the obtained antibodies or using sequence information of the obtained antibodies.
  • the antibody can be labeled by a general method in this field, or can be labeled using a commercially available labeling kit.
  • the labeled antibody or a fragment thereof can be detected by using a device suitable for labeling as appropriate.
  • immunohistochemical staining method a known method can be adopted. For example, a biological sample separated from a patient is fixed in formalin by a conventional method, embedded in paraffin, sliced into tissue pieces, and pasted on a slide glass, and used as a slice sample. According to the characteristics of the antibody, the sectioned sample is appropriately activated by heat treatment with an autoclave or the like, and immunohistologically stained using Vector ABC method or DAKO EnVision detection system. Under the microscope, positive / negative can be determined by color development of EBI3.
  • nucleic acids are amplified by a known method using PCR or the like using appropriately designed primers. Examples of such methods include ARMS (Amplification Refractory Mutation System) method, RT-PCR (Reverse transcriptase-PCR) method and Nested PCR method.
  • the amplified nucleic acid is obtained by using a dot-blot hybridization method, surface plasmon resonance method (SPR method), PCR-RFLP method, In situ RT-PCR method, PCR using a nucleic acid complementary to the nucleic acid.
  • SSO sequence specific oligonucleotide
  • PCR-SSP sequence specific oligonucleotide
  • AMPFLP Amplifiable fragment length polymorphism
  • MVR-PCR MVR-PCR
  • PCRSSCP single strand conformation polymorphism
  • the expression level of EBI3 in the biological sample separated from the pancreatic cancer patient is measured, and the prognosis of the patient is evaluated based on the expression level of EBI3. Specifically, it is assessed by comparing the detected expression level of EBI3 with a control level.
  • the “control level” means, for example, a group of patients with a good prognosis (patient cancer patients whose prognosis was not poor, pancreatic cancer patients who are believed to have a poor prognosis), or pancreatic cancer.
  • the expression level of EBI3 in a group of healthy individuals who do not develop, or the expression level of EBI3 in a group of patients with a poor prognosis (a patient with a poor prognosis, a patient with a known prognosis).
  • the expression level of the target patient's EBI3 is close to, within the range of the expression level or higher than the expression level of the EBI3 in a group of healthy patients or a group of healthy subjects, Prognosis can be evaluated as good.
  • the expression level of EBI3 in a patient to be compared can be known, for example, by measuring the expression level of EBI3 in a sample derived from a patient whose prognosis is already known by the method of the present invention.
  • the prognosis evaluation in the present invention can also be performed by increasing / decreasing the expression level of EBI3.
  • the median value of the expression level of EBI3 gene or EBI3 is set to a threshold level (cutoff value), and the expression level of EBI3 in a patient-derived biological sample is compared with the threshold level.
  • the increase / decrease in the expression level of EBI3 in pancreatic cancer tissue can be used as an index by increasing / decreasing the number of EBI3-expressing cells infiltrating the pancreatic cancer tissue or expanding / reducing the expression region.
  • the threshold level is the sum of the number of EBI3-expressing cells in five fields of the objective lens ⁇ 20 (for example, three times) (Average) is 2 cells or more.
  • the test kit for evaluating the prognosis of pancreatic cancer of the present invention contains an antibody that specifically binds to EBI3, or an oligonucleotide that specifically binds to EBI3 mRNA or the EBI3 gene. That is, the test kit contains a test reagent for measuring the expression level of EBI3 in a biological sample separated from a patient. Specifically, including an antibody that specifically binds to EBI3 (anti-EBI3 antibody), ELISA, Western blot, immunochromatography, reagents for immunohistochemistry, or an oligonucleotide that specifically binds to EBI3 mRNA or EBI3 gene RT-PCR reagents and the like.
  • the nucleic acid specifically binding to the anti-EBI3 antibody, EBI3 gene or mRNA included in the kit can be obtained by a known method as described above.
  • the test kit can contain a labeling reagent, a buffer solution, a chromogenic substrate, a secondary antibody, a blocking agent, instruments and controls necessary for the test, and the like.
  • Example 1 Correlation between EBI3 gene expression and patient prognosis (1) Measurement of EBI3 gene expression level Extracting total RNA from surgically excised pancreatic cancer fresh tissue, and then using single-strand cDNA obtained by reverse transcription reaction as a template Then, using the following PCR primers, the gene was amplified using the quantitative RT-PCR method, and the expression level of the EBI3 gene was measured. Quantitative RT-PCR was performed with the Universal ProbeLibrary system of Roche Applied Science, and the expression level of ACTB gene was measured and compared as a control.
  • EBI3 5 'primer, gAAgTACTggATCCgTTACAAgC (SEQ ID NO: 3) 3 'primer, gAAggACgTggCTTCAATg (SEQ ID NO: 4) probe # 24 ACTB: 5 'primer, CCAACCgCgAgAAgATgA (SEQ ID NO: 5) 3 'primer, TCCATCACgATgCCAgTg (SEQ ID NO: 6) probe # 64 PCR conditions: 95 ° C for 10 seconds, [95 ° C for 15 seconds, 60 ° C for 1 minute] x 40 cycles
  • Tables 1 and 2 show the results of univariate and multivariate analysis using the Cox proportional hazard model.
  • EBI3 gene expression in pancreatic cancer tissue is considered to be an independent prognostic factor.
  • Example 2 Correlation between EBI3 protein expression and patient prognosis (1) Production of monoclonal antibodies against EBI3 Full-length human EBI3 is expressed in mammalian cells and Escherichia coli, purified EBI3 is immunized to mice, and hybridomas are prepared by conventional methods. Then, ELISA, Western blot, monoclonal antibody specific to EBI3 (clone E32-8, IgG1 K; clone E25-2, IgG1 K) using immunohistochemistry on formalin-fixed paraffin-embedded lymph node tissue as a screening method Established.
  • Tables 3 and 4 show the results of univariate and multivariate analysis using the Cox proportional hazard model.
  • the number of EBI3-expressing cells that infiltrate tumors in pancreatic cancer tissue is considered to be an independent prognostic factor.
  • the prognosis can be predicted and an appropriate treatment policy can be established.
  • an aggressive treatment strategy for example, intensive chemotherapy or radiation therapy
  • the need to select an aggressive treatment strategy for example, intensive chemotherapy or radiation therapy
  • the need to select an aggressive treatment strategy for example, intensive chemotherapy or radiation therapy
  • a more relaxed treatment strategy can be selected.
  • the method of the present invention can also be used to monitor the state or progression of pancreatic cancer. That is, EBI3 expression levels can be used to assess changes in the likelihood that a patient will die, recur, or metastasize. For example, by measuring the EBI3 expression level over time, changes in the prognosis of the patient can be known.

Abstract

Provided are a method for identifying markers useful in evaluating the prognosis of pancreatic cancer and for predicting the prognosis of pancreatic cancer using said markers, and a test kit for predicting the prognosis of pancreatic cancer using said markers. A method for evaluating the prognosis of pancreatic cancer which is characterized in that the expression level of EBI3 in a biological sample isolated from a pancreatic cancer patient is measured.

Description

膵がんの予後評価方法Prognostic evaluation method for pancreatic cancer
　本発明は、膵がんの予後を予測する、膵がんの予後評価方法に関する。 The present invention relates to a prognostic evaluation method for pancreatic cancer that predicts the prognosis of pancreatic cancer.
　膵がんは、その発症数と死亡数が殆ど変わらない難治性がんの一つである。すなわち、膵がんは、診断、治療ともに困難で、消化器系悪性腫瘍の中で最も予後が不良である。したがって、膵がんの予後を改善するためには、早期診断のみならず、手術や化学療法などの有効性を評価した上での的確な治療方針の確立が重要である。
　がんの予後を予測するマーカーは、これまでに多くの報告が成されているが、膵がんの予後を特異的に予測するマーカーは未だ殆ど報告されておらず、その予後は生死の結果からの判定を待つのが現状である。したがって、個々の膵がん患者に対する治療法の適切な選択を可能とするバイオマーカーの開発は、早期診断のためのマーカーと共に早急に望まれている。 Pancreatic cancer is one of the intractable cancers whose number of onsets and deaths hardly change. That is, pancreatic cancer is difficult to diagnose and treat and has the poorest prognosis among digestive malignant tumors. Therefore, in order to improve the prognosis of pancreatic cancer, it is important to establish an accurate treatment policy after evaluating the effectiveness of not only early diagnosis but also surgery and chemotherapy.
Many markers for predicting the prognosis of cancer have been reported so far, but few markers for specifically predicting the prognosis of pancreatic cancer have been reported yet. The current situation is to wait for a determination from. Therefore, the development of biomarkers that enable appropriate selection of treatments for individual pancreatic cancer patients is urgently desired along with markers for early diagnosis.
　一方、ＥＢＩ３遺伝子（Epstein-Barr virus induced gene 3）は、ｉｎ　ｖｉｔｒｏでＥｐｓｔｅｉｎ　Ｂａｒｒウイルス（ＥＢＶ）により形質転換されたＢ細胞株において初めて発現され注目された遺伝子である（非特許文献１）。ＥＢＩ３は、ＩＬ－１２のｐ３５関連サブユニットであるｐ２８とヘテロ二量化することにより形成される、ＩＬ－２７の構成要素である（非特許文献２）。またＥＢＩ３は、ＩＬ－１２　ｐ３５とヘテロ二量化することにより形成される、ＩＬ－３５の構成要素である(非特許文献３)。ＩＬ－２７は、ＩＦＮ－γによって誘導される免疫反応に必要なＴｈ１免疫反応開始において重要な役割を果たすと考えられ、一方でＣＤ４＋Ｔ細胞のＴｈ２，Ｔｈ１７，ｉＴｒｅｇへの細胞分化を抑制し、ＩＬ－１０を産生する抑制性Ｔ細胞のＴｒ１分化を誘導する(非特許文献４)。 On the other hand, the EBI3 gene (Epstein-Barr virus induced gene 3) is a gene that was first expressed and attracted attention in a B cell line transformed in vitro with Epstein Barr virus (EBV) (Non-patent Document 1). EBI3 is a component of IL-27 formed by heterodimerization with p28, a p35-related subunit of IL-12 (Non-patent Document 2). EBI3 is a component of IL-35 formed by heterodimerization with IL-12 p35 (Non-patent Document 3). IL-27 is thought to play an important role in initiating the Th1 immune response required for the immune response induced by IFN-γ, while suppressing the differentiation of CD4 + T cells into Th2, Th17, iTreg, and IL Induces Tr1 differentiation of suppressor T cells producing -10 (Non-patent Document 4).
　近年、ヒト造血器腫瘍において、ＥＢＩ３遺伝子が過剰発現されていること（非特許文献５）、肺癌患者の血液においてＥＢＩ３のレベルが上昇し、被験体から得た血液試料においてＥＢＩ３遺伝子の発現のレベルの上昇により、被験体が肺癌の診断や肺癌を発症する危険性を判定できることが（特許文献１）等が報告されている。 In recent years, the EBI3 gene is overexpressed in human hematopoietic tumors (Non-Patent Document 5), the level of EBI3 increases in the blood of lung cancer patients, and the level of EBI3 gene expression in blood samples obtained from subjects (Patent Document 1) and the like have been reported that the subject can diagnose lung cancer and determine the risk of developing lung cancer.
　しかしながら、ＥＢＩ３の発現と膵がんとの関係はこれまでに全く知られていない。 However, the relationship between EBI3 expression and pancreatic cancer has never been known.
特表２０１０－５３６３６６号公報Special table 2010-536366
　本発明は、膵がんの予後を評価するために有用なマーカーを特定し、当該マーカーを用いた膵がんの予後を評価する方法、及び当該マーカーを用いて膵がんの予後を評価するための検査キットを提供することに関する。 The present invention identifies a marker useful for evaluating the prognosis of pancreatic cancer, evaluates the prognosis of pancreatic cancer using the marker, and evaluates the prognosis of pancreatic cancer using the marker It is related with providing the inspection kit for.
　本発明者らは上記課題を解決するために、膵がん外科切除症例の３つのコホートを用いて、膵がん組織に発現するＩＬ－１２ファミリー・ＩＬ－１２リセプターファミリー遺伝子について、転写レベル・蛋白質レベルでの発現を測定し、解析したところ（図１参照）、ＥＢＩ３遺伝子発現の高い症例群は低い症例群に比べて有意に長い生存期間を示すこと、また、がん組織に浸潤するＥＢＩ３発現細胞数の多い症例群が少ない症例群に比して有意に長い生存を示すことを発見し、ＥＢＩ３遺伝子及びＥＢＩ３が膵がんの予後予測のために有意なマーカーとなり得ることを見出した。 In order to solve the above-mentioned problems, the present inventors used three cohorts of surgically resected cases of pancreatic cancer, and analyzed the transcription level, IL-12 family and IL-12 receptor family genes expressed in pancreatic cancer tissue. When the expression at the protein level was measured and analyzed (see FIG. 1), the case group with high EBI3 gene expression showed a significantly longer survival time than the case group with low EBI3 gene expression, and EBI3 infiltrating the cancer tissue It was discovered that the case group with a large number of expressed cells showed significantly longer survival than the case group with a small number of expressed cells, and found that the EBI3 gene and EBI3 can be significant markers for predicting the prognosis of pancreatic cancer.
　すなわち、本発明は、以下の（１）～（６）に係るものである。
（１）膵がん患者から分離された生体試料中のＥＢＩ３の発現レベルを測定することを特徴とする、膵がんの予後を評価する方法。
（２）以下のａ及びｂの工程を含む、（１）に記載の方法。
　ａ）膵がん患者から分離された生体試料中のＥＢＩ３の発現レベルを測定する工程
　ｂ）ＥＢＩ３の発現レベルを対照レベルと比較する工程
（３）ＥＢＩ３の発現レベルが閾値レベルよりも増加している場合に、予後良好であることを示す（１）又は（２）に記載の方法。
（４）生体試料中のＥＢＩ３の発現レベルが、膵がん組織におけるＥＢＩ３遺伝子の発現量である、（１）～（３）のいずれかに記載の方法。
（５）生体試料中のＥＢＩ３の発現レベルが、膵がん組織に浸潤するＥＢＩ３発現細胞数である、（１）～（３）のいずれかに記載の方法。
（６）ＥＢＩ３と特異的に結合する抗体、又はＥＢＩ３ｍＲＮＡ若しくはＥＢＩ３遺伝子と特異的に結合するオリゴヌクレオチドを含有する（１）～（５）の方法に用いる膵がんの予後を評価するための検査用キット。 That is, the present invention relates to the following (1) to (6).
(1) A method for evaluating the prognosis of pancreatic cancer, comprising measuring the expression level of EBI3 in a biological sample isolated from a patient with pancreatic cancer.
(2) The method according to (1), comprising the following steps a and b.
a) measuring the expression level of EBI3 in a biological sample isolated from a patient with pancreatic cancer b) comparing the expression level of EBI3 with a control level (3) the expression level of EBI3 increases above the threshold level The method as described in (1) or (2) which shows that prognosis is favorable when it exists.
(4) The method according to any one of (1) to (3), wherein the expression level of EBI3 in the biological sample is the expression level of the EBI3 gene in pancreatic cancer tissue.
(5) The method according to any one of (1) to (3), wherein the expression level of EBI3 in the biological sample is the number of EBI3-expressing cells infiltrating the pancreatic cancer tissue.
(6) A test for evaluating the prognosis of pancreatic cancer used in the methods (1) to (5), which comprises an antibody that specifically binds to EBI3, or an oligonucleotide that specifically binds to EBI3 mRNA or EBI3 gene. For kit.
　本発明の方法によれば、膵がん患者の予後を予測することが可能になる。すなわち、膵がんと診断された患者について、例えば手術等で切除された膵がん組織を、本発明の方法により評価することで予後を予測することができ、これにより、適切な治療方針を立てることが可能になり、また膵がんの状態や進行をモニターすることもできる。 According to the method of the present invention, it becomes possible to predict the prognosis of pancreatic cancer patients. That is, for patients diagnosed with pancreatic cancer, the prognosis can be predicted by evaluating the pancreatic cancer tissue excised by surgery, for example, by the method of the present invention. It is also possible to monitor the status and progression of pancreatic cancer.
目的遺伝子・分子（マーカー）を同定するための手順を示した模式図。The schematic diagram which showed the procedure for identifying the target gene and molecule | numerator (marker). コホート1を用いたKaplan-Meier生存曲線。左のグラフは生存率(OS)との関係、右のグラフは無再発生存率(DFS)との関係を示したもの。Kaplan-Meier survival curve using cohort 1. The graph on the left shows the relationship with survival rate (OS), and the graph on the right shows the relationship with recurrence-free survival (DFS). (A) リンパ節の免疫組織化学像。茶色：EBI3発現細胞、(B)リンパ節の免疫組織化学二重染色像。赤紫色：EBI3発現細胞、茶色：CD208、倍率：×１００(A) Immunohistochemical image of lymph node. Brown: EBI3-expressing cells, (B) Immunohistochemical double-stained image of lymph node. Magenta: EBI3-expressing cells, brown: CD208, magnification: x100 コホート3を用いたKaplan-Meier生存曲線。左のグラフは生存率(OS)との関係、右のグラフは無再発生存率(DFS)との関係を示したもの。Kaplan-Meier survival curve using cohort 3. The graph on the left shows the relationship with survival rate (OS), and the graph on the right shows the relationship with recurrence-free survival (DFS).
　本発明において、「膵がん」は、膵臓から発生した悪性腫瘍を意味する。 In the present invention, “pancreatic cancer” means a malignant tumor arising from the pancreas.
　本発明において、「予後」とは、膵がんが外科的治療等による腫瘍量の減弱、腫瘍増殖の抑制、軽減後に辿る経過又は結末（例えば、再発・腫瘍増殖の有無、生死等）を意味する。例えば、外科的に腫瘍完全切除後何年或いはそれ以上の時点における生死が挙げられる。
　本発明において、「予後良好」とは、患者の外科的治療等による腫瘍量の減弱、腫瘍増殖の抑制又は軽減後の容態が長期間（例えば、２-５年間）致命的とならないことを意味し、生存、非転移、非回帰、又は、非再発等の場合が挙げられる。すなわち、予後良好の状態としては、再発・転移等何らかの疾患が確認されていてもその悪性度が低く、生存可能な状態も包含される。 In the present invention, “prognosis” means a decrease or decrease in tumor volume due to surgical treatment or the like, or a course or ending after pancreatic cancer is reduced (eg, recurrence / tumor growth, life / death, etc.). To do. For example, life and death at a point in years or more after surgically complete tumor resection.
In the present invention, “good prognosis” means that the condition after attenuation or reduction of tumor growth due to surgical treatment or the like of a patient is not lethal for a long period of time (for example, 2-5 years). And cases of survival, non-metastasis, non-regression, non-recurrence, etc. That is, a state with a good prognosis includes a state in which even if some kind of disease such as recurrence / metastasis has been confirmed, its malignancy is low and it can survive.
　本明細書において、「遺伝子」とは、２本鎖ＤＮＡのみならず、それを構成するセンス鎖およびアンチセンス鎖といった各１本鎖ＤＮＡを包含する趣旨で用いられる。またその長さによって特に制限されるものではない。従って、本明細書において遺伝子(ＤＮＡ)とは、特に言及しない限り、ヒトゲノムＤＮＡを含む２本鎖ＤＮＡおよびｃＤＮＡを含む１本鎖ＤＮＡ（正鎖）並びに該正鎖と相補的な配列を有する１本鎖ＤＮＡ（相補鎖）、およびこれらの断片のいずれもが含まれる。 In the present specification, “gene” is used to include not only double-stranded DNA but also each single-stranded DNA such as sense strand and antisense strand constituting the DNA. The length is not particularly limited. Therefore, in this specification, unless otherwise specified, a gene (DNA) is a double-stranded DNA containing human genomic DNA and a single-stranded DNA containing DNA (positive strand) and a sequence having a sequence complementary to the positive strand. Both double-stranded DNA (complementary strand) and fragments thereof are included.
　本発明の膵がんの予後を評価する方法は、膵がん患者から分離された生体試料中のＥＢＩ３の発現レベルを測定するものである。 The method for evaluating the prognosis of pancreatic cancer according to the present invention measures the expression level of EBI3 in a biological sample isolated from a patient with pancreatic cancer.
　本発明において、「ＥＢＩ３の発現」とは、ＥＢＩ３遺伝子の発現とＥＢＩ３タンパク質の発現の両者を意味する。
　ＥＢＩ３遺伝子（Epstein-Barr virus induced gene 3）は、ＧｅｎＢａｎｋアクセッション番号：ＮＭ＿００５７５５として登録されている公知の遺伝子である。本発明において、「ＥＢＩ３遺伝子」には、当該塩基配列（配列番号１）で示される遺伝子だけでなく、これらによりコードされるタンパク質（「ＥＢＩ３」（配列番号２）と称する）と生物学的活性が等価であるタンパク質をコードする遺伝子が包含される。
　具体的には、配列番号１で表されるＥＢＩ３遺伝子の他に、配列番号１で示される塩基配列の相補配列とストリンジェントな条件下でハイブリダイズする塩基配列からなるＤＮＡが包含される。
　なお、ここでストリンジェントな条件は、例えばハイブリダイズ後の洗浄条件として、通常「１×ＳＳＣ、０．１％ＳＤＳ、３７℃」、「０．５×ＳＳＣ、０．１％ＳＤＳ、４２℃」、「０．１×ＳＳＣ、０．１％ＳＤＳ、６５℃」等を挙げることができる。具体的には、このような相補鎖として、対象の正鎖の塩基配列と完全に相補的な関係にある塩基配列からなる鎖、並びに該鎖と９０％以上、好ましくは９５％以上の同一性を有する塩基配列からなる鎖を例示することができる。
　ここで、塩基配列の同一性は、例えばリップマン－パーソン法（Ｌｉｐｍａｎ－Ｐｅａｒｓｏｎ法；Science, 227, 1435, （1985）)によって計算され、遺伝情報処理ソフトウェアＧｅｎｅｔｙｘ－Ｗｉｎ（Ｖｅｒ.５．１．１；ソフトウェア開発）のホモロジー解析（Ｓｅａｒｃｈ　ｈｏｍｏｌｏｇｙ）プログラムを用いて、Ｕｎｉｔ　ｓｉｚｅ　ｔｏ　ｃｏｍｐａｒｅ（ｋｔｕｐ）を２として解析を行なうことにより算出できる。 In the present invention, “expression of EBI3” means both expression of EBI3 gene and expression of EBI3 protein.
The EBI3 gene (Epstein-Barr virus induced gene 3) is a known gene registered as GenBank accession number: NM_005755. In the present invention, the “EBI3 gene” includes not only the gene represented by the nucleotide sequence (SEQ ID NO: 1) but also a protein encoded by these (referred to as “EBI3” (SEQ ID NO: 2)) and biological activity. Genes encoding proteins for which are equivalent.
Specifically, in addition to the EBI3 gene represented by SEQ ID NO: 1, a DNA comprising a base sequence that hybridizes with a complementary sequence of the base sequence represented by SEQ ID NO: 1 under stringent conditions is included.
Here, the stringent conditions are usually, for example, “1 × SSC, 0.1% SDS, 37 ° C.”, “0.5 × SSC, 0.1% SDS, 42 ° C. as washing conditions after hybridization. And “0.1 × SSC, 0.1% SDS, 65 ° C.”. Specifically, as such a complementary strand, a strand consisting of a base sequence that is completely complementary to the target positive strand base sequence, and 90% or more, preferably 95% or more identity with the strand A chain consisting of a base sequence having can be exemplified.
Here, the identity of the base sequence is calculated by, for example, the Lippmann-Pearson method (Lipman-Pearson method; Science, 227, 1435, (1985)), and genetic information processing software Genetyx-Win (Ver. 5.1.1). It can be calculated by analyzing the unit size to compare (ktup) as 2 using the software development) homology analysis (Search homology) program.
　本発明において用いられる生体試料は、予後の評価対象となる膵がん患者から分離された生体検体であればよく、患者の膵がん組織である。生体試料は、患者から分離・採取された検体をそのままであってもよいが、各種測定に供するために適宜前処理されたものであってもよい。例えば検体を免疫組織化学に供する場合は、患者から得られた検体から調製したパラフィン切片を用いることができる。また、例えば試料をウェスタンブロット法又はＲＴ－ＰＣＲに供する場合は、患者から得られた検体から調製したタンパク質抽出液又はｍＲＮＡ抽出液を用いることができる。 The biological sample used in the present invention may be a biological specimen isolated from a pancreatic cancer patient to be evaluated for prognosis, and is a patient's pancreatic cancer tissue. The biological sample may be the sample separated and collected from the patient as it is, or may be pretreated appropriately for various measurements. For example, when a specimen is subjected to immunohistochemistry, a paraffin section prepared from a specimen obtained from a patient can be used. For example, when the sample is subjected to Western blotting or RT-PCR, a protein extract or mRNA extract prepared from a specimen obtained from a patient can be used.
　本発明の方法において、ＥＢＩ３の発現レベルを測定する方法は、生体試料中のＥＢＩ３を確認可能な方法であれば良く、特に限定されない。すなわち、ＥＢＩ３の発現レベルを検出又は定量できる方法であればよく、またＥＢＩ３の発現量を相対的又は絶対的に決定するものでもよい。ＥＢＩ３の発現は、タンパク質レベルで検出又は定量してもよく、また核酸（ｍＲＮＡ）レベルで検出又は定量してもよい。 In the method of the present invention, the method for measuring the expression level of EBI3 is not particularly limited as long as it can confirm EBI3 in a biological sample. That is, any method capable of detecting or quantifying the expression level of EBI3 may be used, and the expression level of EBI3 may be determined relative or absolutely. EBI3 expression may be detected or quantified at the protein level or may be detected or quantified at the nucleic acid (mRNA) level.
　ＥＢＩ３発現のタンパク質レベルでの検出又は定量は、ＥＢＩ３に特異的に結合する抗体（抗ＥＢＩ３抗体）を用いて、免疫学的に測定する方法が簡便であり好適である。免疫学的測定法としては、例えば、酵素免疫測定法（ＥＬＩＳＡ）、ラジオイムノアッセイ、免疫組織化学染色、ウエスタンブロット、免疫沈降、免疫蛍光、フローサイトメトリー等が挙げられる。
　また、核酸レベルでの検出又は定量としては、例えば、ＲＴ－ＰＣＲ（好ましくはリアルタイムＲＴ－ＰＣＲ）、ノーザン・ブロッティング法、Ｂｒａｎｃｈｅｄ　ＤＮＡアッセイ、in situ hybridization法等が挙げられる。 For the detection or quantification of EBI3 expression at the protein level, an immunological measurement method using an antibody that specifically binds to EBI3 (anti-EBI3 antibody) is simple and preferable. Examples of the immunological measurement method include enzyme immunoassay (ELISA), radioimmunoassay, immunohistochemical staining, Western blot, immunoprecipitation, immunofluorescence, flow cytometry, and the like.
Examples of detection or quantification at the nucleic acid level include RT-PCR (preferably real-time RT-PCR), Northern blotting method, Branched DNA assay, in situ hybridization method and the like.
　ＥＢＩ３と特異的に結合する抗体は、ＥＢＩ３と結合する限り、その構造、大きさ、イムノグロブリンクラスは何れのものでもよく、モノクローナルであってもポリクローナルであってもよく、標識化抗体、キメラ抗体、ヒト化抗体であってもよい。また、抗体の一部分（部分断片）又は抗体の一部分を含むペプチドであって、抗体の抗原（ＥＢＩ３）への結合作用を保持する抗体の断片を用いることもできる。斯かる抗体断片としては、例えば、Ｆ（ａｂ’）２、Ｆａｂ’、Ｆａｂ、一本鎖Ｆｖ（ｓｃＦｖ）等が挙げられる。 The antibody that specifically binds to EBI3 may be of any structure, size, and immunoglobulin class as long as it binds to EBI3, and may be monoclonal or polyclonal, labeled antibody, chimeric antibody It may be a humanized antibody. Moreover, it is also possible to use a part of an antibody (partial fragment) or a peptide containing a part of an antibody, and an antibody fragment that retains the binding action of an antibody to an antigen (EBI3). Examples of such antibody fragments include F (ab ') 2, Fab', Fab, single chain Fv (scFv) and the like.
　抗ＥＢＩ３抗体は、当業者によく知られた方法により得ることができる。例えば、ＥＢＩ３の全部若しくはその一部を有するポリペプチド、又はそれらをコードするポリヌクレオチドを組み込んだ哺乳動物細胞用発現ベクター等を調製しこれを抗原とする。この抗原を用いて動物を免疫後、免疫した動物から得られた免疫細胞と、ミエローマ細胞とを融合させてハイブリドーマを得、当該ハイブリドーマの培養物から抗体を採取する。最後に採取した抗体を、抗原に用いたＥＢＩ３やその一部に一致するポリペプチドを利用した抗原特異精製を行うことによりＥＢＩ３に対するモノクローナル抗体を得ることができる。また、ポリクローナル抗体を作成する場合、上記と同様の抗原で動物を免疫し、免疫した動物から血液を採取し、この血液から血清を分離し、この血清に対して上記抗原を利用した抗原特異精製を行うことにより得ることができる。得られた抗体を酵素処理又は得られた抗体の配列情報を利用することにより、抗体の断片を得ることができる。 Anti-EBI3 antibodies can be obtained by methods well known to those skilled in the art. For example, an expression vector for mammalian cells into which a polypeptide having all or part of EBI3 or a polynucleotide encoding the same is prepared and used as an antigen. After immunization of the animal with this antigen, immune cells obtained from the immunized animal and myeloma cells are fused to obtain a hybridoma, and an antibody is collected from the hybridoma culture. A monoclonal antibody against EBI3 can be obtained by subjecting the last collected antibody to antigen-specific purification using a polypeptide corresponding to EBI3 used as an antigen or a part thereof. In the case of producing a polyclonal antibody, an animal is immunized with the same antigen as described above, blood is collected from the immunized animal, serum is separated from the blood, and antigen-specific purification using the antigen is performed on the serum. Can be obtained. Antibody fragments can be obtained by enzymatic treatment of the obtained antibodies or using sequence information of the obtained antibodies.
　抗体の標識は、当分野において一般的な方法により行うことができ、また市販の標識キットを用いて標識することもできる。また、標識した抗体又はその断片の検出は、適宜標識に適した機器を使用することにより行うことができる。 The antibody can be labeled by a general method in this field, or can be labeled using a commercially available labeling kit. In addition, the labeled antibody or a fragment thereof can be detected by using a device suitable for labeling as appropriate.
　免疫組織化学法（免疫組織染色法）は、公知の方法を採用することができる。例えば、患者から分離した生体試料を常法によりホルマリン固定をした後、パラフィンに包埋をして組織片に薄切し、スライドガラスに貼り付けたものを切片試料として使用する。切片試料は、抗体の特性に応じて適宜オートクレーブによる熱処理等で抗原を賦活化し、Vector社ABC法あるいはDAKO社EnVision検出システム等を用いて免疫組織染色を行う。顕微鏡下において、ＥＢＩ３の発色により陽性・陰性の判定を行うことができる。 As the immunohistochemical method (immunohistological staining method), a known method can be adopted. For example, a biological sample separated from a patient is fixed in formalin by a conventional method, embedded in paraffin, sliced into tissue pieces, and pasted on a slide glass, and used as a slice sample. According to the characteristics of the antibody, the sectioned sample is appropriately activated by heat treatment with an autoclave or the like, and immunohistologically stained using Vector ABC method or DAKO EnVision detection system. Under the microscope, positive / negative can be determined by color development of EBI3.
　ＥＢＩ３をｍＲＮＡレベルで検出又は定量する場合、適宜設計したプライマーを用いて、ＰＣＲ等を利用した公知の方法により核酸を増幅する。このような方法としては、例えば、ＡＲＭＳ（Amplification Refractory Mutation System）法、ＲＴ－ＰＣＲ（Reverse transcriptase-PCR）法、Ｎｅｓｔｅｄ　ＰＣＲ法を挙げることができる。そして、増幅された核酸は、当該核酸と相補的な核酸を使用して、ドット・ブロット・ハイブリダイゼーション法、表面プラズモン共鳴法（ＳＰＲ法）、ＰＣＲ－ＲＦＬＰ法、Ｉｎ　ｓｉｔｕ　ＲＴ－ＰＣＲ法、ＰＣＲ－ＳＳＯ（sequence specific Oligonucleotide）法、ＰＣＲ－ＳＳＰ法、ＡＭＰＦＬＰ（Amplifiable fragment length polymorphism）法、ＭＶＲ－ＰＣＲ法、ＰＣＲＳＳＣＰ（single strand conformation polymorphism）法により検出される。
　ここで使用されるプライマー等は、公知の情報を基に当業者周知の方法により適宜設計し、化学合成により得ることができる When EBI3 is detected or quantified at the mRNA level, nucleic acids are amplified by a known method using PCR or the like using appropriately designed primers. Examples of such methods include ARMS (Amplification Refractory Mutation System) method, RT-PCR (Reverse transcriptase-PCR) method and Nested PCR method. The amplified nucleic acid is obtained by using a dot-blot hybridization method, surface plasmon resonance method (SPR method), PCR-RFLP method, In situ RT-PCR method, PCR using a nucleic acid complementary to the nucleic acid. -Detected by SSO (sequence specific oligonucleotide) method, PCR-SSP method, AMPFLP (Amplifiable fragment length polymorphism) method, MVR-PCR method, PCRSSCP (single strand conformation polymorphism) method.
Primers and the like used here can be appropriately designed by a method well known to those skilled in the art based on known information and obtained by chemical synthesis.
　斯くして、膵がん患者から分離された生体試料中のＥＢＩ３の発現レベルが測定され、当該ＥＢＩ３の発現レベルに基づいて、患者の予後が評価される。具体的には、検出されたＥＢＩ３の発現レベルを対照レベルと比較することによって、評価される。
　ここで、「対照レベル」とは、例えば、予後良好の患者群（予後が不良ではなかった膵がん患者、予後が不良ではないと信じられている膵がん患者）、若しくは膵がんを発症していない健常人群におけるＥＢＩ３の発現レベル、又は予後不良の患者群（予後が不良であった患者、予後が不良であろうことが知られている患者）におけるＥＢＩ３の発現レベルが挙げられる。
　例えば、対象患者のＥＢＩ３の発現レベルが、予後良好の患者群又は健常人群における当該ＥＢＩ３の発現レベルに近い、当該発現レベルの範囲内に属する、或いは当該発現レベルより高い場合には、当該患者の予後は良好であると評価できる。
　尚、比較対象となる患者におけるＥＢＩ３の発現レベルは、例えば、本発明の方法により、又は既に予後が判明している患者由来の試料におけるＥＢＩ３の発現レベルを測定すること等により知ることができる。 Thus, the expression level of EBI3 in the biological sample separated from the pancreatic cancer patient is measured, and the prognosis of the patient is evaluated based on the expression level of EBI3. Specifically, it is assessed by comparing the detected expression level of EBI3 with a control level.
Here, the “control level” means, for example, a group of patients with a good prognosis (patient cancer patients whose prognosis was not poor, pancreatic cancer patients who are believed to have a poor prognosis), or pancreatic cancer. The expression level of EBI3 in a group of healthy individuals who do not develop, or the expression level of EBI3 in a group of patients with a poor prognosis (a patient with a poor prognosis, a patient with a known prognosis).
For example, if the expression level of the target patient's EBI3 is close to, within the range of the expression level or higher than the expression level of the EBI3 in a group of healthy patients or a group of healthy subjects, Prognosis can be evaluated as good.
In addition, the expression level of EBI3 in a patient to be compared can be known, for example, by measuring the expression level of EBI3 in a sample derived from a patient whose prognosis is already known by the method of the present invention.
　また、本発明における予後の評価は、ＥＢＩ３の発現レベルの上昇／減少により行うこともできる。この場合は、対照レベルとして、ＥＢＩ３遺伝子又はＥＢＩ３の発現量の中央値を閾値レベル（カットオフ値）に設定し、患者由来の生体試料におけるＥＢＩ３の発現レベルを閾値レベルと比較することにより行うことができる。また、膵がん組織におけるＥＢＩ３の発現レベルの上昇／減少は、膵がん組織に浸潤するＥＢＩ３発現細胞数の増加／減少、或いは発現領域の拡大／縮小を指標とすることができる。そして、患者由来の生体試料におけるＥＢＩ３の発現レベルが閾値レベルより高い場合に、当該患者の予後が良好であると評価できる。
　閾値レベルは、例えば、膵がん組織に浸潤するＥＢＩ３発現細胞数の増加（発現領域の拡大）を指標とした場合、ＥＢＩ３発現細胞数の対物レンズ×２０の５視野での合計(例えば３回の平均)が２細胞以上、のように設定することができる。 In addition, the prognosis evaluation in the present invention can also be performed by increasing / decreasing the expression level of EBI3. In this case, as a control level, the median value of the expression level of EBI3 gene or EBI3 is set to a threshold level (cutoff value), and the expression level of EBI3 in a patient-derived biological sample is compared with the threshold level. Can do. Further, the increase / decrease in the expression level of EBI3 in pancreatic cancer tissue can be used as an index by increasing / decreasing the number of EBI3-expressing cells infiltrating the pancreatic cancer tissue or expanding / reducing the expression region. And when the expression level of EBI3 in a patient-derived biological sample is higher than the threshold level, it can be evaluated that the prognosis of the patient is good.
For example, when the increase in the number of EBI3-expressing cells infiltrating the pancreatic cancer tissue (expansion of the expression region) is used as an index, the threshold level is the sum of the number of EBI3-expressing cells in five fields of the objective lens × 20 (for example, three times) (Average) is 2 cells or more.
　本発明の膵がんの予後を評価するための検査用キットは、ＥＢＩ３と特異的に結合する抗体、又はＥＢＩ３ｍＲＮＡ若しくはＥＢＩ３遺伝子と特異的に結合するオリゴヌクレオチドを含有するものである。
　すなわち、当該検査用キットは、患者から分離した生体試料におけるＥＢＩ３の発現レベルを測定するための検査試薬を含有するものである。具体的には、ＥＢＩ３と特異的に結合する抗体（抗ＥＢＩ３抗体）を含む、ＥＬＩＳＡ、ウェスタンブロット、イムノクロマト、免疫組織化学用の試薬、又はＥＢＩ３ｍＲＮＡ若しくはＥＢＩ３遺伝子と特異的に結合するオリゴヌクレオチドを含む、ＲＴ－ＰＣＲ用の試薬等が挙げられる。当該キットに包含される抗ＥＢＩ３抗体、ＥＢＩ３遺伝子又はｍＲＮＡと特異的に結合する核酸は、上述したとおり公知の方法により得ることができる。
　また、当該検査用キットには、上記抗体や核酸の他、標識試薬、緩衝液、発色基質、二次抗体、ブロッキング剤や、試験に必要な器具やコントロール等を含むことができる。 The test kit for evaluating the prognosis of pancreatic cancer of the present invention contains an antibody that specifically binds to EBI3, or an oligonucleotide that specifically binds to EBI3 mRNA or the EBI3 gene.
That is, the test kit contains a test reagent for measuring the expression level of EBI3 in a biological sample separated from a patient. Specifically, including an antibody that specifically binds to EBI3 (anti-EBI3 antibody), ELISA, Western blot, immunochromatography, reagents for immunohistochemistry, or an oligonucleotide that specifically binds to EBI3 mRNA or EBI3 gene RT-PCR reagents and the like. The nucleic acid specifically binding to the anti-EBI3 antibody, EBI3 gene or mRNA included in the kit can be obtained by a known method as described above.
In addition to the antibody and nucleic acid, the test kit can contain a labeling reagent, a buffer solution, a chromogenic substrate, a secondary antibody, a blocking agent, instruments and controls necessary for the test, and the like.
　以下、本発明をより詳細に説明するため実施例を示すが、本発明はこれに限定されるものではない。
＜症例＞
　以下に示す膵がん外科切除症例の３つのコホートを用い、実施例１～２に示す膵がん組織に発現するEBI3の転写レベル及び蛋白質レベルでの発現量の測定、並びに生存解析を行った。
　コホート１(スクリーニング用): 2002-2005年国立がん研究センター中央病院で外科的に切除された約120症例。
　コホート２(検証用): 2006-2008年国立がん研究センター中央病院で外科的に切除された約150症例。
　コホート３(検証用): 1990-2001年国立がん研究センター中央病院で外科的に切除された約120症例)。 Hereinafter, examples will be shown to describe the present invention in more detail, but the present invention is not limited to these examples.
<Case>
Using the following three cohorts of surgically resected cases of pancreatic cancer, the expression levels of EBI3 expressed in the pancreatic cancer tissues and the expression levels at the protein level shown in Examples 1 and 2 and survival analysis were performed. .
Cohort 1 (for screening): About 120 cases surgically resected at the National Cancer Center Central Hospital in 2002-2005.
Cohort 2 (for verification): About 150 cases resected surgically at the National Cancer Center Central Hospital in 2006-2008.
Cohort 3 (for verification): About 120 cases surgically removed at the National Cancer Center Hospital in 1990-2001).
実施例１　EBI3遺伝子発現と患者予後の相関
（１）EBI3遺伝子発現量の測定
　外科切除された膵がん新鮮凍結組織から全RNAを抽出後、逆転写反応で得られたシングルストランドcDNAを鋳型にして、以下のPCRプライマーを用い、定量的RT-PCR法を用いて遺伝子を増幅し、EBI3遺伝子の発現量を測定した。定量的RT-PCRはロシュ・アプライド・サイエンス社のUniversal ProbeLibraryシステムにより実施し、コントロールとして、ACTB遺伝子の発現量を測定し、比較した。
EBI3: 5' primer, gAAgTACTggATCCgTTACAAgC（配列番号３）
　　　3' primer, gAAggACgTggCTTCAATg（配列番号４）
　　　probe #24
ACTB: 5' primer, CCAACCgCgAgAAgATgA（配列番号５）
　　　3' primer, TCCATCACgATgCCAgTg（配列番号６）
　　　probe #64
PCRの条件：95℃　10秒、[95℃　15秒、60℃　1分]x40サイクル Example 1 Correlation between EBI3 gene expression and patient prognosis (1) Measurement of EBI3 gene expression level Extracting total RNA from surgically excised pancreatic cancer fresh tissue, and then using single-strand cDNA obtained by reverse transcription reaction as a template Then, using the following PCR primers, the gene was amplified using the quantitative RT-PCR method, and the expression level of the EBI3 gene was measured. Quantitative RT-PCR was performed with the Universal ProbeLibrary system of Roche Applied Science, and the expression level of ACTB gene was measured and compared as a control.
EBI3: 5 'primer, gAAgTACTggATCCgTTACAAgC (SEQ ID NO: 3)
3 'primer, gAAggACgTggCTTCAATg (SEQ ID NO: 4)
probe # 24
ACTB: 5 'primer, CCAACCgCgAgAAgATgA (SEQ ID NO: 5)
3 'primer, TCCATCACgATgCCAgTg (SEQ ID NO: 6)
probe # 64
PCR conditions: 95 ° C for 10 seconds, [95 ° C for 15 seconds, 60 ° C for 1 minute] x 40 cycles
（２）生存解析
　コホート1を用いて、EBI3遺伝子の発現量の中間値を以て、高発現・低発現の２群に患者を分け、Kaplan-Meier法およびCox比例ハザードモデルを用いた生存解析方法により、EBI3遺伝子の発現と患者生命予後との関係を検討した。
　コホート1を用いた解析で得られた結果を、同様の方法にてコホート2を用いて検証した。 (2) Survival analysis Using cohort 1, the patients were divided into two groups of high and low expression with an intermediate value of the expression level of EBI3 gene, and the survival analysis method using Kaplan-Meier method and Cox proportional hazard model was used. We investigated the relationship between EBI3 gene expression and patient prognosis.
The results obtained from the analysis using Cohort 1 were verified using Cohort 2 in the same manner.
（３）結果
　１）コホート1を用いたKaplan-Meier法による生存解析の結果を図２に示す。
　EBI3遺伝子発現の高い症例群は低い症例群に比べて、有意に長い生存率＜overall survival (OS)＞ (P = 0.003)および無再発生存率＜disease-free survival (DFS)＞ (P = 0.016)が示された。また、この結果はコホート2を用いて確認された。[OS, P = 0.0003; DFS, P = 0.003] (3) Results 1) The results of survival analysis by Kaplan-Meier method using cohort 1 are shown in FIG.
Cases with high EBI3 gene expression had significantly longer survival <overall survival (OS)> (P = 0.003) and relapse-free survival <disease-free survival (DFS)> (P = 0.016) )It has been shown. This result was confirmed using Cohort 2. [OS, P = 0.0003; DFS, P = 0.003]
　２）Cox比例ハザードモデルを用いた単変量・多変量解析の結果を表１及び表２に示す。
　表１及び表２より、EBI3遺伝子発現は生存に関わる有意な因子であることが確認された[OS, HR 2.079, 95%CI 1.290-3.349, P = 0.003; DFS, HR 1.659, 95%CI 1.067-2.579, P = 0.025]。また、この結果はコホート2を用いて確認された[OS, HR 2.353, 95%CI 1.484-3.729, P = 0.0002; DFS, HR 1.530, 95%CI 1.016-2.305, P = 0.042]。 2) Tables 1 and 2 show the results of univariate and multivariate analysis using the Cox proportional hazard model.
Table 1 and Table 2 confirmed that EBI3 gene expression was a significant factor related to survival [OS, HR 2.079, 95% CI 1.290-3.349, P = 0.003; DFS, HR 1.659, 95% CI 1.067 -2.579, P = 0.025]. This result was also confirmed using Cohort 2 [OS, HR 2.353, 95% CI 1.484-3.729, P = 0.0002; DFS, HR 1.530, 95% CI 1.016-2.305, P = 0.042].
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
３）以上より、膵がん組織におけるEBI3遺伝子発現は独立した予後因子と考えられる。 3) Based on the above, EBI3 gene expression in pancreatic cancer tissue is considered to be an independent prognostic factor.
実施例２　EBI3の蛋白質発現と患者予後の相関
（１）EBI3に対するモノクローナル抗体の作成
　全長ヒトEBI3を哺乳動物細胞・大腸菌に発現させ、精製したEBI3をマウスに免役し、通常の方法によりハイブリドーマを作成後、ELISA, Western blot, ホルマリン固定パラフィン包埋リンパ節組織に対する免疫組織化学をスクリーニング方法として用いて、EBI3に特異的なモノクローナル抗体(クローンE32-8, IgG1 K; クローンE25-2, IgG1 K)を確立した。 Example 2 Correlation between EBI3 protein expression and patient prognosis (1) Production of monoclonal antibodies against EBI3 Full-length human EBI3 is expressed in mammalian cells and Escherichia coli, purified EBI3 is immunized to mice, and hybridomas are prepared by conventional methods. Then, ELISA, Western blot, monoclonal antibody specific to EBI3 (clone E32-8, IgG1 K; clone E25-2, IgG1 K) using immunohistochemistry on formalin-fixed paraffin-embedded lymph node tissue as a screening method Established.
（２）免疫組織化学
　免疫組織化学は4ミクロン厚のホルマリン固定パラフィン包埋組織切片を使い、Tris/EDTA(pH8.0)バッファー液中で121℃10分間オートクレーブによる抗原賦活化処理の後、抗EBI3抗体(1000倍希釈)を4℃8時間反応し、ビオチン化抗マウスイムノグロブリン抗体と1時間反応、Vector社ABC試薬と30分間反応後、Diaminobenzidine試薬により発色した。EBI3発現陽性コントロール細胞である非腫瘍部リンパ節内の樹状細胞と対比して染色性の有無を判定した。 (2) Immunohistochemistry For immunohistochemistry, 4 micron thick formalin-fixed paraffin-embedded tissue sections were used, and after antigen activation treatment by autoclaving in Tris / EDTA (pH 8.0) buffer solution at 121 ° C for 10 minutes, EBI3 antibody (1000-fold dilution) was reacted at 4 ° C. for 8 hours, reacted with biotinylated anti-mouse immunoglobulin antibody for 1 hour, reacted with Vector ABC reagent for 30 minutes, and then colored with Diaminobenzidine reagent. The presence or absence of staining was determined in comparison with dendritic cells in non-tumor lymph nodes that were EBI3 expression positive control cells.
（３）生存解析
　コホート3を用いて、膵がん組織にEBI3の免疫組織化学を実施した後、腫瘍に浸潤するEBI3発現細胞数を測定し、その中間値を以て、患者をEBI3発現細胞浸潤の多い群・少ない群の２群に分け、Kaplan-Meier法によりEBI3の発現と患者生命予後との関係を検討した。
　＜参考カットオフ値＞
　腫瘍最大割面　対物レンズx20の5視野合計(3回の平均)
　EBI3陽性細胞が2細胞未満：low group
　EBI3陽性細胞が2細胞以上：high group
　また、Cox比例ハザードモデルを用いた生存解析方法により、EBI3の発現と患者生命予後との関係を検討した。 (3) Survival analysis After performing EBI3 immunohistochemistry on pancreatic cancer tissue using Cohort 3, the number of EBI3-expressing cells infiltrating the tumor was measured, and the median of the EBI3-expressing cells The group was divided into two groups, a large group and a small group, and the relationship between EBI3 expression and patient life prognosis was examined by the Kaplan-Meier method.
<Reference cutoff value>
Maximum split surface of tumor Total of 5 fields of objective lens x20 (average of 3 times)
Less than 2 EBI3-positive cells: low group
2 or more EBI3-positive cells: high group
We also investigated the relationship between EBI3 expression and patient life prognosis using a survival analysis method using the Cox proportional hazards model.
（４）結果
　１）（１）で確立された抗EBI3抗体を用いた免疫組織化学の結果、膵がん組織中でEBI3は樹状細胞に強く発現し、一部のマクロファージに弱く発現していた。一方、EBI3は膵がん細胞に発現していなかった（図３）。図３(A)より、EBI3発現細胞(茶色)は、高発現の樹状細胞と低発現のマクロファージが示唆される。また、図３(B)より、EBI3発現細胞(赤紫色)は、成熟骨髄性樹状細胞を示すCD208(茶色)を発現していた。 (4) Results 1) As a result of immunohistochemistry using the anti-EBI3 antibody established in (1), EBI3 is strongly expressed in dendritic cells and weakly expressed in some macrophages in pancreatic cancer tissues. It was. On the other hand, EBI3 was not expressed in pancreatic cancer cells (FIG. 3). FIG. 3 (A) suggests that EBI3-expressing cells (brown) are highly expressed dendritic cells and low-expressing macrophages. From FIG. 3 (B), EBI3-expressing cells (red purple) expressed CD208 (brown) indicating mature myeloid dendritic cells.
　２）コホート3を用いたKaplan-Meier法による生存解析の結果を図４に示す。
　膵がん組織に浸潤するEBI3発現細胞数と患者予後との関係は、EBI3発現細胞の浸潤の多い群が少ない群に比べて、有意に長いOS (P = 0.006)およびDFS (P = 0.004)を示した（図３）。 2) The results of survival analysis by Kaplan-Meier method using cohort 3 are shown in FIG.
The relationship between the number of EBI3-expressing cells infiltrating the pancreatic cancer tissue and the patient prognosis was significantly longer in the OS (P = 0.006) and DFS (P = 0.004) than in the group with fewer EBI3-expressing cells infiltrating (FIG. 3).
　３）Cox比例ハザードモデルを用いた単変量・多変量解析の結果を表３及び表４に示す。
　表３及び表４より、膵がん組織へのEBI3発現細胞数は生存に関わる有意な因子であることが示された[OS, HR 1.765, 95%CI 1.141-2.728, P = 0.011; DFS, HR 2.041, 95%CI 1.314-3.169, P = 0.002]。 3) Tables 3 and 4 show the results of univariate and multivariate analysis using the Cox proportional hazard model.
Table 3 and Table 4 show that the number of EBI3-expressing cells in pancreatic cancer tissue is a significant factor related to survival [OS, HR 1.765, 95% CI 1.141-2.728, P = 0.011; DFS, HR 2.041, 95% CI 1.314-3.169, P = 0.002].
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　４）以上より、膵がん組織における腫瘍に浸潤するEBI3発現細胞数は、独立した予後因子と考えられる。 4) Based on the above, the number of EBI3-expressing cells that infiltrate tumors in pancreatic cancer tissue is considered to be an independent prognostic factor.
　本発明の方法によれば、膵がんと診断された患者について、膵がん検体等を用いることにより、予後を予測することができ、適切な治療方針を立てることが可能になる。すなわち、予後不良と評価された患者については、積極的な治療戦略（例えば、強化化学療法や放射線療法）を選択する必要性が判断でき、予後良好と判定された患者はより緩和された治療戦略を選択できる。また、本発明の方法は、膵がんの状態又は進行をモニターすることにも利用できる。すなわち、ＥＢＩ３発現レベルを利用して、患者が死亡、再発又は転移する可能性の変化を評価することができる。例えば、継時的にＥＢＩ３発現レベルを測定することにより、患者の予後予測の変化を知ることができる。 According to the method of the present invention, by using a pancreatic cancer specimen or the like for a patient diagnosed with pancreatic cancer, the prognosis can be predicted and an appropriate treatment policy can be established. In other words, for patients who are evaluated as having a poor prognosis, the need to select an aggressive treatment strategy (for example, intensive chemotherapy or radiation therapy) can be judged, and for patients who have a good prognosis, a more relaxed treatment strategy. Can be selected. The method of the present invention can also be used to monitor the state or progression of pancreatic cancer. That is, EBI3 expression levels can be used to assess changes in the likelihood that a patient will die, recur, or metastasize. For example, by measuring the EBI3 expression level over time, changes in the prognosis of the patient can be known.

Claims (6)

  1. 　膵がん患者から分離された生体試料中のＥＢＩ３の発現レベルを測定することを特徴とする、膵がんの予後を評価する方法。 A method for evaluating the prognosis of pancreatic cancer, comprising measuring the expression level of EBI3 in a biological sample isolated from a patient with pancreatic cancer.
  2. 　以下のａ及びｂの工程を含む、請求項１に記載の方法。
    　ａ）膵がん患者から分離された生体試料中のＥＢＩ３の発現レベルを測定する工程
    　ｂ）ＥＢＩ３の発現レベルを対照レベルと比較する工程     The method of Claim 1 including the process of the following a and b.
    a) measuring the expression level of EBI3 in a biological sample isolated from a patient with pancreatic cancer b) comparing the expression level of EBI3 with a control level
  3. 　ＥＢＩ３の発現レベルが閾値レベルよりも増加している場合に、予後良好であることを示す請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the prognosis is good when the expression level of EBI3 is higher than a threshold level.
  4. 　生体試料中のＥＢＩ３の発現レベルが、膵がん組織におけるＥＢＩ３遺伝子の発現量である、請求項１～３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein the expression level of EBI3 in the biological sample is the expression level of the EBI3 gene in pancreatic cancer tissue.
  5. 　生体試料中のＥＢＩ３の発現レベルが、膵がん組織に浸潤するＥＢＩ３発現細胞数である、請求項１～３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein the expression level of EBI3 in the biological sample is the number of EBI3-expressing cells infiltrating the pancreatic cancer tissue.
  6. 　ＥＢＩ３と特異的に結合する抗体、又はＥＢＩ３ｍＲＮＡ若しくはＥＢＩ３遺伝子と特異的に結合するオリゴヌクレオチドを含有する請求項１～５記載の方法に用いる膵がんの予後を評価するための検査用キット。
          A test kit for evaluating the prognosis of pancreatic cancer used in the method according to any one of claims 1 to 5, comprising an antibody that specifically binds to EBI3, or an oligonucleotide that specifically binds to EBI3 mRNA or the EBI3 gene.
PCT/JP2014/062875 2013-05-14 2014-05-14 Prognosis evaluation method for pancreatic cancer WO2014185466A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2013-102529 2013-05-14
JP2013102529A JP2016154447A (en) 2013-05-14 2013-05-14 Evaluation method of prognosis of pancreatic cancer

Publications (1)

Publication Number Publication Date
WO2014185466A1 true WO2014185466A1 (en) 2014-11-20

Family

ID=51898442

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2014/062875 WO2014185466A1 (en) 2013-05-14 2014-05-14 Prognosis evaluation method for pancreatic cancer

Country Status (2)

Country Link
JP (1) JP2016154447A (en)
WO (1) WO2014185466A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109402254A (en) * 2018-09-04 2019-03-01 复旦大学附属华山医院 A kind of LncRNA model and detection kit for predicting cancer of pancreas post-operative survival rates
CN114438217A (en) * 2022-03-23 2022-05-06 深圳大学总医院 Kit for detecting pancreatic cancer prognosis

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008260769A (en) * 2001-09-25 2008-10-30 National Cancer Center-Japan Search for cancer marker by novel screening method

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008260769A (en) * 2001-09-25 2008-10-30 National Cancer Center-Japan Search for cancer marker by novel screening method

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
LIU, L. ET AL.: "Effects on apoptosis and cell cycle arrest contribute to the antitumor responses of interleukin-27 mediated by retrovirus in human pancreatic carcinoma cells", ONCOL. REP., vol. 27, 2012, pages 1497 - 1503 *
LONG, J. ET AL.: "IL -35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 430, 2012, pages 364 - 369, XP028962882, doi:10.1016/j.bbrc.2012.11.004 *
NISHINO, R. ET AL.: "Identification of Epstein- Barr virus-induced gene 3 as a novel serum and tissue biomarker and a therapeutic target for lung cancer", CLIN. CANCER RES., vol. 17, no. 19, 2011, pages 6272 - 6286 *
RYOHEI NISHINO: "Epstein-Barr virus-induced gene 3 wa Haigan no Shinki Kessei Soshiki Biomarker Oyobi Chiryo Hyoteki Bunshi de aru", HIROSHIMA UNIVERSITY GAKUI RONBUN YOSHI, 2011, Retrieved from the Internet <URL:http://www.hiroshima-u.ac.jp/upload/0/kyoiku/daigakuin/gakuironbun/h23_kou/k5678_1.pdf> [retrieved on 20140724] *
TAKAMASA TAKAHASHI ET AL.: "TS1 Suigan 47 Shorei no Rinsho Byorigakuteki Tokucho Oyobi Yogo Inshi", JOURNAL OF JAPAN SURGICAL SOCIETY, vol. 112, no. 1·2, 2011, pages 873, PS-212 - 2 *
YATARO DAIGO: "Haigan Shokudogan no Seimei Yogo ni Kiyo suru Jitsuyoteki Byotai Shindan Biomarker no Kaihatsu Kenkyu", DAIWA SECURITIES HEALTH FOUNDATION HEISEI 22 NENDO (DAI 37 KAI) CHOSA KENKYU JOSEI KENKYU RONBUN, 2012, pages 87 - 89, Retrieved from the Internet <URL:http://www.daiwa-grp.jp/dsh/results/37/pdf/19.pdf> [retrieved on 20140724] *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109402254A (en) * 2018-09-04 2019-03-01 复旦大学附属华山医院 A kind of LncRNA model and detection kit for predicting cancer of pancreas post-operative survival rates
CN109402254B (en) * 2018-09-04 2024-03-08 复旦大学附属华山医院 LncRNA model for predicting pancreatic cancer postoperative survival and detection kit
CN114438217A (en) * 2022-03-23 2022-05-06 深圳大学总医院 Kit for detecting pancreatic cancer prognosis

Also Published As

Publication number Publication date
JP2016154447A (en) 2016-09-01

Similar Documents

Publication Publication Date Title
JP6554646B2 (en) Triage of patients with asymptomatic hematuria using genotype and phenotype biomarkers
US20200333344A1 (en) Use of markers including filamin a in the diagnosis and treatment of prostate cancer
KR101566368B1 (en) Urine gene expression ratios for detection of cancer
CA2762082C (en) Markers for detection of gastric cancer
KR101032607B1 (en) Proteinic markers for diagnosing hepatocellular carcinoma
CA2712505C (en) Biomarkers for the diagnosis and treatment of pancreatic cancer
CN114250299A (en) Urine markers for detection of bladder cancer
TW201305350A (en) Novel marker for detection of bladder cancer
US20120277110A1 (en) Parp and adjuvant cisplatin-based chemotherapy in non-small-cell lung cancer
US20120004136A1 (en) Msh2 and adjuvant cisplatin-based chemotherapy in non-small-cell lung cancer
US20200232039A1 (en) Adm2 gene marker for diagnosis or prognosis prediction of thyroid cancer and uses thereof
WO2011111820A1 (en) Diagnostic marker for lung cancer or cervical cancer
WO2014185466A1 (en) Prognosis evaluation method for pancreatic cancer
JP7150018B2 (en) Novel CIP2A variants and uses thereof
US20200018758A1 (en) Methods for differentiating benign prostatic hyperplasia from prostate cancer
WO2019134994A1 (en) Prognostic biomarkers for human papillomavirus positive cancers
JP7114112B2 (en) Prognostic biomarkers for lung cancer
JP2020072705A (en) Urine markers for detection of bladder cancer
KR101917677B1 (en) Usefulness of Methionyl-tRNA synthetase (MRS) as a prognostic biomarker of lung cancer
KR101346038B1 (en) Composition or Kit and Method for predicting prognosis of liver cancer
JP2022032795A (en) Method for predicting onset of endometrial cancer
WO2020264379A1 (en) Markers for the diagnosis of prostate cancer
CA3214819A1 (en) Protein markers for estrogen receptor (er)-positive luminal a(la)-like and luminal b1 (lb1)-like breast cancer
JP2007503826A (en) Diagnosis of breast cancer risk
JPWO2006080192A1 (en) Cancer-specific gene and diagnostic kit using the same

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14797522

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14797522

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP