WO2019049829A1 - Prognostic biomarker for colon cancer - Google Patents

Prognostic biomarker for colon cancer Download PDF

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WO2019049829A1
WO2019049829A1 PCT/JP2018/032628 JP2018032628W WO2019049829A1 WO 2019049829 A1 WO2019049829 A1 WO 2019049829A1 JP 2018032628 W JP2018032628 W JP 2018032628W WO 2019049829 A1 WO2019049829 A1 WO 2019049829A1
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colorectal cancer
galnt6
biomarker
patients
prognosis
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PCT/JP2018/032628
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French (fr)
Japanese (ja)
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洋和 岡山
勝 野田
浩二 河野
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公立大学法人福島県立医科大学
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Priority to JP2019540946A priority Critical patent/JP6833226B2/en
Publication of WO2019049829A1 publication Critical patent/WO2019049829A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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
    • C12Q1/6813—Hybridisation assays
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a biomarker for predicting the prognosis of colorectal cancer patients and / or determining the efficacy of an anticancer agent for colorectal cancer patients.
  • Colorectal cancer is the leading cause of cancer death worldwide, despite major advances in diagnosis and treatment.
  • the only colorectal cancer prognostic classification currently used in the clinical setting is a clinicopathologic staging (stage) classification, and a treatment policy is selected according to the stage.
  • stage clinicopathologic staging
  • clinical outcomes such as drug sensitivity, recurrence of cancer and survival of patients are significantly different even for colorectal cancer that is determined to be at the same stage clinicopathologically.
  • patients with stage III colorectal cancer are generally given adjuvant chemotherapy after radical surgery, but about 30 to 40% of patients recur colon cancer (non-patent documents 1 to 3).
  • An object of the present invention is to provide a biomarker for predicting the prognosis of colorectal cancer patients and / or determining the efficacy of an anti-cancer agent for colorectal cancer patients.
  • the present inventors repeatedly investigated and examined the expression of the glycosyltransferase GALNT6 protein in colorectal cancer tissues excised from colorectal cancer patients.
  • most colon cancer patients regardless of stage were found to be GALNT6 positive expressing the GALNT6 protein, but some (about 15%) patients were found to be GALNT6 negative.
  • the inventors of the present invention show that patients with GALNT6-positive colorectal cancer have a good prognosis after colorectal cancer removal surgery, while patients with GALNT6-negative colorectal cancer have a poor prognosis after colorectal cancer removal surgery.
  • GALNT6-positive colon cancer patients tended to be more effective than anticancer agents than GALNT6-negative colon cancer patients.
  • the inventors have completed the present invention based on these findings.
  • the present invention includes the following.
  • a biomarker for predicting the prognosis of colorectal cancer patients comprising a GALNT6 protein or a peptide fragment thereof, or a transcript of a GALNT6 gene or a nucleic acid fragment thereof.
  • a biomarker for determining the efficacy of an anticancer agent for colorectal cancer patients comprising a GALNT6 protein or a peptide fragment thereof, or a transcript of a GALNT6 gene or a nucleic acid fragment thereof.
  • the GALNT6 protein is any one of the following (a) to (c): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, (b) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and (c) 90% of the amino acid sequence shown in SEQ ID NO: 1 A protein consisting of an amino acid sequence having the above amino acid identity [5] The biomarker according to any one of [1] to [3], wherein the GALNT6 gene encodes the protein shown in [4].
  • [6] Use of the biomarker according to any one of [1] and [4] to [5] for predicting the prognosis of colorectal cancer patients.
  • [7] Use of the biomarker according to any one of [2] to [5] for determining the efficacy of an anticancer agent for colorectal cancer patients.
  • [8] The use according to [6] or [7], wherein the colorectal cancer is stage I-IV colorectal cancer.
  • [9] The use according to [8], wherein the colon cancer is stage III colon cancer.
  • a method for predicting the prognosis of a patient with colorectal cancer Measuring the amount of the biomarker according to any one of [1] and [4] to [5] in a colon cancer cell or tissue obtained from a colon cancer patient, and the measuring step Including a prediction step of predicting the prognosis of a colorectal cancer patient based on the measurement result,
  • the colorectal cancer cells or tissues are negative for the biomarker, then the prognosis of the colorectal cancer patient is predicted to be poor.
  • a method of determining the efficacy of an anti-cancer agent for colorectal cancer patients comprising Based on the measurement result obtained in the measurement step of measuring the amount of the biomarker according to any one of [2] to [5] in colon cancer cells or tissues obtained from a colon cancer patient, and the measurement step And determining the effectiveness of the anti-cancer drug in patients with colorectal cancer,
  • the colon cancer cell or tissue is negative for the biomarker, it is determined that the efficacy of the anticancer agent for colon cancer patients is low.
  • a kit for predicting the prognosis of a colorectal cancer patient comprising a reagent for measuring the amount of the biomarker according to any of [1] and [4] to [5].
  • a kit for determining the efficacy of an anticancer agent for colorectal cancer patients comprising a reagent for measuring the amount of the biomarker according to any one of [2] to [5].
  • the present specification includes the disclosure content of Japanese Patent Application No. 2017-170157 based on which the priority of the present application is based.
  • a biomarker is provided to predict the prognosis of colorectal cancer patients and / or to determine the efficacy of anti-cancer agents for colorectal cancer patients.
  • FIG. 1 is a photograph showing a representative image of immunohistochemical staining for protein expression. Images of (A), (D) and (F) were acquired at 100 ⁇ , (B), (C), (E) and (G) at 400 ⁇ magnification. It is a graph which shows the disease specific survival rate after curative surgery in a GALNT6 positive or negative colon cancer patient determined by immunohistochemical staining.
  • the survival rates of (A) 267 patients in stages I-IV, (B) 195 patients in stages II-III, and (C) 80 patients in stage III are shown. It is a graph which shows the Kaplan-Meier curve of disease-free survival rate in the positive
  • FIG. 4 shows the biological effects of GALNT6 silencing.
  • GALNT6 knockdown was confirmed by qRT-PCR (A) and Western blotting (B) after transfection of GALNT6 targeting siRNA (siGALNT6-1 and siGALNT6-2) or scrambled control into SW480 cells .
  • C Cell growth measured by CCK-8 assay at various time points. The results are expressed as mean ⁇ standard deviation of 3 independent experiments.
  • D shows the dose response effect of 5-FU treatment on cell viability. The results are expressed as mean ⁇ standard deviation of three independent experiments (* P ⁇ 0.05, ** P ⁇ 0.01).
  • E In cells treated with 10 ⁇ M 5-FU, apoptosis was analyzed by flow cytometry labeled with Annexin V-PE and 7-AAD. The results are expressed as mean ⁇ standard deviation of three independent experiments (* P ⁇ 0.05).
  • the present invention provides a biomarker for predicting the prognosis of colorectal cancer patients.
  • the present invention also provides a biomarker for determining the efficacy of an anti-cancer agent for colorectal cancer patients.
  • the biomarker according to the present invention consists of the GALNT6 protein or its peptide fragment, or the transcript of the GALNT6 gene or its nucleic acid fragment.
  • colon cancer refers to cancer that develops in the large intestine, including the colon, rectum and anus.
  • Colorectal cancer includes, in particular, colon cancer and rectal cancer.
  • prognosis refers to the predicted course (e.g., life and death) after resection surgery for colorectal cancer in colorectal cancer patients. “Prognostic” may be prediction of survival time or survival rate after a certain period of time (eg, 1, 3, 5, 10, 15 or 20 years or more after surgery). Predicting prognosis can also be referred to as determining, evaluating, or diagnosing prognosis.
  • Glycosylation ie, glycosylation of proteins
  • Glycosylation is one of the common and important post-translational modifications of proteins that regulate diverse physiological processes. Glycosylation involves the sequential addition of single sugar residues to a target protein, which results in glycan elongation. Additional chemical modifications and branching can ultimately form various glycan structures. Glycosylation generally occurs by the multienzyme reaction of glycosyltransferases (glycosyl transferases).
  • the GALNT6 (polypeptide N-acetylgalactosaminyltransferase 6) protein is a glycosyltransferase belonging to the UDP-N-acetyl- ⁇ -D-galactosamine: polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) family .
  • the GalNAc-T enzyme is known to initiate glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to a serine or threonine residue on a target protein.
  • the GALNT6 protein or its gene transcript derived from an endogenous gene of a colorectal cancer patient can be a biomarker.
  • the patient is a human
  • human GALNT6 protein derived from human GALNT6 gene and transcript (mRNA) of human GALNT6 gene can be a biomarker of the present invention.
  • GALNT6 protein is a human-derived GALNT6 (human GALNT6) protein consisting of the amino acid sequence of 622 residues shown in SEQ ID NO: 1.
  • the GALNT6 protein also includes a GALNT6 variant having functionally equivalent activity to the GALNT6 protein shown by SEQ ID NO: 1 and GALNT6 orthologs of other biological species.
  • a GALNT6 variant having functionally equivalent activity to the GALNT6 protein shown by SEQ ID NO: 1 and GALNT6 orthologs of other biological species.
  • an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, or 90%, 95% or more relative to the amino acid sequence shown in SEQ ID NO: 1 Included are GALNT6 proteins having an amino acid identity of 97% or more, 98% or more, or 99% or more.
  • amino acid substitution is preferably conservative amino acid substitution.
  • Constant amino acid substitution refers to substitution between amino acids of similar properties such as charge, side chain, polarity, aromaticity and the like.
  • Amino acids having similar properties are, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), nonpolar Organic amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched chain amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), etc. it can.
  • basic amino acids arginine, lysine, histidine
  • acidic amino acids aspartic acid, glutamic acid
  • uncharged polar amino acids glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine
  • amino acid identity refers to the sequence number of the amino acid sequence when the two amino acid sequences are aligned (alignment) and a gap is introduced as needed to maximize the amino acid identity between the two.
  • Amino acid identity can be calculated using a protein search system by BLAST or FASTA.
  • the "GALNT6 gene” is a gene encoding the above-mentioned GALNT6 protein.
  • a specific example of the GALNT6 gene is a human GALNT6 gene encoding a human GALNT6 protein consisting of the amino acid sequence shown by SEQ ID NO: 1. More specifically, the GALNT6 gene is a gene consisting of the base sequence shown in SEQ ID NO: 2.
  • the GALNT6 gene also includes a GALNT6 variant having an activity equivalent to that of the GALNT6 protein encoded by the GALNT6 gene shown in SEQ ID NO: 2 and a GALNT6 gene encoding GALNT6 ortholog of other species. Specifically, 90%, 95% or more of the base sequence in which one or more bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2 or the base sequence shown in SEQ ID NO: 2 And GALNT6 genes having a base identity of 97% or more, 98% or more, or 99% or more.
  • nucleotide sequence that hybridizes under high stringency conditions with a nucleic acid fragment consisting of a portion of the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2, and is functionally equivalent to the GALNT6 protein Included are genes encoding proteins having activity.
  • base identity refers to the sequence number when the two base sequences are aligned (aligned), and a gap is introduced as necessary to maximize the degree of base identity between the two.
  • hybridize under highly stringent conditions refers to hybridization and washing under conditions of low salt concentration and / or high temperature. For example, incubate with the probe at 65 ° C. to 68 ° C. in 6 ⁇ SSC, 5 ⁇ Denhardt's reagent, 0.5% SDS, 100 ⁇ g / mL denatured fragmented salmon sperm DNA, then in a wash solution of 2 ⁇ SSC, 0.1% SDS Starting from room temperature, the salt concentration in the wash solution is lowered to 0.1 ⁇ SSC, and the temperature is raised to 68 ° C. to wash until background signal is not detected.
  • the conditions for highly stringent hybridization are described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. It can be helpful.
  • Such nucleotide sequence information of GALNT6 gene can be searched from public databases (GenBank, EMBL, DDBJ). For example, based on the known nucleotide sequence information of the GALNT6 gene shown by SEQ ID NO: 2, genes with high base identity can be searched and obtained from the database.
  • Transcript of GALNT6 gene means GALNT6 mRNA.
  • the mRNA may be a pre-mRNA (pre-mRNA) or a mature mRNA (mature mRNA).
  • pre-mRNA pre-mRNA
  • mature mRNA mature mRNA
  • the transcript of the GALNT6 gene substantially serving as a biomarker of the present invention is a GALNT6 mature mRNA.
  • peptide fragment is a peptide fragment consisting of a part of the amino acid sequence constituting the GALNT6 protein, which can be identified as a fragment of the GALNT6 protein from the amino acid sequence constituting the fragment I say something.
  • nucleic acid fragment is a nucleic acid fragment consisting of a part of the nucleotide sequence constituting GALNT6 mRNA, and it can be identified from the nucleotide sequence constituting the fragment that it is a fragment of GALNT6 mRNA I say something. Generally, it is a nucleic acid consisting of 40 or more and 600 or less, 50 or more and 450 or less, 60 or more and 300 or less, or 70 or more and 200 or less consecutive bases in the full-length base sequence of GALNT6 mRNA. Good.
  • the present invention provides the use of the above-mentioned biomarker according to the present invention for predicting the prognosis of colorectal cancer patients.
  • the present invention also provides the use of the above-mentioned biomarker according to the present invention for determining the efficacy of an anti-cancer agent for colorectal cancer patients.
  • the present invention also provides a method of predicting the prognosis of colorectal cancer patients.
  • This method comprises the steps of measuring the amount of the biomarker according to the present invention described above in a colon cancer cell or tissue obtained from a colon cancer patient, and the large intestine based on the measurement result obtained in the above measuring step.
  • the prediction process which predicts the prognosis of a cancer patient is included.
  • the measuring step can be performed in vitro. Each step is specifically described below.
  • UICC-TNM classification the degree of progression of cancerous lesions is classified by three factors: wall depth (T classification), lymph node metastasis (N classification) and distant metastasis (M classification).
  • T classification wall depth
  • N classification lymph node metastasis
  • M classification distant metastasis
  • stage I colon cancer has its own muscle It is determined as Stage II if the stroma is infiltrated into the subserosal membrane (T3) or the serosa or adjacent organs (T4).
  • no lymph node metastasis is involved (N0). If lymph node metastasis is involved (N1-2), it is judged as stage III.
  • MO distant metastasis
  • M1 cancer with distant metastasis
  • the colorectal cancer which suffers from the subject of the present invention may be stage I to IV, preferably stage II and III, more preferably stage III colon cancer.
  • the colorectal cancer patient in the present invention is preferably a mammal, more preferably a primate, most preferably a human.
  • the colon cancer cells or tissues used in the present invention can be obtained from colon cancer patients by, for example, biopsy or excision surgery, although not particularly limited.
  • the cells or tissues may be used as they are for measurement of a biomarker, but may be appropriately pretreated for measurement.
  • paraffin embedded sections may be prepared from a sample from a patient.
  • a protein extract or mRNA extract may be prepared from a sample derived from a patient.
  • the biomarker to be measured in the present method may be either the GALNT6 protein or its peptide fragment or the transcript of the GALNT6 gene or its nucleic acid fragment.
  • the measurement of the amount (expression amount) includes measuring the presence or absence of expression, or the size of expression amount or expression concentration, and the like.
  • the term "measurement” includes any of detection, qualitative, quantitative and semi-quantitative.
  • the measuring method may be any known method for quantifying proteins, and is not particularly limited, but includes, for example, an immunological detection method.
  • An “immunological detection method” is a method of measuring the amount of a target molecule using an antibody or antibody fragment that specifically binds to a target molecule that is an antigen.
  • the antibodies can be from any animal, including mammals and birds. For example, mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens or humans can be mentioned.
  • the antibody used in the immunological detection method is not particularly limited, but a monoclonal antibody or a polyclonal antibody may be used.
  • monoclonal antibody refers to a clonal group of single immunoglobulins. Each immunoglobulin constituting a monoclonal antibody contains a common framework region and a common complementarity determining region, and can recognize and bind to the same epitope of the same antigen. Monoclonal antibodies can be obtained from single cell derived hybridomas.
  • polyclonal antibody refers to a plurality of immunoglobulin groups that recognize and bind to different epitopes of the same antigen. Polyclonal antibodies can be obtained from the serum of an animal after immunizing the animal with the target molecule as an antigen.
  • each class of immunoglobulin molecule is known: IgG, IgM, IgA, IgE, and IgD, but the antibody of the present invention may be any class. . Preferably it is IgG.
  • a method for producing a polyclonal antibody that recognizes and binds to the GALNT6 protein or a hybridoma that produces a monoclonal antibody may be performed according to a method for producing an antibody known in the art using the GALNT6 protein or a fragment thereof as an antigen.
  • Antibodies may also be obtained from manufacturers such as, for example, Sigma-Aldrich.
  • antibody fragment is a partial fragment of a polyclonal antibody or a monoclonal antibody, and refers to a polypeptide chain or a complex thereof having an activity substantially equivalent to the antigen-specific binding activity of the antibody.
  • an antibody portion that includes at least one antigen binding site ie, a polypeptide chain having at least one set of VL and VH, or a complex thereof, is relevant.
  • Specific examples include many well-characterized antibody fragments and the like generated by cleaving immunoglobulin with various peptidases. More specific examples include Fab, F (ab ') 2 , Fab' and the like. All of these antibody fragments contain an antigen binding site, and have the ability to specifically bind to a target molecule that is an antigen.
  • Immunological detection methods include, for example, immunohistochemical staining, enzyme-linked immunosorbent assay (including ELISA, EIA), western blotting, radioimmunoassay (RIA), immunoprecipitation, or flow cytometry. The law is mentioned.
  • the "immunohistochemical staining method” can adopt a known method. For example, a sample derived from a patient may be formalin-fixed, embedded in paraffin, sliced into tissue pieces, and affixed to a slide glass, and used as a section sample. Section samples may optionally be heat treated to activate the antigen, and then immunohistochemical staining may be performed on the section samples using a commercially available detection system such as the Dako EnVision + System (Agilent).
  • a commercially available detection system such as the Dako EnVision + System (Agilent).
  • the measuring method may be any known nucleic acid quantitative method, and is not particularly limited.
  • Nucleic acid amplification method refers to a method of amplifying a specific region of a target nucleic acid by a nucleic acid polymerase using a forward / reverse primer set.
  • nucleic acid amplification methods include PCR (polymerase chain reaction) methods such as RT-PCR (reverse transcription polymerase chain reaction) method.
  • the “hybridization method” uses a nucleic acid fragment having a base sequence complementary to all or part of the base sequence of a target nucleic acid to be detected as a probe, and utilizes base pairing between the nucleic acid and the probe. It is a method of detecting and quantifying a target nucleic acid or a fragment thereof.
  • the hybridization method several different detection means are known, and for example, Northern hybridization (Northern blot hybridization), in situ hybridization, or microarray method can be mentioned.
  • Nucleic acid chains such as primers and probes can be appropriately designed by methods known to those skilled in the art based on known biomarker sequence information, and obtained by known preparation methods such as chemical synthesis.
  • each of the above-mentioned measurement methods is a technique known in the art. Therefore, the specific measurement method may be performed according to a known method. For example, the methods described in Green, MR and Sambrook, J., 2012 (described above) can be referred to.
  • Step 2 the prognosis of a colorectal cancer patient is predicted based on the measurement result obtained in the measurement step.
  • This step may include determining from the measurement result obtained in the measurement step whether the colon cancer cell or tissue is positive or negative for the biomarker.
  • control samples may be derived from healthy individuals (eg, healthy individuals) or benign colorectal adenoma patients.
  • a "healthy individual” refers to a healthy individual not suffering from cancer of the same species as the test individual.
  • the present inventors have found that the expression level of GALNT6 protein is not in normal colon mucosa or low, while it is high in colon adenoma tissue, as described in the examples below. Therefore, the amount of biomarkers measured in colon cancer cells or tissues obtained from colon cancer patients is higher (eg, statistically higher) than the amount of control measured in control samples derived from healthy individuals If so, the colon cancer cell or tissue can be determined to be positive for the biomarker. In addition, the amount of biomarker measured in colon cancer cells or tissues obtained from patients with colorectal cancer is the same or lower than the amount of control measured in a control sample derived from a healthy individual (eg, statistically significant If so, the colon cancer cells or tissues can be determined to be negative for the biomarker.
  • the amount of biomarker measured in colon cancer cells or tissues obtained from colon cancer patients is the same or higher than that in control samples derived from colon adenoma patients (eg, statistically If it is significantly higher, the colon cancer cell or tissue can be determined to be positive for the biomarker.
  • the amount of biomarkers measured in colon cancer cells or tissues obtained from colon cancer patients is lower than that in control samples derived from colon adenoma patients (eg, statistically significant) If low, the colon cancer cell or tissue may be determined to be negative for the biomarker.
  • “statistically significant” means, specifically, p ⁇ 0.05 (less than 5%), p ⁇ 0.01 (less than 1%) when the risk factor (level of significance) of the obtained value is small. Or p ⁇ 0.001 (less than 0.1%).
  • the statistical test method is not particularly limited as long as a known test method capable of determining the presence or absence of significance can be appropriately used. For example, Student's t test, multiple comparison test, log rank test can be used.
  • the amount of the biomarker may be measured in advance in a control sample, and the cutoff value (threshold) may be determined based on the measurement value. If the cutoff value is used as a reference and the cutoff value is exceeded, it can be judged as positive.
  • the cutoff value can be determined by, for example, ROC (receiver operating characteristic curve) analysis.
  • the present inventors have found that the expression level of GALNT6 protein correlates with the prognosis of colorectal cancer patients, as described in the examples below. Therefore, in this step, if colon cancer cells or tissues are negative for the biomarker, the prognosis of colon cancer patients can be predicted to be bad. On the other hand, if colon cancer cells or tissues are positive for the biomarker, then the prognosis for colon cancer patients can be predicted to be good.
  • a poor prognosis means poor clinical outcome (eg, high recurrence rate after resection surgery for colorectal cancer, low disease (cancer) specific survival rate, or overall survival Rate is low). If the prognosis is poor, the 5-year survival rate after resection surgery for colorectal cancer may be less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65% or less than 60%. In the present invention, survival rate means cumulative survival rate. In the present invention, the survival rate may be disease (cancer) specific survival rate or overall survival rate.
  • good prognosis means that the clinical outcome is good. If the prognosis is good, then the 5-year survival rate after resection surgery for colorectal cancer may be 90% or more or 95% or more.
  • the prognosis of patients with colorectal cancer can be predicted, and based on the result, the treatment plan (for example, type of anticancer agent, dosage, administration interval, etc.) is determined, or the large intestine The interval between examinations for cancer recurrence and metastasis can be determined.
  • the treatment plan for example, type of anticancer agent, dosage, administration interval, etc.
  • the present invention when it is predicted that the prognosis of a colorectal cancer patient is poor, the patient is administered an anticancer agent to prevent recurrence of the colorectal cancer or to improve the prognosis or improve the survival rate. You may Therefore, the present invention also prevents the recurrence of colorectal cancer or improves the prognosis, which comprises administering an anticancer agent to colorectal cancer patients predicted to have a poor prognosis by the method of the present invention, Or provide a method to improve the survival rate.
  • Anti-cancer agents include, but are not limited to, 5-fluorouracil (5-FU), capecitabine, oxaliplatin, irinotecan, bevacizumab, cetuximab, panitumumab and regorafenib.
  • the anticancer agent can be used alone or in combination.
  • the anticancer agent can be administered by routes such as injection, intravenous administration, oral administration and the like.
  • the present invention also provides a method of determining the efficacy of an anti-cancer agent for colorectal cancer patients.
  • the present method is based on the measurement step of measuring the amount of the biomarker according to the present invention described above in a colon cancer cell or tissue obtained from a colon cancer patient, and the measurement result obtained in the measurement step. It includes a determination step of determining the efficacy of the cancer drug.
  • the measurement process is the same as that described for the method of predicting the prognosis of the colorectal cancer patient, and thus the description thereof is omitted.
  • the determination step instead of predicting the prognosis of a colorectal cancer patient based on the measurement results, the prognosis of the above colorectal cancer patient is determined except that the efficacy of the anticancer drug is determined. Since it conforms to the description of the forecasting method, only the differences will be described below.
  • a lower efficacy of the anti-cancer agent in a patient who is negative for a biomarker is less effective than a patient who is positive for the biomarker (eg, a disease-free survival rate is lower Cancer) Low specific survival rate or low overall survival rate).
  • survival rate after resection surgery for colorectal cancer for example, disease-free survival rate is less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, 55% It may be less than or less than 50%.
  • survival rate means cumulative survival rate.
  • the survival rate may be disease free survival rate, disease (cancer) specific survival rate or overall survival rate.
  • the efficacy of the anti-cancer drug is high means that the efficacy is high compared to the patient who is positive for the biomarker, for example, the disease-free survival rate after 5 years of resection for colorectal cancer is It may be 60% or more or 70% or more.
  • the anticancer agent when it is judged that the anticancer agent for colorectal cancer patients is high, the anticancer agent is administered to the patients in order to prevent the recurrence of colorectal cancer or improve the prognosis or improve the survival rate. You may Therefore, the present invention also prevents the recurrence of colorectal cancer, which comprises administering an anticancer agent to a colorectal cancer patient who is judged to have a high anticancer agent for a colorectal cancer patient by the method of the present invention, or Provided is a method of improving prognosis or improving survival rate.
  • Anti-cancer agents include, but are not limited to, 5-fluorouracil (5-FU), capecitabine, oxaliplatin, irinotecan, bevacizumab, cetuximab, panitumumab and regorafenib.
  • the anticancer agent can be used alone or in combination.
  • the anticancer agent can be administered by routes such as injection, intravenous administration, oral administration and the like.
  • the present invention when it is judged that the anticancer agent for colorectal cancer patients is low, in order to prevent the recurrence of colorectal cancer or to improve the prognosis or to improve the survival rate, the patient other than the anticancer agent is used.
  • a therapy eg, radiation therapy etc.
  • the present invention also prevents the recurrence of colorectal cancer, which comprises applying a therapy other than an anticancer drug to a colorectal cancer patient who is judged to have a high anticancer agent for a colorectal cancer patient by the method of the present invention. Or provide a method of improving prognosis or improving survival rate.
  • kits The present invention also provides a kit for predicting the prognosis of colorectal cancer patients, comprising a reagent for measuring the amount of the biomarker according to the present invention described above.
  • the present invention further provides a kit for determining the efficacy of an anti-cancer agent for colorectal cancer patients, comprising a reagent for measuring the amount of the biomarker according to the present invention described above.
  • the reagent for measuring the amount of the biomarker includes, for example, an antibody or antibody fragment as described above, or a probe or a primer.
  • the kit includes known immunohistochemical staining, ELISA, Western blot, or reagents for RT-PCR, etc., for example, labeling reagents, buffers, chromogenic substrates, secondary antibodies, blocking agents, and devices and controls required for testing. And the like may be further included.
  • Example 1 Immunohistochemical staining of GALNT6 protein in colorectal cancer tissue
  • Immunohistochemical staining for GALNT6 protein expression was performed.
  • the stage of the tumor was determined according to TNM classification of malignant tumor (UICC 7th Edition; Sobin LH, Gospodarowicz MK, Wittekind C, International Union against Cancer. TNM classification of malignant tumors, 7th edn. Wiley-Blackwell (2010)).
  • FFPE Formalin-fixed, paraffin-embedded
  • the 4 ⁇ m thick sections were deparaffinized, rehydrated and endogenous peroxidase blocked with 0.3% hydrogen peroxide in methanol.
  • the sections were autoclaved in 10 mM citrate buffer (pH 6.0) for 5 minutes at 105 ° C. to recover the antigen.
  • Primary rabbit polyclonal anti-GALNT6 antibody (HPA011762, Sigma-Aldrich, St. Louis, MO, USA) diluted 500 fold in 10 mM phosphate buffered saline (PBS) containing Tween 20 (Sigma-Aldrich)
  • An anti-GALNT6 antibody solution was prepared, and the sections were incubated overnight at 4 ° C. with the antibody solution.
  • FIG. 1A and FIG. 1B Representative images of immunohistochemical staining of GALNT6 protein are shown in FIG. Of the 304 normal colonic mucosa sections, 92.8% were considered negative for GALNT 6 (arrowheads in FIG. 1A and FIG. 1B), indicating that GALNT 6 protein expression was not detected in most normal colonic mucosal cells .
  • GALNT6 protein expression was not seen in 49 (14.6%) of the 335 colon cancer sections (FIGS. 1F and G). Of these 49 sections, 10 were from stage I, 19 from stage II, 15 from stage III, and 5 from stage IV colorectal cancer patients.
  • GALNT6 is not expressed in normal colon mucosa, expression of GALNT6 is high in precancerous and preinvasive states, and that expression of GALNT6 is most in patients with various stages of colorectal cancer. Although high, it has been shown that GALNT6 expression is reduced or eliminated in a part of colon cancer patients (about 15%).
  • Example 2 Providestic evaluation after resection surgery for colorectal cancer patients.
  • GALNT6 negative or positive was determined based on the results of immunohistochemical staining as described in Example 1. The patient's clinical information was obtained retrospectively by reviewing the medical records.
  • Cumulative survival rates were estimated by the Kaplan-Meier method. As survival rates, disease-specific survival rates and overall survival rates were used. Disease specific survival rates and overall survival rates are defined as the time from the day of surgery to death from cancer and the time from the day of surgery to death from any cause. Survival rates between the two groups (GALNT6 positive group and GALNT6 negative group) were compared by the log rank test. All statistical analyzes were two-sided and were performed using Graphpad Prism v6.0 (Graphpad Software, La Jolla, CA, USA) and SPSS Statistics version 24 (IBM, NY, USA) . All P values were two-sided, and P values less than 0.05 were considered statistically significant.
  • stage I-IV colon cancer patients with GALNT6-positive tumors showed high disease-specific survival (FIG. 2A; 5-year survival 95.7%, 10-year survival 90.8%).
  • GALNT6-positive colon cancer patients show high disease-specific survival (Fig. 2C; 5-year survival rate 95.4%, 10-year survival rate 90.7%), while GALNT 6 negative colon Cancer patients showed significantly lower disease-specific survival rates (P ⁇ 0.0001) compared to GALNT6-positive patients (FIG. 2C; 5-year survival rate 57.1%, 10-year survival rate 57.1%).
  • the difference in survival rates between GALNT6-positive patients and negative patients was remarkable especially in stage III colon cancer patients.
  • the overall survival rate also showed the same tendency as the disease-specific survival rate.
  • Example 3 (Efficacy of chemotherapy in colorectal cancer patients) We evaluated the prognosis of 190 primary colorectal cancer patients in stages II-III who underwent curative resection between 1990 and 2010 at Fukushima Medical University Hospital. The stage of the tumor was determined as described in Example 1. For colorectal cancer tissues excised from patients, GALNT6 negative or positive was determined based on the results of immunohistochemical staining as described in Example 1. The patient's clinical information was obtained retrospectively by reviewing the medical records.
  • SW480 cells were purchased from American Typed Culture Collection (ATCC, Manassas, Va., USA).
  • ATCC American Typed Culture Collection
  • SW480 is a humidified atmosphere containing 5% CO 2 in RPMI-1640 medium (Promega, Madison, WI, USA) with 10% fetal bovine serum and penicillin / streptomycin (ThermoFisher Scientific, Waltham, MA, USA) Maintained below.
  • Quantitative RT-PCR was performed as follows. Total RNA was extracted using TRIzol reagent, and 1 ⁇ g of total RNA was reverse transcribed using SuperScript III First-Strand Synthesis System (ThermoFisher Scientific) according to the manufacturer's instructions. QRT-PCR targeting GALNT6 (Assay ID Hs00926629_m1) and ACTB (Hs99999903_m1) was performed by TaqMan in triplicate in a 7500 real time PCR system using TaqMan Gene Expression Master Mix (ThermoFisher Scientific). Relative expression levels were determined by the 2- ⁇ Ct method, by SDS software according to the manufacturer's instructions, and ACTB was used as a calibration gene.
  • the membrane is blocked with 5% non-fat dry skimmed milk powder (Cell signaling Technology), and rabbit anti-GALNT6 antibody ((# HPA011762, 1: 250, Atlas Antibodies) or mouse anti- ⁇ -actin antibody ((# SC-69879, as primary antibody)
  • the membrane was incubated with goat anti-rabbit or anti-mouse HRP secondary antibody (Santa Cruz Biotechnology) and developed with SuperSignal West Pico luminescent Substrate (ThermoFisher Scientific), LAS 4000 imager. It observed by (GE Healthcare).
  • SiRNA transfection was performed as follows. In the logarithmic phase, cells are seeded in 6-well plates and Lipofectamine RNAiMAX reagent (ThermoFisher) using siRNA oligonucleotide or scrambled control (Ambion (R) Silencer Select, s22154, s22155 and negative control # 1, ThermoFisher Scientific). Transfection was performed according to the manufacturer's instructions. After 48 hours, cells were harvested. The experiments were performed at least three times and knockdown efficiency was assessed by qRT-PCR and Western blot analysis.
  • Cell proliferation assays were performed using Cell Counting Kit-8 (CCK-8, Dojin Kagaku) according to the manufacturer's instructions. Briefly, 24 h after transfection, three groups of SW480 cells were harvested, resuspended and seeded in 96 well plates at a density of 4 ⁇ 10 3 cells / well. After incubation for 24, 48 and 72 hours after transfection in complete medium, 10 ⁇ l of CCK-8 reagent was added and incubated for 3 hours in a humidified atmosphere containing 5% CO 2 . Absorbance at 450 nm was measured using a microplate reader. The experiment was performed three times in triplicate.
  • Apoptotic cells were performed using the Annexin V-PE / 7-AAD Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Briefly, cells transfected with GALNT6 siRNA and control siRNA were seeded in 6 well plates. After 48 hours of incubation, cells were treated with Annexin V and 7-AAD labeling and flow cytometry was performed. Annexin V positive cells were considered as apoptotic cells. The 5-FU cytotoxicity assay was performed as follows. SW480 cells transfected with GALNT6 siRNA and control siRNA were seeded at a density of 5 ⁇ 10 3 / well in 96 well plates.
  • SW480 cell line was used in the following experiments. In addition, it has been confirmed in advance that SW480 cells show relatively high GALNT6 mRNA expression and protein expression (data not shown).
  • GALNT6 expression contributes to the presence or absence of sensitivity to 5-FU treatment.
  • a significant increase in 5-FU resistance was observed in GALNT6 knockdown cells relative to cells treated with control siRNA (FIG. 4D).
  • apoptosis was significantly suppressed in 5-FU-treated GALNT6 knockdown cells (FIG. 4E).

Abstract

Provided is a biomarker for predicting the prognosis of a colon cancer patient, and/or for determining the efficacy of an anticancer agent in a colon cancer patient. Provided is a biomarker for predicting the prognosis of a colon cancer patient and/or for determining the efficacy of an anticancer agent in a colon cancer patient, the biomarker comprising a GALNT6 protein or peptide fragment thereof, or a GALNT6 gene transcription product or a nucleic acid fragment thereof.

Description

大腸がんの予後バイオマーカーPrognostic biomarkers for colorectal cancer
　本発明は、大腸がん患者の予後を予測するための及び/又は大腸がん患者に対する抗がん剤の有効性を判定するためのバイオマーカーに関する。 The present invention relates to a biomarker for predicting the prognosis of colorectal cancer patients and / or determining the efficacy of an anticancer agent for colorectal cancer patients.
　大腸がん(colorectal cancer、CRC)は、診断及び治療が大きく進歩しているにもかかわらず、世界中のがん死の主要な原因である。臨床現場で現在使用されている唯一の大腸がん予後分類は、臨床病理学的な病期(ステージ)分類であり、ステージに応じて治療方針が選択される。しかし、臨床病理学的に同一ステージと判定された大腸がんでも、薬剤感受性やがんの再発及び患者の生存などの臨床転帰は著しく異なる。例えば、ステージIII大腸がん患者は、根治手術後に補助化学療法を行うことが標準的であるが、約30～40％の患者で大腸がんが再発する(非特許文献1～3)。したがって、大腸がん患者の予後を予測することは重要であり、予後予測に基づく個別的な大腸がん患者のマネージメントが求められている。しかし、大腸がんの予後予測に臨床的に使用することができる臨床因子及び分子バイオマーカーは知られていない。また、大腸がん患者に対する抗がん剤の有効性を判定することができれば大腸がんの治療に役立ち得るため、抗がん剤の有効性の判定に有効なバイオマーカーが求められている。 Colorectal cancer (CRC) is the leading cause of cancer death worldwide, despite major advances in diagnosis and treatment. The only colorectal cancer prognostic classification currently used in the clinical setting is a clinicopathologic staging (stage) classification, and a treatment policy is selected according to the stage. However, clinical outcomes such as drug sensitivity, recurrence of cancer and survival of patients are significantly different even for colorectal cancer that is determined to be at the same stage clinicopathologically. For example, patients with stage III colorectal cancer are generally given adjuvant chemotherapy after radical surgery, but about 30 to 40% of patients recur colon cancer (non-patent documents 1 to 3). Therefore, it is important to predict the prognosis of colorectal cancer patients, and management of individual colorectal cancer patients based on the prognosis is required. However, there are no known clinical factors and molecular biomarkers that can be used clinically for prognosis of colorectal cancer. In addition, since it can be useful for the treatment of colon cancer if the efficacy of the anticancer agent for colon cancer patients can be determined, a biomarker effective for the assessment of the efficacy of the anticancer agent is required.
　本発明は、大腸がん患者の予後を予測するための、及び/又は大腸がん患者に対する抗がん剤の有効性を判定するためのバイオマーカーを提供することを課題とする。 An object of the present invention is to provide a biomarker for predicting the prognosis of colorectal cancer patients and / or determining the efficacy of an anti-cancer agent for colorectal cancer patients.
　本発明者らは、上記課題を解決するため、鋭意検討を重ね、大腸がん患者から切除された大腸がん組織の糖転移酵素GALNT6タンパク質の発現を調べた。その結果、ステージに関係なくほとんどの大腸がん患者は、GALNT6タンパク質を発現しているGALNT6陽性であったが、一部(約15％)の患者はGALNT6陰性であることを見出した。さらに、本発明者らは、GALNT6陽性の大腸がん患者は、大腸がん切除手術後の予後が良いのに対し、GALNT6陰性の大腸がん患者は、大腸がん切除手術後の予後が悪い傾向があること、及びGALNT6陽性の大腸がん患者は、GALNT6陰性の大腸がん患者よりも抗癌剤の有効性が高い傾向があることを見出した。本発明者は、これらの発見に基づいて本発明を完成するに至った。 Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors repeatedly investigated and examined the expression of the glycosyltransferase GALNT6 protein in colorectal cancer tissues excised from colorectal cancer patients. As a result, most colon cancer patients regardless of stage were found to be GALNT6 positive expressing the GALNT6 protein, but some (about 15%) patients were found to be GALNT6 negative. Furthermore, the inventors of the present invention show that patients with GALNT6-positive colorectal cancer have a good prognosis after colorectal cancer removal surgery, while patients with GALNT6-negative colorectal cancer have a poor prognosis after colorectal cancer removal surgery. We found that there was a tendency and that GALNT6-positive colon cancer patients tended to be more effective than anticancer agents than GALNT6-negative colon cancer patients. The inventors have completed the present invention based on these findings.
　すなわち本発明は以下を包含する。
　[１]GALNT6タンパク質若しくはそのペプチド断片、又はGALNT6遺伝子の転写産物若しくはその核酸断片からなる、大腸がん患者の予後を予測するためのバイオマーカー。
　[２]GALNT6タンパク質若しくはそのペプチド断片、又はGALNT6遺伝子の転写産物若しくはその核酸断片からなる、大腸がん患者に対する抗がん剤の有効性を判定するためのバイオマーカー。
　[３]抗がん剤が5-FUである、[２]に記載のバイオマーカー。
　[４]前記GALNT6タンパク質が、以下の(a)～(c)のいずれかのタンパク質である、[１]～[３]のいずれかに記載のバイオマーカー。
　(a)配列番号1で示されるアミノ酸配列からなるタンパク質、
　(b)配列番号1で示されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなるタンパク質、及び
　(c)配列番号1で示されるアミノ酸配列に対して90％以上のアミノ酸同一性を有するアミノ酸配列からなるタンパク質
　[５]前記GALNT6遺伝子が、[４]に示すタンパク質をコードする、[１]～[３]のいずれかに記載のバイオマーカー。
　[６]大腸がん患者の予後を予測するための、[１]及び[４]～[５]のいずれかに記載のバイオマーカーの使用。
　[７]大腸がん患者に対する抗がん剤の有効性を判定するための、[２]～[５]のいずれかに記載のバイオマーカーの使用。
　[８]前記大腸がんが、ステージI～IVの大腸がんである、[６]又は[７]に記載の使用。
　[９]前記大腸がんが、ステージIIIの大腸がんである、[８]に記載の使用。
　[１０]大腸がん患者の予後を予測する方法であって、
　大腸がん患者から得た大腸がん細胞又は組織における、[１]及び[４]～[５]のいずれかに記載のバイオマーカーの量を測定する測定工程、及び
　前記測定工程で得られた測定結果に基づいて、大腸がん患者の予後を予測する予測工程を含み、
　ここで、前記大腸がん細胞又は組織が前記バイオマーカーについて陰性である場合、前記大腸がん患者の予後は悪いと予測する、方法。
　［１１］大腸がん患者に対する抗がん剤の有効性を判定する方法であって、
　大腸がん患者から得た大腸がん細胞又は組織における、[２]～[５]のいずれかに記載のバイオマーカーの量を測定する測定工程、及び
　前記測定工程で得られた測定結果に基づいて、大腸がん患者に対する抗がん剤の有効性を判定する判定工程を含み、
　ここで、前記大腸がん細胞又は組織が前記バイオマーカーについて陰性である場合、大腸がん患者に対する抗がん剤の有効性は低いと判定する、方法。
　[１２][１]及び[４]～[５]のいずれかに記載のバイオマーカーの量を測定するための試薬を含む、大腸がん患者の予後を予測するためのキット。
　[１３][２]～[５]のいずれかに記載のバイオマーカーの量を測定するための試薬を含む、大腸がん患者に対する抗がん剤の有効性を判定するためのキット。 That is, the present invention includes the following.
[1] A biomarker for predicting the prognosis of colorectal cancer patients, comprising a GALNT6 protein or a peptide fragment thereof, or a transcript of a GALNT6 gene or a nucleic acid fragment thereof.
[2] A biomarker for determining the efficacy of an anticancer agent for colorectal cancer patients, comprising a GALNT6 protein or a peptide fragment thereof, or a transcript of a GALNT6 gene or a nucleic acid fragment thereof.
[3] The biomarker according to [2], wherein the anticancer agent is 5-FU.
[4] The biomarker according to any one of [1] to [3], wherein the GALNT6 protein is any one of the following (a) to (c):
(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,
(b) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and (c) 90% of the amino acid sequence shown in SEQ ID NO: 1 A protein consisting of an amino acid sequence having the above amino acid identity [5] The biomarker according to any one of [1] to [3], wherein the GALNT6 gene encodes the protein shown in [4].
[6] Use of the biomarker according to any one of [1] and [4] to [5] for predicting the prognosis of colorectal cancer patients.
[7] Use of the biomarker according to any one of [2] to [5] for determining the efficacy of an anticancer agent for colorectal cancer patients.
[8] The use according to [6] or [7], wherein the colorectal cancer is stage I-IV colorectal cancer.
[9] The use according to [8], wherein the colon cancer is stage III colon cancer.
[10] A method for predicting the prognosis of a patient with colorectal cancer,
Measuring the amount of the biomarker according to any one of [1] and [4] to [5] in a colon cancer cell or tissue obtained from a colon cancer patient, and the measuring step Including a prediction step of predicting the prognosis of a colorectal cancer patient based on the measurement result,
Here, if the colorectal cancer cells or tissues are negative for the biomarker, then the prognosis of the colorectal cancer patient is predicted to be poor.
[11] A method of determining the efficacy of an anti-cancer agent for colorectal cancer patients, comprising
Based on the measurement result obtained in the measurement step of measuring the amount of the biomarker according to any one of [2] to [5] in colon cancer cells or tissues obtained from a colon cancer patient, and the measurement step And determining the effectiveness of the anti-cancer drug in patients with colorectal cancer,
Here, when the colon cancer cell or tissue is negative for the biomarker, it is determined that the efficacy of the anticancer agent for colon cancer patients is low.
[12] A kit for predicting the prognosis of a colorectal cancer patient, comprising a reagent for measuring the amount of the biomarker according to any of [1] and [4] to [5].
[13] A kit for determining the efficacy of an anticancer agent for colorectal cancer patients, comprising a reagent for measuring the amount of the biomarker according to any one of [2] to [5].
　本明細書は本願の優先権の基礎となる日本国特許出願番号2017-170157号の開示内容を包含する。 The present specification includes the disclosure content of Japanese Patent Application No. 2017-170157 based on which the priority of the present application is based.
　本発明により、大腸がん患者の予後を予測するための、及び/又は大腸がん患者に対する抗がん剤の有効性を判定するためのバイオマーカーが提供される。 According to the present invention, a biomarker is provided to predict the prognosis of colorectal cancer patients and / or to determine the efficacy of anti-cancer agents for colorectal cancer patients.
(A)大腸腺腫(矢印で示される)、及び隣接する正常大腸粘膜(矢頭で示される)、(B)正常大腸粘膜、(C)大腸腺腫、並びに(D～G)大腸がんにおける、GALNT6タンパク質発現についての免疫組織化学染色の代表的な画像を示す写真である。(A)、(D)及び(F)は100倍、(B)、(C)、(E)、及び(G)は400倍の倍率で画像を取得した。GALNT 6 in (A) colorectal adenomas (indicated by arrows) and adjacent normal colon mucosa (indicated by arrowheads), (B) normal colon mucosa, (C) colon adenomas, and (D to G) colon cancer FIG. 1 is a photograph showing a representative image of immunohistochemical staining for protein expression. Images of (A), (D) and (F) were acquired at 100 ×, (B), (C), (E) and (G) at 400 × magnification. 免疫組織化学染色で判定したGALNT6陽性又は陰性の大腸がん患者における、治癒手術後の疾患特異的生存率を示すグラフである。(A)ステージI～IVの267人の患者、(B)ステージII～IIIの195人の患者、及び(C)ステージIIIの80人の患者の生存率を示す。It is a graph which shows the disease specific survival rate after curative surgery in a GALNT6 positive or negative colon cancer patient determined by immunohistochemical staining. The survival rates of (A) 267 patients in stages I-IV, (B) 195 patients in stages II-III, and (C) 80 patients in stage III are shown. 免疫組織化学によるGALNT6の発現陽性患者と陰性患者における無病生存率のカプランマイヤー曲線を示すグラフである。結果は手術後に化学療法を処置した患者と、手術のみを行った患者に分けて示す。(A)外科手術後にアジュバント化学療法を処置したステージII～IIIの114人の患者、(B)外科手術のみで処置したステージIII～IIIの76人の患者の結果を示す。(C)外科手術後にアジュバント化学療法を処置したステージIIIの56人の患者、(D)外科手術のみで処置したステージIIIの22人の患者の結果を示す。It is a graph which shows the Kaplan-Meier curve of disease-free survival rate in the positive | positive patient and negative patient of the expression of GALNT6 by immunohistochemistry. The results are shown separately for patients treated with chemotherapy after surgery and patients who received surgery alone. (A) Results of 114 patients of stages II-III treated with adjuvant chemotherapy after surgery, (B) of 76 patients of stages III-III treated with surgery only. (C) Results of 56 patients in stage III treated with adjuvant chemotherapy after surgery, (D) 22 patients in stage III treated with surgery alone. 図４は、GALNT6サイレンシングの生物学的効果を示す。GALNT6を標的とするsiRNA（siGALNT6-1及びsiGALNT6-2）又はスクランブル対照のSW480細胞へのトランスフェクション後の、GALNT6ノックダウンの効果を、qRT-PCR(A)及びウエスタンブロッティング(B)により確かめた。(C)様々な時点においてCCK-8アッセイで測定した細胞増殖を示す。結果は、3回の独立した実験の平均値±標準偏差として表す。(D)細胞生存性に対する5-FU処置の用量応答効果を示す。結果は、3回の独立した実験の平均値±標準偏差として表す(*P < 0.05、**P <0.01)。(E)10μMの5-FUで処置した細胞において、アポトーシスをAnnexin V-PE及び7-AADで標識したフローサイトメトリーによって解析した。結果は、3回の独立した実験の平均値±標準偏差として表す(*P < 0.05)。FIG. 4 shows the biological effects of GALNT6 silencing. The effect of GALNT6 knockdown was confirmed by qRT-PCR (A) and Western blotting (B) after transfection of GALNT6 targeting siRNA (siGALNT6-1 and siGALNT6-2) or scrambled control into SW480 cells . (C) Cell growth measured by CCK-8 assay at various time points. The results are expressed as mean ± standard deviation of 3 independent experiments. (D) shows the dose response effect of 5-FU treatment on cell viability. The results are expressed as mean ± standard deviation of three independent experiments (* P <0.05, ** P <0.01). (E) In cells treated with 10 μM 5-FU, apoptosis was analyzed by flow cytometry labeled with Annexin V-PE and 7-AAD. The results are expressed as mean ± standard deviation of three independent experiments (* P <0.05).
　以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
(大腸がん患者の予後を予測するための及び/又は抗がん剤の有効性を判定するためのバイオマーカー)
　本発明は、大腸がん患者の予後を予測するためのバイオマーカーを提供する。本発明はまた、大腸がん患者に対する抗がん剤の有効性を判定するためのバイオマーカーを提供する。本発明に係るバイオマーカーは、GALNT6タンパク質若しくはそのペプチド断片、又はGALNT6遺伝子の転写産物若しくはその核酸断片からなる。 (Biomarkers for predicting the prognosis of colorectal cancer patients and / or determining the efficacy of anticancer drugs)
The present invention provides a biomarker for predicting the prognosis of colorectal cancer patients. The present invention also provides a biomarker for determining the efficacy of an anti-cancer agent for colorectal cancer patients. The biomarker according to the present invention consists of the GALNT6 protein or its peptide fragment, or the transcript of the GALNT6 gene or its nucleic acid fragment.
　本明細書において、「大腸がん」は、結腸、直腸及び肛門を含む大腸に発生するがんを指す。大腸がんは、特に、結腸がん及び直腸がんを含む。 As used herein, "colorectal cancer" refers to cancer that develops in the large intestine, including the colon, rectum and anus. Colorectal cancer includes, in particular, colon cancer and rectal cancer.
　本明細書において、「予後」は、大腸がん患者において、大腸がんの切除手術後に予測される経過(例えば、生死)を指す。「予後の予測」は、生存期間、又は手術から一定期間後(例えば、1、3、5、10、15若しくは20年後又はそれ以上の時点)の生存率の予測であってもよい。予後の予測は、予後の判定、評価、又は診断ということもできる。 As used herein, "prognosis" refers to the predicted course (e.g., life and death) after resection surgery for colorectal cancer in colorectal cancer patients. "Prognostic" may be prediction of survival time or survival rate after a certain period of time (eg, 1, 3, 5, 10, 15 or 20 years or more after surgery). Predicting prognosis can also be referred to as determining, evaluating, or diagnosing prognosis.
　グリコシル化、すなわちタンパク質の糖鎖付加は、多様な生理学的過程を調節する、一般的かつ重要なタンパク質翻訳後修飾の1つである。グリコシル化は、標的タンパク質へ単一の糖残基を逐次的に付加することを伴い、その結果、グリカンが伸長する。さらなる化学修飾及び分枝化により、最終的に、様々なグリカン構造が形成され得る。グリコシル化は、一般に、糖転移酵素(グリコシルトランスフェラーゼ)の多酵素反応によって生じる。 Glycosylation, ie, glycosylation of proteins, is one of the common and important post-translational modifications of proteins that regulate diverse physiological processes. Glycosylation involves the sequential addition of single sugar residues to a target protein, which results in glycan elongation. Additional chemical modifications and branching can ultimately form various glycan structures. Glycosylation generally occurs by the multienzyme reaction of glycosyltransferases (glycosyl transferases).
　GALNT6(ポリペプチドN-アセチルガラクトサミニルトランスフェラーゼ6)タンパク質は、UDP-N-アセチル-α-D-ガラクトサミン:ポリペプチドN-アセチルガラクトサミニルトランスフェラーゼ(GalNAc-T)ファミリーに属する糖転移酵素である。GalNAc-T酵素は、N-アセチルガラクトサミン(GalNAc)の、標的タンパク質上のセリン又はトレオニン残基への糖転移を触媒することによって、グリコシル化を開始することが知られている。 The GALNT6 (polypeptide N-acetylgalactosaminyltransferase 6) protein is a glycosyltransferase belonging to the UDP-N-acetyl-α-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) family . The GalNAc-T enzyme is known to initiate glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to a serine or threonine residue on a target protein.
　本発明では、原則として、大腸がん患者の内在遺伝子に由来するGALNT6タンパク質又はその遺伝子転写産物がバイオマーカーとなり得る。例えば、前記患者がヒトであれば、ヒトGALNT6遺伝子に由来するヒトGALNT6タンパク質及びヒトGALNT6遺伝子の転写産物(mRNA)が本発明のバイオマーカーとなり得る。 In the present invention, in principle, the GALNT6 protein or its gene transcript derived from an endogenous gene of a colorectal cancer patient can be a biomarker. For example, if the patient is a human, human GALNT6 protein derived from human GALNT6 gene and transcript (mRNA) of human GALNT6 gene can be a biomarker of the present invention.
　GALNT6タンパク質の具体例として、配列番号1で示される622残基のアミノ酸配列からなるヒト由来のGALNT6(ヒトGALNT6)タンパク質が挙げられる。 A specific example of the GALNT6 protein is a human-derived GALNT6 (human GALNT6) protein consisting of the amino acid sequence of 622 residues shown in SEQ ID NO: 1.
　また、GALNT6タンパク質には、配列番号1で示されるGALNT6タンパク質と機能的に同等の活性を有するGALNT6バリアントや他生物種のGALNT6オルソログも包含される。具体的には、配列番号1で示されるアミノ酸配列において1若しくは複数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、あるいは配列番号1で示されるアミノ酸配列に対して90％、95％以上、97％以上、98％以上又は99％以上のアミノ酸同一性を有するGALNT6タンパク質が包含される。 In addition, the GALNT6 protein also includes a GALNT6 variant having functionally equivalent activity to the GALNT6 protein shown by SEQ ID NO: 1 and GALNT6 orthologs of other biological species. Specifically, an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, or 90%, 95% or more relative to the amino acid sequence shown in SEQ ID NO: 1 Included are GALNT6 proteins having an amino acid identity of 97% or more, 98% or more, or 99% or more.
　本明細書において「複数個」とは、例えば、2～20個、2～15個、2～10個、2～7個、2～5個、2～4個又は2～3個をいう。また、アミノ酸の置換は、保存的アミノ酸置換が望ましい。「保存的アミノ酸置換」とは、電荷、側鎖、極性、芳香族性等の性質の類似するアミノ酸間の置換をいう。性質の類似するアミノ酸は、例えば、塩基性アミノ酸(アルギニン、リジン、ヒスチジン)、酸性アミノ酸(アスパラギン酸、グルタミン酸)、無電荷極性アミノ酸(グリシン、アスパラギン、グルタミン、セリン、トレオニン、システイン、チロシン)、無極性アミノ酸(ロイシン、イソロイシン、アラニン、バリン、プロリン、フェニルアラニン、トリプトファン、メチオニン)、分枝鎖アミノ酸(ロイシン、バリン、イソロイシン)、芳香族アミノ酸(フェニルアラニン、チロシン、トリプトファン、ヒスチジン)等に分類することができる。 In the present specification, the term "plural" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Also, amino acid substitution is preferably conservative amino acid substitution. "Conservative amino acid substitution" refers to substitution between amino acids of similar properties such as charge, side chain, polarity, aromaticity and the like. Amino acids having similar properties are, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), nonpolar Organic amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched chain amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), etc. it can.
　本明細書において「アミノ酸同一性」とは、2つのアミノ酸配列を整列(アラインメント)し、必要に応じてギャップを導入して、両者のアミノ酸一致度が最も高くなるようにしたときに、配列番号1で示されるアミノ酸配列からなるGALNT6タンパク質の全アミノ酸残基に対する2つのアミノ酸配列間で同一アミノ酸残基の割合(％)をいう。アミノ酸同一性は、BLASTやFASTAによるタンパク質の検索システムを用いて算出することができる。 In the present specification, “amino acid identity” refers to the sequence number of the amino acid sequence when the two amino acid sequences are aligned (alignment) and a gap is introduced as needed to maximize the amino acid identity between the two. The ratio (%) of identical amino acid residues between two amino acid sequences relative to all amino acid residues of the GALNT6 protein consisting of the amino acid sequence represented by 1. Amino acid identity can be calculated using a protein search system by BLAST or FASTA.
　「GALNT6遺伝子」は、前記GALNT6タンパク質をコードする遺伝子である。GALNT6遺伝子の具体例として、配列番号1で示されるアミノ酸配列からなるヒトGALNT6タンパク質をコードするヒトGALNT6遺伝子が挙げられる。より具体的には、GALNT6遺伝子は、配列番号2で示される塩基配列からなる遺伝子である。 The "GALNT6 gene" is a gene encoding the above-mentioned GALNT6 protein. A specific example of the GALNT6 gene is a human GALNT6 gene encoding a human GALNT6 protein consisting of the amino acid sequence shown by SEQ ID NO: 1. More specifically, the GALNT6 gene is a gene consisting of the base sequence shown in SEQ ID NO: 2.
　また、GALNT6遺伝子には、配列番号2で示されるGALNT6遺伝子がコードするGALNT6タンパク質と機能的に同等の活性を有するGALNT6バリアントや他生物種のGALNT6オルソログをコードするGALNT6遺伝子も包含される。具体的には、配列番号2で示される塩基配列において1若しくは複数個の塩基が欠失、置換又は付加された塩基配列、あるいは配列番号2で示される塩基配列に対して90％、95％以上、97％以上、98％以上又は99％以上の塩基同一性を有するGALNT6遺伝子が包含される。さらに、配列番号2で示される塩基配列に対して相補的な塩基配列の一部からなる核酸断片と高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り、かつGALNT6タンパク質と機能的に同等の活性を有するタンパク質をコードする遺伝子が包含される。 In addition, the GALNT6 gene also includes a GALNT6 variant having an activity equivalent to that of the GALNT6 protein encoded by the GALNT6 gene shown in SEQ ID NO: 2 and a GALNT6 gene encoding GALNT6 ortholog of other species. Specifically, 90%, 95% or more of the base sequence in which one or more bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2 or the base sequence shown in SEQ ID NO: 2 And GALNT6 genes having a base identity of 97% or more, 98% or more, or 99% or more. Furthermore, it comprises a nucleotide sequence that hybridizes under high stringency conditions with a nucleic acid fragment consisting of a portion of the nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2, and is functionally equivalent to the GALNT6 protein Included are genes encoding proteins having activity.
　本明細書において「塩基同一性」とは、2つの塩基配列を整列(アラインメント)し、必要に応じてギャップを導入して、両者の塩基一致度が最も高くなるようにしたときに、配列番号2で示される塩基配列からなるGALNT6遺伝子の全塩基に対する2つの塩基配列間で同一塩基の割合(％)をいう。 In the present specification, “base identity” refers to the sequence number when the two base sequences are aligned (aligned), and a gap is introduced as necessary to maximize the degree of base identity between the two. The ratio (%) of identical bases between two base sequences to all bases of the GALNT6 gene consisting of the base sequence shown in 2.
　本明細書において「高ストリンジェントな条件下でハイブリダイズする」とは、低塩濃度及び/又は高温の条件下でハイブリダイゼーションと洗浄を行うことをいう。例えば、6×SSC、5×Denhardt試薬、0.5％SDS、100μg/mL変性断片化サケ***DNA中で65℃～68℃にてプローブと共にインキュベートし、その後、2×SSC、0.1％SDSの洗浄液中で室温から開始して、洗浄液中の塩濃度を0.1×SSCまで下げ、かつ温度を68℃まで上げて、バックグラウンドシグナルが検出されなくなるまで洗浄することが例示される。高ストリンジェントなハイブリダイゼーションの条件については、Green, M.R. and Sambrook, J., 2012, Molecular Cloning： A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New Yorkに記載されているので参考にすることができる。 As used herein, “hybridize under highly stringent conditions” refers to hybridization and washing under conditions of low salt concentration and / or high temperature. For example, incubate with the probe at 65 ° C. to 68 ° C. in 6 × SSC, 5 × Denhardt's reagent, 0.5% SDS, 100 μg / mL denatured fragmented salmon sperm DNA, then in a wash solution of 2 × SSC, 0.1% SDS Starting from room temperature, the salt concentration in the wash solution is lowered to 0.1 × SSC, and the temperature is raised to 68 ° C. to wash until background signal is not detected. The conditions for highly stringent hybridization are described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. It can be helpful.
　このようなGALNT6遺伝子の塩基配列情報は、公共のデータベース(GenBank、EMBL、DDBJ)より検索可能である。例えば、配列番号2で示されるGALNT6遺伝子の既知塩基配列情報に基づいて、塩基同一性の高い遺伝子をデータベースから検索し、入手することができる。 Such nucleotide sequence information of GALNT6 gene can be searched from public databases (GenBank, EMBL, DDBJ). For example, based on the known nucleotide sequence information of the GALNT6 gene shown by SEQ ID NO: 2, genes with high base identity can be searched and obtained from the database.
　「GALNT6遺伝子の転写産物」とは、GALNT6 mRNAを意味する。mRNAは、mRNA前駆体(pre-mRNA)及び成熟mRNA(mature mRNA)を問わない。通常、mRNA前駆体は、核内において直ちにスプライシングされて、成熟mRNA成熟体となることから、実質的に本発明のバイオマーカーとなるGALNT6遺伝子の転写産物は、GALNT6成熟mRNAである。 "Transcript of GALNT6 gene" means GALNT6 mRNA. The mRNA may be a pre-mRNA (pre-mRNA) or a mature mRNA (mature mRNA). Usually, since the pre-mRNA is immediately spliced in the nucleus to become a mature mRNA mature body, the transcript of the GALNT6 gene substantially serving as a biomarker of the present invention is a GALNT6 mature mRNA.
　本明細書において「ペプチド断片」とは、GALNT6タンパク質を構成するアミノ酸配列の一部からなるペプチド断片であって、その断片を構成するアミノ酸配列からGALNT6タンパク質の断片であることを同定することができるものをいう。通常は、GALNT6タンパク質の全長アミノ酸配列のうちの20個以上200個以下、30個以上150個以下、40個以上100個以下、又は50個以上80個以下の連続するアミノ酸残基からなるペプチドであればよい。 In the present specification, "peptide fragment" is a peptide fragment consisting of a part of the amino acid sequence constituting the GALNT6 protein, which can be identified as a fragment of the GALNT6 protein from the amino acid sequence constituting the fragment I say something. Usually, a peptide consisting of 20 or more and 200 or less, 30 or more and 150 or less, 40 or more and 100 or less, or 50 or more and 80 or less consecutive amino acid residues in the full-length amino acid sequence of GALNT6 protein I hope there is.
　本明細書において「核酸断片」とは、GALNT6 mRNAを構成する塩基配列の一部からなる核酸断片であって、その断片を構成する塩基配列からGALNT6 mRNAの断片であることを同定することができるものをいう。通常は、GALNT6 mRNAの全長塩基配列のうちの40個以上600個以下、50個以上450個以下、60個以上300個以下、又は70個以上200個以下の連続する塩基からなる核酸であればよい。 In the present specification, a "nucleic acid fragment" is a nucleic acid fragment consisting of a part of the nucleotide sequence constituting GALNT6 mRNA, and it can be identified from the nucleotide sequence constituting the fragment that it is a fragment of GALNT6 mRNA I say something. Generally, it is a nucleic acid consisting of 40 or more and 600 or less, 50 or more and 450 or less, 60 or more and 300 or less, or 70 or more and 200 or less consecutive bases in the full-length base sequence of GALNT6 mRNA. Good.
(バイオマーカーを指標とした予後の予測及び/又は抗がん剤の有効性の判定)
　本発明は、大腸がん患者の予後を予測するための、上述の本発明に係るバイオマーカーの使用を提供する。本発明はまた、大腸がん患者に対する抗がん剤の有効性を判定するための、上述の本発明に係るバイオマーカーの使用を提供する。 (Prognostic prediction of prognosis using biomarker as index and / or judgment of efficacy of anticancer agent)
The present invention provides the use of the above-mentioned biomarker according to the present invention for predicting the prognosis of colorectal cancer patients. The present invention also provides the use of the above-mentioned biomarker according to the present invention for determining the efficacy of an anti-cancer agent for colorectal cancer patients.
　本発明はまた、大腸がん患者の予後を予測する方法を提供する。本方法は、大腸がん患者から得た大腸がん細胞又は組織における、上述の本発明に係るバイオマーカーの量を測定する測定工程、及び前記測定工程で得られた測定結果に基づいて、大腸がん患者の予後を予測する予測工程を含む。測定工程は、インビトロで行うことができる。
　以下に各工程について具体的に説明する。 The present invention also provides a method of predicting the prognosis of colorectal cancer patients. This method comprises the steps of measuring the amount of the biomarker according to the present invention described above in a colon cancer cell or tissue obtained from a colon cancer patient, and the large intestine based on the measurement result obtained in the above measuring step. The prediction process which predicts the prognosis of a cancer patient is included. The measuring step can be performed in vitro.
Each step is specifically described below.
(1)測定工程
　大腸がんは、進行度の低い方からステージ0、I、II、III及びIVに分類される。本発明では、病期(ステージ)の決定は、国際対がん連合(UICC)のTNM分類(UICC第7版; Sobin LH, Gospodarowicz MK, Wittekind C, International Union against Cancer. TNM classification of malignant tumours, 7th edn. Wiley-Blackwell (2010))に基づいて行う。上記の国際対がん連合(UICC)のTNM分類を、UICC-TNM分類と本明細書で称する。UICC-TNM分類では、壁深達度(T分類)、リンパ節転移(N分類)及び遠隔転移(M分類)の3つの因子により、がん病変の進行度を分類する。UICC-TNM分類に基づく病期の決定は当業者の通常の知識に従って行うことができる。 (1) Measurement process Colorectal cancer is classified into stages 0, I, II, III and IV in ascending order of progression. In the present invention, the determination of stages is based on the International Association of Cancer and Cancer (UICC) TNM classification (UICC 7th edition; Sobin LH, Gospodarowicz MK, Wittekind C, International Union against Cancer. TNM classification of malignant tumors, 7th edn Conduct based on Wiley-Blackwell (2010). The above mentioned International Anti-Cancer Union (UICC) TNM classification is referred to herein as UICC-TNM classification. In UICC-TNM classification, the degree of progression of cancerous lesions is classified by three factors: wall depth (T classification), lymph node metastasis (N classification) and distant metastasis (M classification). The determination of the stage based on the UICC-TNM classification can be performed according to the ordinary knowledge of the person skilled in the art.
　具体的には、大腸がんが粘膜内に留まる(Tis)場合をステージ0、大腸がんが粘膜下層(T1)又は固有筋層(T2)に留まる場合をステージI、大腸がんが固有筋層を超え漿膜下層(T3)又は漿膜若しくは隣接臓器(T4)に浸潤する場合をステージIIと判定する。なおステージ0～IIではいずれもリンパ節転移を伴わない(N0)。リンパ節転移を伴う(N1～2)場合はステージIIIと判定する。ステージ0～IIIはいずれも遠隔転移のない(M0)がんであり、遠隔転移を伴う(M1)がんはステージIVと判定する。 Specifically, stage 0 when colon cancer stays in the mucosa (Tis), stage I when colon cancer stays in the submucosal layer (T1) or proper muscle layer (T2), stage I, colon cancer has its own muscle It is determined as Stage II if the stroma is infiltrated into the subserosal membrane (T3) or the serosa or adjacent organs (T4). In stages 0 to II, no lymph node metastasis is involved (N0). If lymph node metastasis is involved (N1-2), it is judged as stage III. Stages 0 to III all have distant metastasis (MO) and cancer with distant metastasis (M1) is classified as stage IV.
　本発明の対象となる患者が患う大腸がんは、ステージI～IV、好ましくはステージII及びIII、より好ましくはステージIIIの大腸がんであってよい。 The colorectal cancer which suffers from the subject of the present invention may be stage I to IV, preferably stage II and III, more preferably stage III colon cancer.
　本発明における大腸がん患者は、好ましくは哺乳動物、より好ましくは霊長類、最も好ましくはヒトである。 The colorectal cancer patient in the present invention is preferably a mammal, more preferably a primate, most preferably a human.
　本発明において使用する大腸がん細胞又は組織は、特に限定されないが、例えば、生検又は切除手術によって大腸がん患者から得ることができる。当該細胞又は組織は、そのままバイオマーカーの測定に用いてもよいが、測定のために適宜前処理してもよい。例えば、免疫組織化学染色法でバイオマーカーを検出する場合は、患者由来の試料からパラフィン包埋切片を調製してもよい。また、例えば、ウエスタンブロット法又はRT-PCR法によってバイオマーカーを検出する場合は、患者由来の試料からタンパク質抽出液又はmRNA抽出液を調製してもよい。 The colon cancer cells or tissues used in the present invention can be obtained from colon cancer patients by, for example, biopsy or excision surgery, although not particularly limited. The cells or tissues may be used as they are for measurement of a biomarker, but may be appropriately pretreated for measurement. For example, when detecting a biomarker by immunohistochemical staining, paraffin embedded sections may be prepared from a sample from a patient. Also, for example, in the case of detecting a biomarker by Western blotting or RT-PCR, a protein extract or mRNA extract may be prepared from a sample derived from a patient.
　本方法で測定すべきバイオマーカーは、GALNT6タンパク質若しくはそのペプチド断片又はGALNT6遺伝子の転写産物若しくはその核酸断片のいずれであってもよい。量(発現量)の測定は、発現の有無、又は発現量若しくは発現濃度の大小等を測定することを包含する。本発明において、「測定」という用語には、検出、定性、定量及び半定量のいずれもが包含される。 The biomarker to be measured in the present method may be either the GALNT6 protein or its peptide fragment or the transcript of the GALNT6 gene or its nucleic acid fragment. The measurement of the amount (expression amount) includes measuring the presence or absence of expression, or the size of expression amount or expression concentration, and the like. In the present invention, the term "measurement" includes any of detection, qualitative, quantitative and semi-quantitative.
　測定すべきバイオマーカーがGALNT6タンパク質又はそのペプチド断片の場合、その測定方法は、公知のタンパク質定量方法であればよく、特に限定はしないが、例えば、免疫学的検出法が挙げられる。 When the biomarker to be measured is the GALNT6 protein or its peptide fragment, the measuring method may be any known method for quantifying proteins, and is not particularly limited, but includes, for example, an immunological detection method.
　「免疫学的検出法」は、抗原である標的分子と特異的に結合する抗体又は抗体断片を用いて、標的分子の量を測定する方法である。 An "immunological detection method" is a method of measuring the amount of a target molecule using an antibody or antibody fragment that specifically binds to a target molecule that is an antigen.
　抗体は、哺乳動物及び鳥を含めたいずれの動物由来とすることができる。例えば、マウス、ラット、モルモット、ウサギ、ヤギ、ロバ、ヒツジ、ラクダ、ウマ、ニワトリ又はヒト等が挙げられる。 The antibodies can be from any animal, including mammals and birds. For example, mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens or humans can be mentioned.
　免疫学的検出法で使用する抗体は、特に限定されないが、モノクローナル抗体又はポリクローナル抗体を使用してよい。 The antibody used in the immunological detection method is not particularly limited, but a monoclonal antibody or a polyclonal antibody may be used.
　本明細書において「モノクローナル抗体」とは、単一免疫グロブリンのクローン群をいう。モノクローナル抗体を構成する各免疫グロブリンは、共通するフレームワーク領域及び共通する相補性決定領域を含み、同一抗原の同一エピトープを認識し、それに結合することができる。モノクローナル抗体は、単一細胞由来のハイブリドーマから得ることができる。 As used herein, "monoclonal antibody" refers to a clonal group of single immunoglobulins. Each immunoglobulin constituting a monoclonal antibody contains a common framework region and a common complementarity determining region, and can recognize and bind to the same epitope of the same antigen. Monoclonal antibodies can be obtained from single cell derived hybridomas.
　本明細書において「ポリクローナル抗体」とは、同一抗原の異なるエピトープを認識し結合する複数種の免疫グロブリン群をいう。ポリクローナル抗体は、標的分子を抗原として動物に免疫後、その動物の血清から得ることができる。 As used herein, "polyclonal antibody" refers to a plurality of immunoglobulin groups that recognize and bind to different epitopes of the same antigen. Polyclonal antibodies can be obtained from the serum of an animal after immunizing the animal with the target molecule as an antigen.
　抗体がポリクローナル抗体又はモノクローナル抗体の場合、免疫グロブリン分子には、IgG、IgM、IgA、IgE、及びIgDの各クラスが知られているが、本発明の抗体は、いずれのクラスであってもよい。好ましくはIgGである。 When the antibody is a polyclonal antibody or a monoclonal antibody, each class of immunoglobulin molecule is known: IgG, IgM, IgA, IgE, and IgD, but the antibody of the present invention may be any class. . Preferably it is IgG.
　GALNT6タンパク質を認識し結合するポリクローナル抗体、又はモノクローナル抗体を産生するハイブリドーマを作製する方法は、GALNT6タンパク質又はその断片を抗原として当該分野で公知の抗体作製方法に準じて行えばよい。抗体はまた、例えばSigma-Aldrich社等の製造業者から得てもよい。 A method for producing a polyclonal antibody that recognizes and binds to the GALNT6 protein or a hybridoma that produces a monoclonal antibody may be performed according to a method for producing an antibody known in the art using the GALNT6 protein or a fragment thereof as an antigen. Antibodies may also be obtained from manufacturers such as, for example, Sigma-Aldrich.
　本明細書において「抗体断片」とは、ポリクローナル抗体又はモノクローナル抗体の部分断片であって、該抗体が有する抗原特異的結合活性と実質的に同等の活性を有するポリペプチド鎖又はその複合体をいう。例えば、抗原結合部位を少なくとも1つ包含する抗体部分、すなわち、少なくとも1組のVLとVHを有するポリペプチド鎖、又はその複合体が該当する。具体例としては、免疫グロブリンを様々なペプチダーゼで切断することによって生じる多数の十分に特徴付けられた抗体断片等が挙げられる。より具体的な例としては、Fab、F(ab')2、Fab'等が挙げられる。これらの抗体断片は、いずれも抗原結合部位を包含しており、抗原である標的分子と特異的に結合する能力を有している。 As used herein, “antibody fragment” is a partial fragment of a polyclonal antibody or a monoclonal antibody, and refers to a polypeptide chain or a complex thereof having an activity substantially equivalent to the antigen-specific binding activity of the antibody. . For example, an antibody portion that includes at least one antigen binding site, ie, a polypeptide chain having at least one set of VL and VH, or a complex thereof, is relevant. Specific examples include many well-characterized antibody fragments and the like generated by cleaving immunoglobulin with various peptidases. More specific examples include Fab, F (ab ') 2 , Fab' and the like. All of these antibody fragments contain an antigen binding site, and have the ability to specifically bind to a target molecule that is an antigen.
　免疫学的検出法としては、例えば、免疫組織化学染色法、酵素免疫測定法(ELISA法、EIA法を含む)、ウエスタンブロット法、放射免疫測定(RIA)法、免疫沈降法、又はフローサイトメトリー法が挙げられる。 Immunological detection methods include, for example, immunohistochemical staining, enzyme-linked immunosorbent assay (including ELISA, EIA), western blotting, radioimmunoassay (RIA), immunoprecipitation, or flow cytometry. The law is mentioned.
　「免疫組織化学染色法」は、公知の方法を採用することができる。例えば、患者由来の試料をホルマリン固定後、パラフィンに包埋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してよい。切片試料は、場合により熱処理して抗原を賦活化し、その後、Dako EnVision+ System(Agilent社)などの市販の検出システムを用いて切片試料について免疫組織化学染色を行ってよい。 The "immunohistochemical staining method" can adopt a known method. For example, a sample derived from a patient may be formalin-fixed, embedded in paraffin, sliced into tissue pieces, and affixed to a slide glass, and used as a section sample. Section samples may optionally be heat treated to activate the antigen, and then immunohistochemical staining may be performed on the section samples using a commercially available detection system such as the Dako EnVision + System (Agilent).
　また、測定すべきバイオマーカーがGALNT6遺伝子転写産物又はその核酸断片の場合、その測定方法は、公知の核酸定量方法であればよく、特に限定はしないが、例えば、プライマーを用いる核酸増幅法又はプローブを用いるハイブリダイゼーション法が挙げられる。 In addition, when the biomarker to be measured is the GALNT6 gene transcript or its nucleic acid fragment, the measuring method may be any known nucleic acid quantitative method, and is not particularly limited. For example, a nucleic acid amplification method or a probe using a primer And hybridization methods using
　「核酸増幅法」は、フォワード/リバースプライマーセット用いて、標的核酸の特定の領域を核酸ポリメラーゼによって増幅させる方法をいう。核酸増幅法としては、例えば、RT-PCR(逆転写ポリメラーゼ連鎖反応)法などのPCR(ポリメラーゼ連鎖反応)法が挙げられる。 "Nucleic acid amplification method" refers to a method of amplifying a specific region of a target nucleic acid by a nucleic acid polymerase using a forward / reverse primer set. Examples of nucleic acid amplification methods include PCR (polymerase chain reaction) methods such as RT-PCR (reverse transcription polymerase chain reaction) method.
　「ハイブリダイゼーション法」は、検出すべき標的核酸の塩基配列の全部又は一部に相補的な塩基配列を有する核酸断片をプローブとして用い、その核酸と該プローブ間の塩基対合を利用して、標的核酸若しくはその断片を検出、定量する方法である。ハイブリダイゼーション法には、検出手段の異なるいくつかの方法が知られているが、例えば、ノザンハイブリダイゼーション法(ノザンブロットハイブリダイゼーション法)、in situハイブリダイゼーション法、又はマイクロアレイ法が挙げられる。 The “hybridization method” uses a nucleic acid fragment having a base sequence complementary to all or part of the base sequence of a target nucleic acid to be detected as a probe, and utilizes base pairing between the nucleic acid and the probe. It is a method of detecting and quantifying a target nucleic acid or a fragment thereof. As the hybridization method, several different detection means are known, and for example, Northern hybridization (Northern blot hybridization), in situ hybridization, or microarray method can be mentioned.
　プライマー及びプローブ等の核酸鎖は、公知のバイオマーカー配列情報を基に当業者に公知の方法により適宜設計し、化学合成などの公知の作製方法により得ることができる。 Nucleic acid chains such as primers and probes can be appropriately designed by methods known to those skilled in the art based on known biomarker sequence information, and obtained by known preparation methods such as chemical synthesis.
　上述の各測定法は、いずれも当該分野に公知の技術である。したがって、具体的な測定方法については、公知の方法に準じて行えばよい。例えば、Green, M.R. and Sambrook, J., 2012(前述)に記載の方法を参考にすることができる。 Each of the above-mentioned measurement methods is a technique known in the art. Therefore, the specific measurement method may be performed according to a known method. For example, the methods described in Green, MR and Sambrook, J., 2012 (described above) can be referred to.
(2)予測工程
　本工程では、前記測定工程で得られた測定結果に基づいて、大腸がん患者の予後を予測する。本工程は、前記測定工程で得られた測定結果から、前記大腸がん細胞又は組織がバイオマーカーについて陽性であるか又は陰性であるかを判定することを包含し得る。 (2) Prediction Step In this step, the prognosis of a colorectal cancer patient is predicted based on the measurement result obtained in the measurement step. This step may include determining from the measurement result obtained in the measurement step whether the colon cancer cell or tissue is positive or negative for the biomarker.
　免疫組織化学染色法を用いる場合、例えば、腫瘍細胞総数に対して染色された腫瘍細胞数が一定割合(例えば10％、15％又は20％)を超える場合、陽性と判定し、腫瘍細胞総数に対して染色された腫瘍細胞数が前記一定割合以下の場合、陰性と判定してもよい。 When using immunohistochemical staining, for example, when the number of tumor cells stained relative to the total number of tumor cells exceeds a certain percentage (for example, 10%, 15% or 20%), it is judged as positive and If the number of tumor cells stained against the above-mentioned fixed ratio or less, it may be judged as negative.
　あるいは、大腸がん患者から得た大腸がん細胞又は組織において測定したバイオマーカーの量を、対照試料(対照細胞又は組織)において測定した対照量と比較して、陽性又は陰性を判定してもよい。対照試料は、健常個体(例えば健常人)、又は良性の大腸腺腫患者に由来してもよい。本発明において「健常個体」とは、被験個体と同じ生物種の、がんに罹患していない健康な個体をいう。 Alternatively, the amount of the biomarker measured in colon cancer cells or tissues obtained from colon cancer patients is compared with the amount of control measured in a control sample (control cells or tissues) to determine positive or negative. Good. Control samples may be derived from healthy individuals (eg, healthy individuals) or benign colorectal adenoma patients. In the present invention, a "healthy individual" refers to a healthy individual not suffering from cancer of the same species as the test individual.
　本発明者らは、後述の実施例で述べるとおり、GALNT6タンパク質の発現量が、正常大腸粘膜で無く又は低く、一方、大腸腺腫組織で高いことを見出した。したがって、大腸がん患者から得た大腸がん細胞又は組織において測定したバイオマーカーの量が、健常個体に由来する対照試料において測定した対照量と比べて高い(例えば、統計学的に有意に高い)場合、当該大腸がん細胞又は組織はバイオマーカーについて陽性であると判定し得る。また、大腸がん患者から得た大腸がん細胞又は組織において測定したバイオマーカーの量が、健常個体に由来する対照試料において測定した対照量と比べて同じ又は低い(例えば、統計学的に有意に低い)場合、当該大腸がん細胞又は組織はバイオマーカーについて陰性であると判定し得る。 The present inventors have found that the expression level of GALNT6 protein is not in normal colon mucosa or low, while it is high in colon adenoma tissue, as described in the examples below. Therefore, the amount of biomarkers measured in colon cancer cells or tissues obtained from colon cancer patients is higher (eg, statistically higher) than the amount of control measured in control samples derived from healthy individuals If so, the colon cancer cell or tissue can be determined to be positive for the biomarker. In addition, the amount of biomarker measured in colon cancer cells or tissues obtained from patients with colorectal cancer is the same or lower than the amount of control measured in a control sample derived from a healthy individual (eg, statistically significant If so, the colon cancer cells or tissues can be determined to be negative for the biomarker.
　あるいは、大腸がん患者から得た大腸がん細胞又は組織において測定したバイオマーカーの量が、大腸腺腫患者に由来する対照試料において測定した対照量と比べて同じ又は高い(例えば、統計学的に有意に高い)場合、当該大腸がん細胞又は組織はバイオマーカーについて陽性であると判定し得る。また、大腸がん患者から得た大腸がん細胞又は組織において測定したバイオマーカーの量が、大腸腺腫患者に由来する対照試料において測定した対照量と比べて低い(例えば、統計学的に有意に低い)場合、当該大腸がん細胞又は組織はバイオマーカーについて陰性であると判定し得る。 Alternatively, the amount of biomarker measured in colon cancer cells or tissues obtained from colon cancer patients is the same or higher than that in control samples derived from colon adenoma patients (eg, statistically If it is significantly higher, the colon cancer cell or tissue can be determined to be positive for the biomarker. In addition, the amount of biomarkers measured in colon cancer cells or tissues obtained from colon cancer patients is lower than that in control samples derived from colon adenoma patients (eg, statistically significant) If low, the colon cancer cell or tissue may be determined to be negative for the biomarker.
　本明細書において「統計学的に有意」とは、得られた値の危険率(有意水準)が小さい場合、具体的には、p＜0.05(5％未満)、p＜0.01(1％未満)又はp＜0.001(0.1％未満)の場合を指す。統計学的検定方法は、有意性の有無を判断可能な公知の検定方法を適宜使用すればよく、特に限定しない。例えば、スチューデントt検定法、多重比較検定法、ログランク検定法を用いることができる。 In the present specification, “statistically significant” means, specifically, p <0.05 (less than 5%), p <0.01 (less than 1%) when the risk factor (level of significance) of the obtained value is small. Or p <0.001 (less than 0.1%). The statistical test method is not particularly limited as long as a known test method capable of determining the presence or absence of significance can be appropriately used. For example, Student's t test, multiple comparison test, log rank test can be used.
　また、あらかじめ対照試料においてバイオマーカーの量を測定しておき、該測定値に基づいてカットオフ値(閾値)を定めておいてもよい。該カットオフ値を基準としカットオフ値を超えた場合に、陽性であると判断することができる。カットオフ値は、例えば、ROC(receiver operating characteristic curve：受信者動作特性曲線)解析により定めることができる。 Alternatively, the amount of the biomarker may be measured in advance in a control sample, and the cutoff value (threshold) may be determined based on the measurement value. If the cutoff value is used as a reference and the cutoff value is exceeded, it can be judged as positive. The cutoff value can be determined by, for example, ROC (receiver operating characteristic curve) analysis.
　本発明者らは、後述の実施例で述べるとおり、GALNT6タンパク質の発現量が大腸がん患者の予後と相関することを見出した。したがって、本工程では、大腸がん細胞又は組織がバイオマーカーについて陰性である場合、大腸がん患者の予後は悪いと予測し得る。一方、大腸がん細胞又は組織がバイオマーカーについて陽性である場合、大腸がん患者の予後は良いと予測し得る。 The present inventors have found that the expression level of GALNT6 protein correlates with the prognosis of colorectal cancer patients, as described in the examples below. Therefore, in this step, if colon cancer cells or tissues are negative for the biomarker, the prognosis of colon cancer patients can be predicted to be bad. On the other hand, if colon cancer cells or tissues are positive for the biomarker, then the prognosis for colon cancer patients can be predicted to be good.
　本明細書において、「予後が悪い」とは、臨床転帰が不良である(例えば、大腸がんの切除手術後の再発率が高い、疾患(がん)特異的生存率が低い、又は全生存率が低い)ことをいう。予後が悪い場合、大腸がんの切除手術後の5年生存率は90％未満、85％未満、80％未満、75％未満、70％未満、65％未満又は60％未満であり得る。本発明では生存率は、累積生存率を意味する。本発明では生存率は、疾患(がん)特異的生存率又は全生存率であってもよい。 As used herein, “a poor prognosis” means poor clinical outcome (eg, high recurrence rate after resection surgery for colorectal cancer, low disease (cancer) specific survival rate, or overall survival Rate is low). If the prognosis is poor, the 5-year survival rate after resection surgery for colorectal cancer may be less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65% or less than 60%. In the present invention, survival rate means cumulative survival rate. In the present invention, the survival rate may be disease (cancer) specific survival rate or overall survival rate.
　本明細書において、「予後が良い」とは、臨床転帰が良好であることをいう。予後が良い場合、大腸がんの切除手術後の5年生存率は90％以上又は95％以上であり得る。 As used herein, "good prognosis" means that the clinical outcome is good. If the prognosis is good, then the 5-year survival rate after resection surgery for colorectal cancer may be 90% or more or 95% or more.
　本発明によれば、大腸がん患者の予後を予測することができ、その結果に基づき、治療方針(例えば、抗がん剤の種類、投与量、投与間隔など)を決定し、又は大腸がんの再発及び転移の検査の間隔を決定することができる。 According to the present invention, the prognosis of patients with colorectal cancer can be predicted, and based on the result, the treatment plan (for example, type of anticancer agent, dosage, administration interval, etc.) is determined, or the large intestine The interval between examinations for cancer recurrence and metastasis can be determined.
　本発明により、大腸がん患者の予後が悪いと予測された場合、大腸がんの再発を防止し、又は予後を改善し、又は生存率を改善するために、患者に抗がん剤を投与してもよい。したがって、本発明はまた、本発明の方法により予後が悪いと予測された大腸がん患者に抗がん剤を投与することを含む、大腸がんの再発を防止し、又は予後を改善し、又は生存率を改善する方法を提供する。 According to the present invention, when it is predicted that the prognosis of a colorectal cancer patient is poor, the patient is administered an anticancer agent to prevent recurrence of the colorectal cancer or to improve the prognosis or improve the survival rate. You may Therefore, the present invention also prevents the recurrence of colorectal cancer or improves the prognosis, which comprises administering an anticancer agent to colorectal cancer patients predicted to have a poor prognosis by the method of the present invention, Or provide a method to improve the survival rate.
　抗がん剤としては、以下に限定されないが、5-フルオロウラシル(5-FU)、カペシタビン、オキサリプラチン、イリノテカン、ベバシズマブ、セツキシマブ、パニツムマブ及びレゴラフェニブなどが挙げられる。抗がん剤は、単独で又は組みあわせて使用できる。抗がん剤は、注射、静脈内投与、経口投与などの経路で投与され得る。 Anti-cancer agents include, but are not limited to, 5-fluorouracil (5-FU), capecitabine, oxaliplatin, irinotecan, bevacizumab, cetuximab, panitumumab and regorafenib. The anticancer agent can be used alone or in combination. The anticancer agent can be administered by routes such as injection, intravenous administration, oral administration and the like.
　本発明はまた、大腸がん患者に対する抗がん剤の有効性を判定する方法を提供する。本方法は、大腸がん患者から得た大腸がん細胞又は組織における、上述の本発明に係るバイオマーカーの量を測定する測定工程、及び前記測定工程で得られた測定結果に基づいて、抗がん剤の有効性を判定する判定工程を含む。 The present invention also provides a method of determining the efficacy of an anti-cancer agent for colorectal cancer patients. The present method is based on the measurement step of measuring the amount of the biomarker according to the present invention described above in a colon cancer cell or tissue obtained from a colon cancer patient, and the measurement result obtained in the measurement step. It includes a determination step of determining the efficacy of the cancer drug.
　測定工程については、上記大腸がん患者の予後を予測する方法について記載したのと同様であるから記載を省略する。また、判定工程についても、測定結果に基づいて、大腸がん患者の予後を予測するのにかえて、抗がん剤の有効性を判定することを除いては上記大腸がん患者の予後を予測する方法について記載したのに準ずるから、異なる部分についてのみ以下記載する。 The measurement process is the same as that described for the method of predicting the prognosis of the colorectal cancer patient, and thus the description thereof is omitted. In addition, as to the determination step, instead of predicting the prognosis of a colorectal cancer patient based on the measurement results, the prognosis of the above colorectal cancer patient is determined except that the efficacy of the anticancer drug is determined. Since it conforms to the description of the forecasting method, only the differences will be described below.
　本工程では、大腸がん細胞又は組織がバイオマーカーについて陰性である場合、大腸がん患者に対する抗がん剤の効果は低いと判定し得る。一方、大腸がん細胞又は組織がバイオマーカーについて陽性である場合、大腸がん患者に対する抗がん剤の効果は高いと判定し得る。 In this step, when colon cancer cells or tissues are negative for a biomarker, it can be determined that the effect of the anticancer agent on colon cancer patients is low. On the other hand, when colon cancer cells or tissues are positive for the biomarker, the effect of the anticancer agent on colon cancer patients can be judged to be high.
　本明細書において、バイオマーカーについて陰性である患者における「抗がん剤の効果が低い」とは、バイオマーカーについて陽性である患者に比べて効果が低い(例えば、無病生存率が低い、疾患(がん)特異的生存率が低い、又は全生存率が低い)ことをいう。抗がん剤の効果が低い場合、大腸がんの切除手術5年後の生存率、例えば無病生存率は80％未満、75％未満、70％未満、65％未満、60％未満、55％未満又は50％未満であり得る。本発明では生存率は、累積生存率を意味する。本発明では生存率は、無病生存率、疾患(がん)特異的生存率又は全生存率であってもよい。 In the present specification, “a lower efficacy of the anti-cancer agent” in a patient who is negative for a biomarker is less effective than a patient who is positive for the biomarker (eg, a disease-free survival rate is lower Cancer) Low specific survival rate or low overall survival rate). If the effect of the anticancer drug is low, survival rate after resection surgery for colorectal cancer, for example, disease-free survival rate is less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, 55% It may be less than or less than 50%. In the present invention, survival rate means cumulative survival rate. In the present invention, the survival rate may be disease free survival rate, disease (cancer) specific survival rate or overall survival rate.
　本明細書において、「抗がん剤の効果が高い」とバイオマーカーについて陽性である患者に比べて効果が高いことを意味し、例えば、大腸がんの切除手術5年後の無病生存率は60％以上又は70％以上であり得る。 In the present specification, "the efficacy of the anti-cancer drug is high" means that the efficacy is high compared to the patient who is positive for the biomarker, for example, the disease-free survival rate after 5 years of resection for colorectal cancer is It may be 60% or more or 70% or more.
　本発明により、大腸がん患者に対する抗癌剤が高いと判定された場合、大腸がんの再発を防止し、又は予後を改善し、又は生存率を改善するために、患者に抗がん剤を投与してもよい。したがって、本発明はまた、本発明の方法により大腸がん患者に対する抗癌剤が高いと判定された大腸がん患者に抗がん剤を投与することを含む、大腸がんの再発を防止し、又は予後を改善し、又は生存率を改善する方法を提供する。 According to the present invention, when it is judged that the anticancer agent for colorectal cancer patients is high, the anticancer agent is administered to the patients in order to prevent the recurrence of colorectal cancer or improve the prognosis or improve the survival rate. You may Therefore, the present invention also prevents the recurrence of colorectal cancer, which comprises administering an anticancer agent to a colorectal cancer patient who is judged to have a high anticancer agent for a colorectal cancer patient by the method of the present invention, or Provided is a method of improving prognosis or improving survival rate.
　抗がん剤としては、以下に限定されないが、5-フルオロウラシル(5-FU)、カペシタビン、オキサリプラチン、イリノテカン、ベバシズマブ、セツキシマブ、パニツムマブ及びレゴラフェニブなどが挙げられる。抗がん剤は、単独で又は組みあわせて使用できる。抗がん剤は、注射、静脈内投与、経口投与などの経路で投与され得る。 Anti-cancer agents include, but are not limited to, 5-fluorouracil (5-FU), capecitabine, oxaliplatin, irinotecan, bevacizumab, cetuximab, panitumumab and regorafenib. The anticancer agent can be used alone or in combination. The anticancer agent can be administered by routes such as injection, intravenous administration, oral administration and the like.
　本発明により、大腸がん患者に対する抗癌剤が低いと判定された場合、大腸がんの再発を防止し、又は予後を改善し、又は生存率を改善するために、患者に抗がん剤以外の療法（例えば放射線療法等）を適用してもよい。したがって、本発明はまた、本発明の方法により大腸がん患者に対する抗癌剤が高いと判定された大腸がん患者に抗がん剤以外の療法を適用することを含む、大腸がんの再発を防止し、又は予後を改善し、又は生存率を改善する方法を提供する。 According to the present invention, when it is judged that the anticancer agent for colorectal cancer patients is low, in order to prevent the recurrence of colorectal cancer or to improve the prognosis or to improve the survival rate, the patient other than the anticancer agent is used. A therapy (eg, radiation therapy etc.) may be applied. Therefore, the present invention also prevents the recurrence of colorectal cancer, which comprises applying a therapy other than an anticancer drug to a colorectal cancer patient who is judged to have a high anticancer agent for a colorectal cancer patient by the method of the present invention. Or provide a method of improving prognosis or improving survival rate.
(キット)
　本発明はまた、上述の本発明に係るバイオマーカーの量を測定するための試薬を含む、大腸がん患者の予後を予測するためのキットも提供する。本発明はさらに、上述の本発明に係るバイオマーカーの量を測定するための試薬を含む、大腸がん患者に対する抗がん剤の有効性を判定するためのキットも提供する。 (kit)
The present invention also provides a kit for predicting the prognosis of colorectal cancer patients, comprising a reagent for measuring the amount of the biomarker according to the present invention described above. The present invention further provides a kit for determining the efficacy of an anti-cancer agent for colorectal cancer patients, comprising a reagent for measuring the amount of the biomarker according to the present invention described above.
　バイオマーカーの量を測定するための試薬としては、例えば、上述のような抗体若しくは抗体断片、又はプローブ若しくはプライマーが挙げられる。キットは、公知の免疫組織化学染色、ELISA、ウエスタンブロット、又はRT-PCR用の試薬等、例えば、標識試薬、緩衝液、発色基質、二次抗体、ブロッキング剤、並びに試験に必要な器具及びコントロール等をさらに含んでもよい。 The reagent for measuring the amount of the biomarker includes, for example, an antibody or antibody fragment as described above, or a probe or a primer. The kit includes known immunohistochemical staining, ELISA, Western blot, or reagents for RT-PCR, etc., for example, labeling reagents, buffers, chromogenic substrates, secondary antibodies, blocking agents, and devices and controls required for testing. And the like may be further included.
　以下、実施例を用いて本発明をさらに具体的に説明する。但し、本発明の技術的範囲はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be more specifically described using examples. However, the technical scope of the present invention is not limited to these examples.
[実施例1]
(大腸がん組織におけるGALNT6タンパク質の免疫組織化学染色)
　GALNT6タンパク質発現のための免疫組織化学染色を行った。
　福島県立医科大学附属病院で1990～2010年の間に治癒的切除手術を受けた原発性大腸がん(primary colorectal cancer)患者368人を本研究に登録した。各患者からはインフォームドコンセントを得るか、又は患者の拒否機会を保障した(オプトアウト)。腫瘍のステージを、悪性腫瘍のTNM分類(UICC第7版; Sobin LH, Gospodarowicz MK, Wittekind C, International Union against Cancer. TNM classification of malignant tumours, 7th edn. Wiley-Blackwell (2010))に従って決定した。手術前に化学療法又は放射線療法を受けた患者を除外し、ステージ0～IVの335人の患者から切除された大腸がん組織のホルマリン固定パラフィン包埋(formalin-fixed, paraffin-embedded; FFPE)切片を用いた。これらのうち304個の切片では、がん細胞に隣接する正常粘膜も評価のために利用可能であった。また、40例の大腸腺腫組織のFFPE切片も用いた。なお、本研究は、ヘルシンキ宣言に従って実施され、福島県立医科大学の施設内審査委員会の承認を受けた。 Example 1
(Immunohistochemical staining of GALNT6 protein in colorectal cancer tissue)
Immunohistochemical staining for GALNT6 protein expression was performed.
We enrolled 368 primary colorectal cancer patients who underwent curative surgery between 1990 and 2010 at Fukushima Medical University Hospital in this study. Informed consent was obtained from each patient or the patient's refusal opportunity was guaranteed (opt-out). The stage of the tumor was determined according to TNM classification of malignant tumor (UICC 7th Edition; Sobin LH, Gospodarowicz MK, Wittekind C, International Union against Cancer. TNM classification of malignant tumors, 7th edn. Wiley-Blackwell (2010)). Formalin-fixed, paraffin-embedded (FFPE) colorectal cancer tissues resected from 335 patients with stages 0-IV excluding patients who received chemotherapy or radiation prior to surgery The sections were used. Of these 304 sections, normal mucosa adjacent to the cancer cells was also available for evaluation. In addition, FFPE sections of 40 colorectal adenoma tissues were also used. This research was conducted in accordance with the Helsinki Declaration and was approved by the Institutional Review Board of Fukushima Medical University.
　4μm厚の切片を脱パラフィン処理し、再水和し、内在性ペルオキシダーゼをメタノール中の0.3％過酸化水素でブロックした。切片を、10mMクエン酸緩衝液(pH6.0)中で105℃にて5分間オートクレーブ処理し、抗原を回復した。一次ウサギポリクローナル抗GALNT6抗体(HPA011762、Sigma-Aldrich社、セントルイス、ミズーリ州、米国)を、Tween 20(Sigma-Aldrich社)を含有する10mMリン酸緩衝生理食塩水(PBS)中で500倍に希釈して抗GALNT6抗体溶液を調製し、切片を抗体溶液と4℃で一晩インキュベートした。次いで、Dako EnVision+ System(Agilent社(旧Dako社))を用いて、切片を、ホースラディッシュペルオキシダーゼ(HRP)結合抗ウサギポリマーとインキュベートし、その後、ジアミノベンジジンとインキュベートして、抗GALNT6抗体を可視化した。切片をヘマトキシリンで対比染色した。陰性対照は、一次抗体をPBSで置き換えることによって行った。陽性対照として、肺、膵臓、***及び胃に由来するいくつかの腺がん組織を用いた。 The 4 μm thick sections were deparaffinized, rehydrated and endogenous peroxidase blocked with 0.3% hydrogen peroxide in methanol. The sections were autoclaved in 10 mM citrate buffer (pH 6.0) for 5 minutes at 105 ° C. to recover the antigen. Primary rabbit polyclonal anti-GALNT6 antibody (HPA011762, Sigma-Aldrich, St. Louis, MO, USA) diluted 500 fold in 10 mM phosphate buffered saline (PBS) containing Tween 20 (Sigma-Aldrich) An anti-GALNT6 antibody solution was prepared, and the sections were incubated overnight at 4 ° C. with the antibody solution. The sections were then incubated with horseradish peroxidase (HRP) -conjugated anti-rabbit polymer using the Dako EnVision + System (Agilent (formerly Dako)) followed by incubation with diaminobenzidine to visualize the anti-GALNT6 antibody . Sections were counterstained with hematoxylin. Negative controls were performed by replacing the primary antibody with PBS. Several positive adenocarcinoma tissues from lung, pancreas, breast and stomach were used as positive controls.
　免疫組織化学スライドを、患者の臨床情報を知らない2人の独立した観察者によって評価し、10％を超える腫瘍細胞が細胞質において染色された場合、切片をGALNT6陽性とみなした。 Immunohistochemistry slides were evaluated by two independent observers blinded to the patient's clinical information, and sections were considered positive for GALNT6 if more than 10% of the tumor cells were stained in the cytoplasm.
　GALNT6タンパク質の免疫組織化学染色の代表的な画像を、図1に示す。304個の正常大腸粘膜を示す切片のうち92.8％がGALNT6陰性とみなされ(図1Aの矢頭、及び図1B)、GALNT6タンパク質発現は、大部分の正常大腸粘膜細胞では検出されないことが示された。 Representative images of immunohistochemical staining of GALNT6 protein are shown in FIG. Of the 304 normal colonic mucosa sections, 92.8% were considered negative for GALNT 6 (arrowheads in FIG. 1A and FIG. 1B), indicating that GALNT 6 protein expression was not detected in most normal colonic mucosal cells .
　一方、大腸腺腫のほぼ全て(97.5％)において、実質的に腫瘍領域全体にわたってGALNT6の強い顆粒状細胞質染色が観察された(図1Aの矢印、及び図1C)。 On the other hand, in almost all (97.5%) of colorectal adenomas, strong granular cytoplasmic staining of GALNT6 was observed substantially over the entire tumor area (arrows in FIG. 1A and FIG. 1C).
　さらに、335個の大腸がん切片のうち286個(85.4％)において、がん細胞内に強いGALNT6染色が散在して見られた(図1D及びEの茶色染色)。これらの286個の切片のうち、17個はステージ0、52個はステージI、103個はステージII、74個はステージIII、40個はステージIVの大腸がん患者に由来した。 In addition, in 286 (85.4%) of 335 colon cancer sections, strong GALNT6 staining was scattered in the cancer cells (brown staining in FIGS. 1D and E). Of these 286 sections, 17 were from stage 0, 52 from stage I, 103 from stage II, 74 from stage III, and 40 from stage IV colorectal cancer patients.
　しかし、335個の大腸がん切片のうち49個(14.6％)では、GALNT6タンパク質発現が見られなかった(図1F及びG)。これらの49個の切片のうち、10個はステージI、19個はステージII、15個はステージIII、5個はステージIVの大腸がん患者に由来した。 However, GALNT6 protein expression was not seen in 49 (14.6%) of the 335 colon cancer sections (FIGS. 1F and G). Of these 49 sections, 10 were from stage I, 19 from stage II, 15 from stage III, and 5 from stage IV colorectal cancer patients.
　これらの結果から、正常大腸粘膜ではGALNT6は発現せず、前がん状態及び前浸潤状態でGALNT6の発現が高くなること、さらに、様々なステージの大腸がん患者の大部分ではGALNT6の発現が高いが、大腸がんの患者の一部(約15％)においてGALNT6発現が低下又は消失することが示された。 From these results, it is shown that GALNT6 is not expressed in normal colon mucosa, expression of GALNT6 is high in precancerous and preinvasive states, and that expression of GALNT6 is most in patients with various stages of colorectal cancer. Although high, it has been shown that GALNT6 expression is reduced or eliminated in a part of colon cancer patients (about 15%).
[実施例2]
(大腸がん患者の切除手術後の予後評価)
　福島県立医科大学附属病院で1990～2010年の間に治癒的切除手術を受けたステージI～IVの267人の原発性大腸がん患者の予後を評価した。腫瘍のステージを、実施例1に記載したように決定した。患者から切除した大腸がん組織について、実施例1に記載したように免疫組織化学染色の結果に基づいてGALNT6陰性又は陽性を決定した。患者の臨床情報は、医療記録を再調査することによって遡及的に得た。 Example 2
(Prognostic evaluation after resection surgery for colorectal cancer patients)
We evaluated the prognosis of 267 primary colorectal cancer patients in stages I to IV who underwent curative resection between 1990 and 2010 at Fukushima Medical University Hospital. The stage of the tumor was determined as described in Example 1. For colorectal cancer tissues excised from patients, GALNT6 negative or positive was determined based on the results of immunohistochemical staining as described in Example 1. The patient's clinical information was obtained retrospectively by reviewing the medical records.
　カプラン-マイヤー(Kaplan-Meier)法により累積生存率を推定した。生存率として、疾患特異的生存率(disease-specific survival)及び全生存率(overall survival)を用いた。疾患特異的生存率及び全生存率は、それぞれ、手術日からがんによる死亡までの期間、及び手術日から任意の原因による死亡までの期間と定義される。2群(GALNT6陽性群及びGALNT6陰性群)間の生存率をログランク検定により比較した。全ての統計分析は両側(two-sided)であり、Graphpad Prism v6.0(Graphpad Software社、ラホヤ、カリフォルニア州、米国)及びSPSS Statisticsバージョン24(IBM社、ニューヨーク州、米国)を用いて行った。全てのP値は両側であり、0.05未満のP値を統計学的に有意であるとみなした。 Cumulative survival rates were estimated by the Kaplan-Meier method. As survival rates, disease-specific survival rates and overall survival rates were used. Disease specific survival rates and overall survival rates are defined as the time from the day of surgery to death from cancer and the time from the day of surgery to death from any cause. Survival rates between the two groups (GALNT6 positive group and GALNT6 negative group) were compared by the log rank test. All statistical analyzes were two-sided and were performed using Graphpad Prism v6.0 (Graphpad Software, La Jolla, CA, USA) and SPSS Statistics version 24 (IBM, NY, USA) . All P values were two-sided, and P values less than 0.05 were considered statistically significant.
　疾患特異的生存率の結果を図2に示す。GALNT6陽性の腫瘍を有するステージI～IVの大腸がん患者は、高い疾患特異的生存率を示した(図2A；5年生存率95.7％、10年生存率90.8％)。一方、GALNT6陰性の腫瘍を有するステージI～IVの大腸がん患者は、GALNT6陽性患者と比較して、有意に低い(P=0.0038)疾患特異的生存率を示した(図2A；5年生存率84.4％、10年生存率75.7％)。 The results of disease specific survival rates are shown in FIG. Stage I-IV colon cancer patients with GALNT6-positive tumors showed high disease-specific survival (FIG. 2A; 5-year survival 95.7%, 10-year survival 90.8%). On the other hand, stage I-IV colorectal cancer patients with GALNT6-negative tumors showed significantly lower (P = 0.0038) disease-specific survival compared to GALNT6-positive patients (FIG. 2A; 5-year survival Rate 84.4%, 10-year survival rate 75.7%).
　ステージII及びIIIのみについても、GALNT6陽性の大腸がん患者は、高い疾患特異的生存率を示し(図2B；5年生存率96.4％、10年生存率91.3％)、一方、GALNT6陰性の大腸がん患者は、GALNT6陽性患者と比較して有意に低い(P=0.0008)疾患特異的生存率を示した(図2B；5年生存率78.8％、10年生存率73.1％)。 Also for stage II and III alone, GALNT6-positive colon cancer patients show high disease-specific survival (Fig. 2B; 5-year survival 96.4%, 10-year survival 91.3%) while GALNT 6-negative colon Cancer patients showed significantly lower disease-specific survival rates (P = 0.0008) compared to GALNT6-positive patients (FIG. 2B; 5-year survival rate 78.8%, 10-year survival rate 73.1%).
　さらに、ステージIIIのみにおいても、GALNT6陽性の大腸がん患者は、高い疾患特異的生存率を示し(図2C；5年生存率95.4％、10年生存率90.7％)、一方、GALNT6陰性の大腸がん患者は、GALNT6陽性患者と比較して有意に低い(P<0.0001)疾患特異的生存率を示した(図2C；5年生存率57.1％、10年生存率57.1％)。特にステージIIIの大腸がん患者において、GALNT6陽性患者と陰性患者の生存率の違いが顕著であった。
　また、全生存率でも、疾患特異的生存率と同様の傾向が示された。 Furthermore, even in stage III alone, GALNT6-positive colon cancer patients show high disease-specific survival (Fig. 2C; 5-year survival rate 95.4%, 10-year survival rate 90.7%), while GALNT 6 negative colon Cancer patients showed significantly lower disease-specific survival rates (P <0.0001) compared to GALNT6-positive patients (FIG. 2C; 5-year survival rate 57.1%, 10-year survival rate 57.1%). The difference in survival rates between GALNT6-positive patients and negative patients was remarkable especially in stage III colon cancer patients.
The overall survival rate also showed the same tendency as the disease-specific survival rate.
　以上の結果から、GALNT6陽性の大腸がん患者は、大腸がん切除手術後の予後が良いのに対し、GALNT6陰性の大腸がん患者は、大腸がん切除手術後の予後が悪い傾向があることが示された。 From the above results, patients with GALNT6-positive colorectal cancer have a good prognosis after colorectal cancer removal surgery, while patients with GALNT6-negative colorectal cancer tend to have a poor prognosis after colorectal cancer removal surgery It was shown.
[実施例3]
(大腸がん患者における化学療法の有効性)
　福島県立医科大学附属病院で1990～2010年の間に治癒的切除手術を受けたステージII～IIIの190人の原発性大腸がん患者の予後を評価した。腫瘍のステージを、実施例1に記載したように決定した。患者から切除した大腸がん組織について、実施例1に記載したように免疫組織化学染色の結果に基づいてGALNT6陰性又は陽性を決定した。患者の臨床情報は、医療記録を再調査することによって遡及的に得た。 [Example 3]
(Efficacy of chemotherapy in colorectal cancer patients)
We evaluated the prognosis of 190 primary colorectal cancer patients in stages II-III who underwent curative resection between 1990 and 2010 at Fukushima Medical University Hospital. The stage of the tumor was determined as described in Example 1. For colorectal cancer tissues excised from patients, GALNT6 negative or positive was determined based on the results of immunohistochemical staining as described in Example 1. The patient's clinical information was obtained retrospectively by reviewing the medical records.
　カプラン-マイヤー(Kaplan-Meier)法により累積生存率を推定した。生存率として、無病生存率(disease-free survival)を用いた。無病生存率は、手術日から疾患の再発までの期間と定義される。統計的手法は実施例2に従った。 Cumulative survival rates were estimated by the Kaplan-Meier method. Disease-free survival was used as the survival rate. Disease-free survival is defined as the time from the date of surgery to the recurrence of the disease. The statistical procedure followed Example 2.
　本実施例では、GALNT6の発現が、アジュバント化学療法に対する応答性と関係しているかを調べた。アジュバント化学療法の適用の有無に関する情報が利用可能である、190人のステージII及びIIIの患者において、免疫組織化学の結果と無病生存率の相関を調べた。本実施例で解析した患者のうち114人は切除手術後に静脈内又は経口5-FUベースの化学療法で処置されており、76人は切除手術のみで処置されていた。 In this example, it was examined whether expression of GALNT6 was associated with responsiveness to adjuvant chemotherapy. The correlation between immunohistochemistry results and disease-free survival was examined in 190 stage II and III patients for whom information on the presence or absence of adjuvant chemotherapy was available. Of the patients analyzed in this example, 114 were treated with intravenous or oral 5-FU based chemotherapy after excision and 76 were treated with excision only.
　図3に結果を示す。化学療法で処置したステージII～IIIの患者のうち、GALNT6陰性の患者は、GALNT6陽性患者より低い無病生存率を示した（図3A）。また、化学療法で処置したステージIIIの患者のうち、GALNT6陰性の患者は、GALNT6陽性患者より統計的に有意に低い治療結果を示した（図3C）。このような結果は、切除手術のみで処置した患者では認められなかった（ステージII～III：図3B、ステージIII：3D）。 The results are shown in FIG. Of the stage II-III patients treated with chemotherapy, GALNT6-negative patients showed lower disease-free survival than GALNT6-positive patients (FIG. 3A). Also, among the stage III patients treated with chemotherapy, GALNT6-negative patients showed statistically significantly lower treatment results than GALNT6-positive patients (FIG. 3C). Such results were not observed in patients treated with resection surgery alone (Stages II-III: FIG. 3B, Stage III: 3D).
　以上の結果から、GALNT6陽性の大腸がん患者は、化学療法の有効性が高いのに対し、GALNT6陰性の大腸がん患者は、化学療法の有効性が低い傾向があることが示された。 From the above results, it has been shown that patients with GALNT6-positive colorectal cancer tend to be more effective in chemotherapy, while patients with GALNT6-negative colon cancer tend to be less effective.
[実施例4]
(GALNT6発現と5-FUに対する抵抗性の関連)
＜材料と方法＞
　SW480細胞は、American Typed Culture Collection（ATCC、マナサス、バージニア州、米国）から購入した。SW480は、10%ウシ胎児血清及びペニシリン/ストレプトマイシン（ThermoFisher Scientific、ウォルサム、マサチューセッツ州、米国）を含むRPMI-1640培地（Promega、マディソン、ウィスコンシン州、米国）において、5%CO2を含む加湿した雰囲気下で維持した。 Example 4
(Relationship between GALNT6 expression and resistance to 5-FU)
<Materials and Methods>
SW480 cells were purchased from American Typed Culture Collection (ATCC, Manassas, Va., USA). SW480 is a humidified atmosphere containing 5% CO 2 in RPMI-1640 medium (Promega, Madison, WI, USA) with 10% fetal bovine serum and penicillin / streptomycin (ThermoFisher Scientific, Waltham, MA, USA) Maintained below.
　定量RT-PCRは、以下の通り行った。全RNAをTRIzol試薬を用いて抽出し、全RNAの1μgをSuperScript III First-Strand Synthesis System（ThermoFisher Scientific）を用いて、製造業者の指示に従って逆転写した。GALNT6（Assay ID Hs00926629_m1）、及びACTB（Hs99999903_m1）を対象とするqRT-PCRは、TaqMan Gene Expression Master Mixを用いて、7500 real time PCR systemにて、三連でTaqManにより行った（ThermoFisher Scientific）。相対発現レベルを2-ΔΔCt法により、SDSソフトウェアによって製造業者の指示に従って測定し、ACTBを校正遺伝子として用いた。 Quantitative RT-PCR was performed as follows. Total RNA was extracted using TRIzol reagent, and 1 μg of total RNA was reverse transcribed using SuperScript III First-Strand Synthesis System (ThermoFisher Scientific) according to the manufacturer's instructions. QRT-PCR targeting GALNT6 (Assay ID Hs00926629_m1) and ACTB (Hs99999903_m1) was performed by TaqMan in triplicate in a 7500 real time PCR system using TaqMan Gene Expression Master Mix (ThermoFisher Scientific). Relative expression levels were determined by the 2- ΔΔCt method, by SDS software according to the manufacturer's instructions, and ACTB was used as a calibration gene.
　ウエスタンブロッティングは、以下の通り行った。全タンパク質を、Halt Protease Inhibitor Cocktail（ThermoFisher Scientific）を加えたRIPA溶解バッファーを用いて抽出し、Tris-Glycine SDSサンプルバッファー（ThermoFisher Scientific）中で加熱した。等量のタンパク質を10% SDS-PAGEゲルに供して分離し、PVDF膜（ThermoFisher Scientific）に転写した。膜を5%の脱脂乾燥スキムミルク粉末（Cell signaling Technology）でブロッキングし、一次抗体としてウサギ抗GALNT6抗体（(#HPA011762、1:250、Atlas Antibodies）又はマウス抗βアクチン抗体（(#SC-69879、1:2000、Santa Cruz Biotechnology）とインキュベートした。続いて膜をヤギ抗ウサギ又は抗マウスHRP二次抗体（Santa Cruz Biotechnology）とインキュベートし、SuperSignal West Pico chemiluminescent Substrate（ThermoFisher Scientific）により発色させ、LAS4000 imager（GE Healthcare）により観察した。 Western blotting was performed as follows. Total protein was extracted using RIPA lysis buffer with Halt Protease Inhibitor Cocktail (ThermoFisher Scientific) and heated in Tris-Glycine SDS sample buffer (ThermoFisher Scientific). Equal amounts of protein were applied to a 10% SDS-PAGE gel, separated and transferred to a PVDF membrane (ThermoFisher Scientific). The membrane is blocked with 5% non-fat dry skimmed milk powder (Cell signaling Technology), and rabbit anti-GALNT6 antibody ((# HPA011762, 1: 250, Atlas Antibodies) or mouse anti-β-actin antibody ((# SC-69879, as primary antibody) The membrane was incubated with goat anti-rabbit or anti-mouse HRP secondary antibody (Santa Cruz Biotechnology) and developed with SuperSignal West Pico luminescent Substrate (ThermoFisher Scientific), LAS 4000 imager. It observed by (GE Healthcare).
　siRNAトランスフェクションは、以下の通り行った。対数増殖期において、細胞を6ウェルプレートに播種し、siRNAオリゴヌクレオチド又はスクランブル対照（Ambion（登録商標） Silencer Select、s22154、s22155及び陰性対照#1、ThermoFisher Scientific）を用いて、Lipofectamine RNAiMAX試薬（ThermoFisher Scientific）により、製造業者の指示に従ってトランスフェクションした。48時間後、細胞を回収した。実験は少なくとも3回行い、ノックダウン効率をqRT-PCR及びウエスタンブロット解析により評価した。 SiRNA transfection was performed as follows. In the logarithmic phase, cells are seeded in 6-well plates and Lipofectamine RNAiMAX reagent (ThermoFisher) using siRNA oligonucleotide or scrambled control (Ambion (R) Silencer Select, s22154, s22155 and negative control # 1, ThermoFisher Scientific). Transfection was performed according to the manufacturer's instructions. After 48 hours, cells were harvested. The experiments were performed at least three times and knockdown efficiency was assessed by qRT-PCR and Western blot analysis.
　細胞増殖アッセイは、Cell Counting Kit-8（CCK-8、同仁化学）を用いて製造業者の指示に従って行った。手短には、トランスフェクションの24時間後、3つの群のSW480細胞を回収し、再懸濁して4×103細胞/ウェルの密度で96ウェルプレートに播種した。完全培地においてトランスフェクションの後24時間、48時間及び72時間インキュベーションした後、10μlのCCK-8試薬を加え、5%CO2を含む加湿した雰囲気下で3時間インキュベートした。450nmでの吸光度をマイクロプレートリーダーを用いて測定した。実験は各回3連で、3回行った。 Cell proliferation assays were performed using Cell Counting Kit-8 (CCK-8, Dojin Kagaku) according to the manufacturer's instructions. Briefly, 24 h after transfection, three groups of SW480 cells were harvested, resuspended and seeded in 96 well plates at a density of 4 × 10 3 cells / well. After incubation for 24, 48 and 72 hours after transfection in complete medium, 10 μl of CCK-8 reagent was added and incubated for 3 hours in a humidified atmosphere containing 5% CO 2 . Absorbance at 450 nm was measured using a microplate reader. The experiment was performed three times in triplicate.
　アポトーシス細胞は、Annexin V-PE/7-AAD Apoptosis Detection Kit（BD Biosciences、フランクリン・レイクス、ニュージャージー州、米国）を用いて、製造業者のプロトコルに従って行った。手短には、GALNT6 siRNA及び対照siRNAをトランスフェクトした細胞を6ウェルプレートに播種した。48時間のインキュベーション後、細胞をAnnexin V及び7-AADによる標識で処理し、フローサイトメトリーを行った。Annexin V陽性細胞を、アポトーシス細胞とみなした。
　5-FUの細胞毒性アッセイは以下の通り行った。GALNT6 siRNA及び対照siRNAをトランスフェクトしたSW480細胞を、96ウェルプレートに5×103/ウェルの密度で播種した。24時間インキュベーション後、細胞を様々な濃度の5-FU（Sigma-Aldrich）で処理し、5%CO2下で37℃にて72時間インキュベートした。細胞毒性は、CCK-8により上記の通り評価した。まず、0.1～1000μg/mlの範囲の5-FU濃度又はビヒクル単独で予備的に処理し、用量応答曲線を作成した。続いて、1、5、10、50、及び100μg/mlの5-FUを実験に用いた。実験は各回3連で、少なくとも3回行った。 Apoptotic cells were performed using the Annexin V-PE / 7-AAD Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Briefly, cells transfected with GALNT6 siRNA and control siRNA were seeded in 6 well plates. After 48 hours of incubation, cells were treated with Annexin V and 7-AAD labeling and flow cytometry was performed. Annexin V positive cells were considered as apoptotic cells.
The 5-FU cytotoxicity assay was performed as follows. SW480 cells transfected with GALNT6 siRNA and control siRNA were seeded at a density of 5 × 10 3 / well in 96 well plates. After 24 hours incubation, cells were treated with various concentrations of 5-FU (Sigma-Aldrich) and incubated for 72 hours at 37 ° C. under 5% CO 2 . Cytotoxicity was assessed as above by CCK-8. First, dose response curves were generated by preliminary treatment with 5-FU concentrations ranging from 0.1 to 1000 μg / ml or vehicle alone. Subsequently, 1, 5, 10, 50, and 100 μg / ml 5-FU were used in the experiments. The experiment was performed at least three times in triplicate.
＜結果＞
　GALNT6の生物学的機能を明らかにするため、SW480細胞株を用いて以下の実験で用いた。なお、SW480細胞が比較亭高いGALNT6 mRNA発現及びタンパク質発現を示すことは事前に確認している（データ示さず）。 <Result>
In order to clarify the biological function of GALNT6, SW480 cell line was used in the following experiments. In addition, it has been confirmed in advance that SW480 cells show relatively high GALNT6 mRNA expression and protein expression (data not shown).
　GALNT6を標的とする2つの異なるsiRNAを用い、GALNT6が有効にサイレンシングされたことを、qRT-PCR及びウエスタンブロッティングによって確かめた（図4A、B）。GALNT6のサイレンシングは、細胞増殖に対しては有意な影響を示さなかった（図4C）。 Effective silencing of GALNT6 was confirmed by qRT-PCR and Western blotting using two different siRNAs targeting GALNT6 (Figure 4A, B). Silencing of GALNT6 had no significant effect on cell proliferation (FIG. 4C).
　続いて、GALNT6発現が5-FU処置に対する感受性の有無に寄与するか否かをインビトロで調べた。GALNT6ノックダウン細胞において、コントロールsiRNAで処置した細胞に対して、有意な5-FU抵抗性の増加が認められた（図4D）。これと対応して、5-FUで処置したGALNT6ノックダウン細胞において、アポトーシスが有意に抑制されていた（図4E）。 Subsequently, it was examined in vitro whether GALNT6 expression contributes to the presence or absence of sensitivity to 5-FU treatment. A significant increase in 5-FU resistance was observed in GALNT6 knockdown cells relative to cells treated with control siRNA (FIG. 4D). Correspondingly, apoptosis was significantly suppressed in 5-FU-treated GALNT6 knockdown cells (FIG. 4E).
　以上の結果から、GALNT6の発現を低減させると、5-FUに対する抵抗性が増加することが示された。この結果は、実施例3において認められた、GALNT6陰性の大腸がん患者における化学療法の有効性が低い傾向を裏付けるものである。 From the above results, it was shown that when the expression of GALNT6 is reduced, the resistance to 5-FU is increased. This result supports the tendency of the chemotherapy to be less effective in patients with GALNT 6 negative colon cancer observed in Example 3.
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (13)

  1. 　GALNT6タンパク質若しくはそのペプチド断片、又はGALNT6遺伝子の転写産物若しくはその核酸断片からなる、大腸がん患者の予後を予測するためのバイオマーカー。 A biomarker for predicting the prognosis of colorectal cancer patients, comprising a GALNT6 protein or a peptide fragment thereof, or a transcript of a GALNT6 gene or a nucleic acid fragment thereof.
  2. 　GALNT6タンパク質若しくはそのペプチド断片、又はGALNT6遺伝子の転写産物若しくはその核酸断片からなる、大腸がん患者に対する抗がん剤の有効性を判定するためのバイオマーカー。 A biomarker for determining the efficacy of an anticancer agent for colorectal cancer patients, comprising a GALNT6 protein or a peptide fragment thereof, or a transcript of a GALNT6 gene or a nucleic acid fragment thereof.
  3. 　抗がん剤が5-FUである、請求項２に記載のバイオマーカー。 The biomarker according to claim 2, wherein the anticancer agent is 5-FU.
  4. 　前記GALNT6タンパク質が、以下の(a)～(c)のいずれかのタンパク質である、請求項１～３のいずれか一項に記載のバイオマーカー。
    　(a)配列番号1で示されるアミノ酸配列からなるタンパク質、
    　(b)配列番号1で示されるアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列からなるタンパク質、及び
    　(c)配列番号1で示されるアミノ酸配列に対して90％以上のアミノ酸同一性を有するアミノ酸配列からなるタンパク質     The biomarker according to any one of claims 1 to 3, wherein the GALNT6 protein is any of the following proteins (a) to (c):
    (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,
    (b) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and (c) 90% of the amino acid sequence shown in SEQ ID NO: 1 A protein consisting of an amino acid sequence having the above amino acid identity
  5. 　前記GALNT6遺伝子が、請求項４に示すタンパク質をコードする、請求項１～４のいずれか一項に記載のバイオマーカー。 The biomarker according to any one of claims 1 to 4, wherein the GALNT6 gene encodes the protein described in claim 4.
  6. 　大腸がん患者の予後を予測するための、請求項１及び４～５のいずれか一項に記載のバイオマーカーの使用。 Use of the biomarker according to any one of claims 1 and 4 for predicting the prognosis of a colorectal cancer patient.
  7. 　大腸がん患者に対する抗がん剤の有効性を判定するための、請求項２～５のいずれか一項に記載のバイオマーカーの使用。 The use of the biomarker according to any one of claims 2 to 5 for determining the efficacy of an anti-cancer agent for colorectal cancer patients.
  8. 　前記大腸がんが、ステージI～IVの大腸がんである、請求項６又は７に記載の使用。 The use according to claim 6 or 7, wherein said colorectal cancer is stage I-IV colorectal cancer.
  9. 　前記大腸がんが、ステージIIIの大腸がんである、請求項８に記載の使用。 The use according to claim 8, wherein the colorectal cancer is stage III colorectal cancer.
  10. 　大腸がん患者の予後を予測する方法であって、
    　大腸がん患者から得た大腸がん細胞又は組織における、請求項１及び４～５のいずれか一項に記載のバイオマーカーの量を測定する測定工程、及び
    　前記測定工程で得られた測定結果に基づいて、大腸がん患者の予後を予測する予測工程を含み、
    　ここで、前記大腸がん細胞又は組織が前記バイオマーカーについて陰性である場合、前記大腸がん患者の予後は悪いと予測する、方法。     It is a method of predicting the prognosis of colon cancer patients, and
    A measurement process for measuring the amount of the biomarker according to any one of claims 1 and 4 in colon cancer cells or tissues obtained from a colon cancer patient, and the measurement result obtained in the measurement process Including a prediction step to predict the prognosis of patients with colorectal cancer based on
    Here, if the colorectal cancer cells or tissues are negative for the biomarker, then the prognosis of the colorectal cancer patient is predicted to be poor.
  11. 　大腸がん患者に対する抗がん剤の有効性を判定する方法であって、
    　大腸がん患者から得た大腸がん細胞又は組織における、請求項２～５のいずれか一項に記載のバイオマーカーの量を測定する測定工程、及び
    　前記測定工程で得られた測定結果に基づいて、大腸がん患者に対する抗がん剤の有効性を判定する判定工程を含み、
    　ここで、前記大腸がん細胞又は組織が前記バイオマーカーについて陰性である場合、大腸がん患者に対する抗がん剤の有効性は低いと判定する、方法。     A method of determining the efficacy of an anti-cancer agent for colorectal cancer patients, comprising
    A measurement process of measuring the amount of the biomarker according to any one of claims 2 to 5 in colon cancer cells or tissues obtained from a colon cancer patient, and the measurement result obtained in the measurement process. And determining the effectiveness of the anti-cancer drug in patients with colorectal cancer,
    Here, when the colon cancer cell or tissue is negative for the biomarker, it is determined that the efficacy of the anticancer agent for colon cancer patients is low.
  12. 　請求項１及び４～５のいずれか一項に記載のバイオマーカーの量を測定するための試薬を含む、大腸がん患者の予後を予測するためのキット。 A kit for predicting the prognosis of a colorectal cancer patient, comprising a reagent for measuring the amount of the biomarker according to any one of claims 1 and 4.
  13. 　請求項２～５のいずれか一項に記載のバイオマーカーの量を測定するための試薬を含む、大腸がん患者に対する抗がん剤の有効性を判定するためのキット。 A kit for determining the efficacy of an anticancer agent for colorectal cancer patients, comprising a reagent for measuring the amount of the biomarker according to any one of claims 2 to 5.
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JAE-HYUN PARK ET AL.: "Identification of an 0-type glycosyltransferase(GALNT6) as a novel molecular target for breast cancer therapy", vol. 69 th, 23 August 2010 (2010-08-23), pages 274 *
MASARU NODA ET AL.: "Glycosyltransferase Gene Expression Identifies a Poor Prognostic Colorectal Cancer Subtype Associated with Mismatch Repair Deficiency and Incomplete Glycan Synthesis", CLINICAL CANCER RESEARCH, vol. 24, no. 18, 15 September 2018 (2018-09-15), pages 4468 - 4481, XP055581339, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-17-3533 *
NAKAMORI SHOJI ET AL.: "Development of tumor marker helpful in early diagnosis and prognosis of cancer", ANNUAL REPORT OF THE CANCER RESEARCH MINISTRY OF HEALTH- LABOUR AND WELFARE, vol. 2006, September 2007 (2007-09-01), pages 196 - 200 *

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