WO2019049829A1 - Biomarqueur de pronostic de cancer du côlon - Google Patents

Biomarqueur de pronostic de cancer du côlon 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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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/ja
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    • C—CHEMISTRY; METALLURGY
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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).

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Abstract

L'invention concerne un biomarqueur permettant de prédire le pronostic d'un patient atteint d'un cancer du côlon, et/ou de déterminer l'efficacité d'un agent anticancéreux chez un patient atteint d'un cancer du côlon. L'invention concerne un biomarqueur permettant de prédire le pronostic d'un patient atteint d'un cancer du côlon et/ou de déterminer l'efficacité d'un agent anticancéreux chez un patient atteint d'un cancer du côlon, le biomarqueur comprenant une protéine GALNT6 ou un fragment peptidique de cette dernière, ou un produit de transcription de gène GALNT6 ou un fragment d'acide nucléique de ce dernier.
PCT/JP2018/032628 2017-09-05 2018-09-03 Biomarqueur de pronostic de cancer du côlon WO2019049829A1 (fr)

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
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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