WO2020162523A1 - Biomarqueur de pronostic du cancer du côlon - Google Patents

Biomarqueur de pronostic du cancer du côlon Download PDF

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WO2020162523A1
WO2020162523A1 PCT/JP2020/004474 JP2020004474W WO2020162523A1 WO 2020162523 A1 WO2020162523 A1 WO 2020162523A1 JP 2020004474 W JP2020004474 W JP 2020004474W WO 2020162523 A1 WO2020162523 A1 WO 2020162523A1
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colorectal cancer
prognosis
krt17
biomarker
protein
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PCT/JP2020/004474
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Japanese (ja)
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大輔 氏家
洋和 岡山
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 JP2020571249A priority Critical patent/JP7065539B2/ja
Publication of WO2020162523A1 publication Critical patent/WO2020162523A1/fr

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    • 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
    • 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
    • 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

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  • the present invention includes a biomarker for predicting the prognosis of colorectal cancer patients, a method for predicting the prognosis of colorectal cancer patients using the biomarker, and a reagent for measuring the amount of the biomarker, colon
  • the present invention relates to a kit for predicting the prognosis of cancer patients.
  • Colorectal cancer is a major cause of cancer death worldwide, despite significant advances in diagnosis and treatment.
  • the only colorectal cancer prognostic classification currently used in clinical practice is clinicopathological staging (stage) classification, and a treatment policy is selected according to the stage.
  • stage I colorectal cancer shows good results after surgery alone
  • stage III colorectal cancer patients can be expected to improve their prognosis by performing adjuvant chemotherapy after radical surgery ( Non-Patent Document 1).
  • predicting the prognosis of colorectal cancer patients is important for individual colorectal cancer patient management based on the prognosis prediction.
  • biomarkers that can be clinically used to predict the prognosis of colorectal cancer have not always been sufficient in terms of their types and properties.
  • an object of the present invention is to provide a biomarker for predicting the prognosis of colorectal cancer patients.
  • the present invention aims to provide a biomarker for predicting the prognosis of stage II colorectal cancer patients.
  • the present inventors have conducted extensive studies, and as a result, in a patient group having a high expression level of KRT17, the prognosis after colorectal cancer resection surgery is poor, whereas the expression level of KRT17 is low. In the patient group, they found that the prognosis after colorectal cancer resection surgery tended to be good, and completed the present invention.
  • the present invention includes the following embodiments.
  • a biomarker for predicting the prognosis of colorectal cancer patients which comprises a KRT17 protein or a peptide fragment thereof, or a transcription product of the KRT17 gene or a nucleic acid fragment thereof.
  • a method of predicting the prognosis of a colorectal cancer patient comprising: Based on the measurement step of measuring the amount of the biomarker according to any one of (1) to (5) in colorectal cancer cells or tissues obtained from colorectal cancer patients, and the measurement result obtained in the measurement step , Including a prediction step to predict the prognosis of colorectal cancer patients,
  • the colon cancer cells or tissues are negative for the biomarker, or when the expression level is low, it is predicted that the prognosis of the colon cancer patient is good, and/or the colon cancer cells or A method of predicting a poor prognosis of the colorectal cancer patient if the tissue is positive for the biomarker or has a high expression level.
  • a kit for predicting the prognosis of colorectal cancer patients which comprises a reagent for measuring the amount of the biomarker according to any one of (1) to (5).
  • the present specification includes the disclosure content of Japanese Patent Application No. 2019-021987, which is the basis of priority of the present application.
  • the present invention provides a biomarker for predicting the prognosis of colorectal cancer patients.
  • the present invention provides biomarkers for predicting the prognosis of stage II colorectal cancer patients, which may aid in the selection and determination of whether adjuvant chemotherapy is given.
  • FIG. 1A shows the immunohistochemistry results of KRT17 protein expression in normal mammary tissue as a positive control.
  • 1B-D show representative immunohistochemistry results for IHC validation set 1.
  • FIG. 2 shows the results of Kaplan-Meier analysis of the KRT17 protein expression positive group and the negative group in the IHC verification set.
  • the present invention relates to biomarkers for predicting the prognosis of colorectal cancer patients.
  • the biomarker according to the present invention comprises a KRT17 protein or a peptide fragment thereof, or a transcription product of a KRT17 gene or a nucleic acid fragment thereof.
  • colon cancer refers to cancer that develops in the large intestine, including the colon, the rectum, and the anus.
  • Colorectal cancer includes in particular colon cancer and rectal cancer.
  • prognosis refers to a predicted course (for example, recurrence or no recurrence or life or death) in a colon cancer patient after excision surgery of the colon cancer.
  • Prediction of prognosis is the risk of recurrence, survival, or after a period of time (e.g., 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years or 20 years or more) It may be a recurrence-free survival rate or a prediction of survival rate.
  • prognostic prediction comprises predicting recurrence risk (eg, recurrence-free survival).
  • recurrence-free survival rate refers to the proportion of patients who do not reappear from colon cancer.
  • Prognosis prediction can also be referred to as prognosis determination, evaluation, or diagnosis.
  • KRT17 (keratin 17) protein (also called cytokeratin 17) is a type I type cytokeratin, and is an intermediate found in basal cells and myoepithelial cells of various glands such as mammary gland, respiratory epithelium, and urothelium. Diameter filament.
  • the keratin 17 protein is not expressed in normal skin, but is rapidly induced in skin epithelial cells in a stress environment such as a wound and causes cell proliferation. Keratin 17 protein is known to have a positive effect on the regulation of epithelial cell size and proliferation by binding to the adapter protein 14-3-3 ⁇ and stimulating the Akt/mTOR pathway.
  • the KRT17 protein derived from the endogenous gene of a colorectal cancer patient or its gene transcription product can serve as a biomarker.
  • the human KRT17 protein derived from the human KRT17 gene and the transcription product (mRNA) of the human KRT17 gene can be the biomarker of the present invention.
  • KRT17 protein examples include human-derived KRT17 (human KRT17) protein containing or consisting of the amino acid sequence shown by SEQ ID NO: 1.
  • the KRT17 protein also includes a KRT17 variant having a functionally equivalent activity to the KRT17 protein represented by SEQ ID NO: 1 and a KRT17 ortholog of another species. Specifically, one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 have been deleted, substituted or added, or 80% or more, 90% of the amino acid sequence represented by SEQ ID NO: 1 Thus, KRT17 proteins having 95% or more, 97% or more, 98% or more or 99% or more amino acid identity are included.
  • “several” means, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4 or 2 to 3.
  • Conservative amino acid substitution is desirable for amino acid substitution.
  • “Conservative amino acid substitution” refers to a substitution between amino acids having similar properties such as charge, side chain, polarity and aromaticity.
  • Amino acids with similar properties include, 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), apolar.
  • 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.
  • amino acid identity refers to when the two amino acid sequences are aligned (aligned) and a gap is introduced, if necessary, so that the amino acid coincidences between the two are the highest.
  • Amino acid identity can be calculated using a protein search system based on BLAST or FASTA. For details on the method for determining identity, see, for example, Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al, J. Mol. Biol. 215, 403-410, 1990. ..
  • KRT17 gene is a gene encoding the KRT17 protein.
  • a specific example of the KRT17 gene is a human KRT17 gene encoding a human KRT17 protein containing the amino acid sequence represented by SEQ ID NO: 1. More specifically, the KRT17 gene is a gene containing or consisting of the nucleotide sequence represented by SEQ ID NO:2.
  • the KRT17 gene also includes a KRT17 variant that has an activity that is functionally equivalent to the KRT17 protein encoded by the KRT17 gene shown in SEQ ID NO: 2 and the KRT17 gene encoding a KRT17 ortholog of another species. Specifically, one or several bases in the base sequence represented by SEQ ID NO: 2 have been deleted, substituted or added, or 80% or more, 90% relative to the base sequence represented by SEQ ID NO: 2. As mentioned above, the KRT17 gene having a base identity of 95% or more, 97% or more, 98% or more or 99% or more is included.
  • nucleotide sequence that hybridizes under high stringent conditions with a nucleic acid fragment containing a part of the nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 2, and is functionally equivalent to the KRT17 protein.
  • a gene encoding a protein having activity is included.
  • base identity means that when two base sequences are aligned (aligned) and a gap is introduced as necessary so that the base coincidence between the two becomes the highest, The ratio (%) of the same bases between two base sequences to all the bases of the KRT17 gene including the base sequence shown in 2.
  • hybridize under high stringent conditions means that hybridization and washing are performed under conditions of low salt concentration and/or high temperature.
  • the conditions for high stringent hybridization are described in Green, MR and Sambrook, J., 2012,Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Laboratory Laboratory Press, Cold Spring Harbor, New York. Can be used as a reference.
  • the nucleotide sequence information of the KRT17 gene can be searched from public databases (GenBank, EMBL, DDBJ). For example, based on the known base sequence information of the KRT17 gene shown in SEQ ID NO: 2, a gene with high base identity can be searched and obtained from a database.
  • the transcription product of the KRT17 gene means KRT17 mRNA.
  • the mRNA may be a mRNA precursor (pre-mRNA) or a mature mRNA (mature mRNA).
  • pre-mRNA mRNA precursor
  • mature mRNA mature mRNA
  • the transcript of the KRT17 gene that is the biomarker of the present invention may be KRT17 mature mRNA.
  • the "peptide fragment” is a peptide fragment containing or consisting of a part of the amino acid sequence constituting the KRT17 protein, and the amino acid sequence constituting the fragment was identified as a fragment of the KRT17 protein. What can be done.
  • a “peptide fragment” may be 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more consecutive amino acid acid residues in the full-length amino acid sequence of KRT17 protein, and , 200 or less, 150 or less, 120 or less, 100 or less, or 80 or less contiguous amino acid residues.
  • a "peptide fragment” may be a peptide consisting of 10 to 200, 30 to 120, 50 to 80 consecutive amino acid residues.
  • nucleic acid fragment refers to a nucleic acid fragment containing or consisting of a part of the base sequence that constitutes KRT17 mRNA, which is identified as a KRT17 mRNA fragment from the base sequence that constitutes the fragment. What can be done.
  • a "nucleic acid fragment” may be a nucleic acid consisting of 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more contiguous bases in the full-length base sequence of KRT17 mRNA. It may be a nucleic acid consisting of 600 or less, 450 or less, 300 or less, 200 or less, or 100 or less consecutive bases.
  • the “nucleic acid fragment” may be a nucleic acid consisting of 30 to 600, 50 to 300, or 70 to 100 contiguous bases in the full-length base sequence of KRT17 mRNA.
  • the present invention relates to the use of the above-described biomarker according to the present invention for predicting the prognosis of colorectal cancer patients.
  • the present invention relates to a method for predicting the prognosis of colorectal cancer patients.
  • This method in a colon cancer cell or tissue obtained from a colon cancer patient, a measurement step of measuring the amount of the biomarker according to the present invention described above, and the measurement result obtained in the measurement step, the colon It includes a prediction step of predicting the prognosis of cancer patients.
  • the measurement step can be performed in vitro.
  • stage TNM classification of the International Union for Cancer (UICC) (UICC 7th Edition; Sobin LH, Gospodarowicz MK, Wittekind C, International Union against Cancer.TNM classification of malignant tumours, 7th edn Wiley-Blackwell (2010)).
  • UICC-TNM classification International Union for Cancer
  • the progression of cancer lesions is classified according to three factors: depth of wall penetration (T classification), lymph node metastasis (N classification) and distant metastasis (M classification).
  • T classification depth of wall penetration
  • N classification lymph node metastasis
  • M classification distant metastasis
  • the stage determination based on the UICC-TNM classification can be performed according to the ordinary knowledge of those skilled in the art.
  • stage I when colon cancer stays in the submucosa (T1) or proper muscle layer (T2)
  • colon cancer is proper muscle.
  • Stage II is judged when it penetrates the subserosal layer (T3) or the serosa or adjacent organs (T4). Note that none of stage 0 to II is accompanied by lymph node metastasis (N0). If lymph node metastasis is involved (N1 to 2), it is judged to be stage III. Stages 0 to III are all cancers without distant metastasis (M0), and cancer with distant metastasis (M1) is judged as stage IV.
  • the colorectal cancer afflicted by the patient targeted by the present invention may be stage I to IV, for example stage II colorectal cancer.
  • stage II colorectal cancer Approximately 15% to 20% of patients with stage II colorectal cancer have recurrence after surgery, but the prognosis does not improve even if adjuvant chemotherapy is applied to all patients, and the conventional choice is whether adjuvant chemotherapy is used or not. There was no clear standard in.
  • INDUSTRIAL APPLICABILITY The prognostic marker of the present invention can be useful for management of colorectal cancer patients based on prognosis, such as selection and determination of whether or not to perform adjuvant chemotherapy.
  • the colorectal cancer patient in the present invention is preferably a mammal, more preferably a primate, and most preferably a human.
  • the colorectal cancer cell or tissue used in the present invention is not particularly limited, but can be obtained from a colorectal cancer patient by, for example, biopsy or excision surgery.
  • the cell or tissue may be used as it is for the measurement of the biomarker, but may be appropriately pretreated for the measurement.
  • paraffin-embedded sections may be prepared from a patient-derived sample.
  • a protein extract or mRNA extract may be prepared from a patient-derived sample.
  • the biomarker to be measured by this method may be either the KRT17 protein or its peptide fragment, the transcription product of the KRT17 gene or its nucleic acid fragment.
  • the measurement of the amount (expression amount) includes measuring the presence or absence of expression, the magnitude of the expression amount or the expression concentration, and the like.
  • the term “measurement” includes any of detection, qualitative, quantitative and semi-quantitative.
  • the measurement method may be any known protein quantification method and is not particularly limited, but examples include immunological detection methods.
  • the immunological detection method is a method of measuring the amount of a target molecule using an antibody or an antibody fragment that specifically binds to a target molecule that is an antigen.
  • the antibody can be derived from any animal, including mammals and birds. Examples thereof include mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens and humans.
  • 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 group of clones of a single immunoglobulin. 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 group of immunoglobulins that recognize and bind to different epitopes of the same antigen.
  • the polyclonal antibody can be obtained from the serum of an animal after immunizing the animal with the target molecule as an antigen.
  • immunoglobulin molecules are known to have IgG, IgM, IgA, IgE, and IgD classes, but the antibody of the present invention may be of any class. , For example IgG.
  • the method for producing a polyclonal antibody that recognizes and binds to the KRT17 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 KRT17 protein or a fragment thereof as an antigen. Antibodies may also be obtained from the manufacturer.
  • antibody fragment refers to a partial fragment of a polyclonal antibody or a monoclonal antibody, and refers to a polypeptide chain having an activity substantially equivalent to the antigen-specific binding activity of the antibody or a complex thereof. ..
  • an antibody portion including at least one antigen-binding site that is, a polypeptide chain having at least one pair of VL and VH, or a complex thereof is applicable.
  • Specific examples include a number of well characterized antibody fragments generated by cleaving immunoglobulins with various peptidases. More specific examples include Fab, F(ab') 2 and Fab'. All of these antibody fragments include an antigen-binding site and have the ability to specifically bind to a target molecule that is an antigen.
  • immunological detection method examples include immunohistochemical staining method, enzyme immunoassay method (including ELISA method and EIA method), western blotting method, radioimmunoassay (RIA) method, immunoprecipitation method, or flow cytometry.
  • enzyme immunoassay method including ELISA method and EIA method
  • western blotting method examples include western blotting method, radioimmunoassay (RIA) method, immunoprecipitation method, or flow cytometry.
  • RIA radioimmunoassay
  • immunohistochemical staining method a known method can be adopted. For example, a sample derived from a patient may be fixed with formalin, embedded in paraffin, sliced into tissue pieces, and attached to a slide glass to be used as a section sample. The section sample may be optionally heat-treated to activate the antigen, and then immunohistochemical staining may be performed on the section sample using a commercially available detection system such as Dako EnVision+System (Agilent).
  • the measurement method may be a known nucleic acid quantification method, and is not particularly limited, and for example, a nucleic acid amplification method using a primer or a probe. And a hybridization method using.
  • Nucleic acid amplification method refers to a method in which a specific region of a target nucleic acid is amplified by a nucleic acid polymerase using a forward/reverse primer set.
  • examples of the nucleic acid amplification method include a PCR (polymerase chain reaction) method such as an RT-PCR (reverse transcription polymerase chain reaction) method.
  • Hybridization method is a nucleic acid fragment having a base sequence complementary to all or part of the base sequence of the target nucleic acid to be detected as a probe, utilizing the nucleic acid and the base pairing between the probe, It is a method of detecting and quantifying a target nucleic acid or a fragment thereof.
  • the hybridization method several methods having different detection means are known, and examples thereof include a Northern hybridization method (Northern blot hybridization method), an in situ hybridization method, and a microarray method.
  • a nucleic acid chain such as a primer and a probe can be appropriately designed by a method known to those skilled in the art based on known biomarker sequence information, and can be obtained by a known production method such as chemical synthesis.
  • Each of the above-mentioned measurement methods is a technique known in the art. Therefore, a specific measuring method may be performed according to a known method. For example, the method described in Green, M.R. and Sambrook, J., 2012 (mentioned above) can be referred to.
  • this step includes determining from the measurement result obtained in the measurement step whether the colon cancer cell or tissue is positive or negative for the biomarker. If the colorectal cancer cells or tissues are negative for the biomarker, the prognosis for colorectal cancer patients may be predicted to be good. On the other hand, if the colorectal cancer cells or tissues are positive for the biomarker, the prognosis for colorectal cancer patients may be predicted to be poor.
  • an immunohistochemical staining method when one or more cells or cell clusters are stained, it can be determined as positive, and when the number of stained tumor cells does not exist, it can be determined as negative.
  • a certain ratio for example, 10%, 15%, or 20%
  • the number of tumor cells stained with respect to the total number of tumor cells is If the ratio is less than the predetermined ratio, it may be determined as negative.
  • the predicting step includes determining whether the expression level of the biomarker in the colon cancer cell or tissue obtained in the measuring step is higher or lower (for example, than a predetermined threshold value). If the expression level of the biomarker in colon cancer cells or tissues is lower than a predetermined threshold (e.g., statistically significantly lower), the colon (e.g., relative to a population having an expression level higher than the predetermined threshold) The prognosis for cancer patients can be expected to be good. On the other hand, if the expression level of the biomarker in colorectal cancer cells or tissues is higher than a predetermined threshold value (for example, statistically significantly higher), (for example, for a population having an expression level lower than the predetermined threshold value). The prognosis for colorectal cancer patients can be predicted to be poor.
  • a predetermined threshold value for example, statistically significantly lower
  • the predetermined threshold value may be a control amount measured in a control sample (control cells or tissues, such as control colon cells or colon tissues).
  • the control sample may be derived from a healthy individual (eg, healthy person), a benign colorectal adenoma patient, or a colorectal cancer patient (eg, stage II colorectal cancer patient).
  • the “healthy individual” refers to a healthy individual of the same species as the test individual who does not suffer from cancer.
  • the expression level in these individuals or the median value, average value, upper limit level, lower limit level, or value in a certain range of expression amounts in multiple individuals can be used as the predetermined threshold value.
  • the threshold value can be appropriately set according to the accuracy of prediction and the like, and can be determined by, for example, ROC (receiver operating characteristic curve) analysis.
  • “statistically significant” means that when the obtained value has a low risk rate (significance level), specifically, p ⁇ 0.05 (less than 5%), p ⁇ 0.01 (less than 1%). ) Or p ⁇ 0.001 (less than 0.1%).
  • a publicly known test method capable of determining the presence or absence of significance may be used as appropriate, and is not particularly limited. For example, Student's t test, multiple comparison test, log rank test can be used.
  • ⁇ poor prognosis'' is a poor clinical outcome (e.g., high recurrence risk or recurrence rate after excision surgery of colorectal cancer, disease (cancer) specific survival rate is low, Or the overall survival rate is low). If the prognosis is poor, recurrence-free survival or survival 5 years after resection surgery for colorectal cancer is ⁇ 90%, ⁇ 85%, ⁇ 80%, ⁇ 75%, ⁇ 70%, ⁇ 65%, or 60. Can be less than %.
  • survival rate means cumulative survival rate.
  • the survival rate may be a disease (cancer)-specific survival rate or an overall survival rate.
  • “good prognosis” means that the clinical outcome is good. With a good prognosis, recurrence-free survival or survival 5 years after resection surgery for colorectal cancer can be ⁇ 90% or ⁇ 95%.
  • the present invention it is possible to predict the prognosis of colorectal cancer patients, based on the results, determine the treatment policy (for example, the type of anticancer agent, dose, administration interval, etc.), or Intervals between tests for cancer recurrence and metastasis can be determined.
  • the treatment policy for example, the type of anticancer agent, dose, administration interval, etc.
  • the present invention when the prognosis of a colorectal cancer patient is predicted to be poor, an anticancer agent is administered to the patient in order to prevent the recurrence of colorectal cancer, improve the prognosis, or improve the survival rate. You may. Therefore, the present invention also comprises administering an anticancer agent to a colorectal cancer patient having a poor prognosis by the method of the present invention, preventing recurrence of colorectal cancer, or improving the prognosis, Alternatively, a method for improving survival rate is provided.
  • Anti-cancer agents include, but are not limited to, 5-fluorouracil (5-FU), capecitabine, oxaliplatin, irinotecan, bevacizumab, cetuximab, panitumumab and regorafenib.
  • the anticancer agents 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 kit for predicting the prognosis of colorectal cancer patients, which comprises a reagent for measuring the amount of the above-described biomarker according to the present invention.
  • a reagent for measuring the amount of biomarker include the above-mentioned antibody or antibody fragment, or probe or primer.
  • the kit includes known immunohistochemical staining, ELISA, Western blot, or RT-PCR reagents, such as labeling reagents, buffers, chromogenic substrates, secondary antibodies, blocking agents, and instruments and controls necessary for the test. Etc. may be further included.
  • IHC Validation Set 1 available for FFPE sections of stage II patients who had not undergone preoperative chemotherapy or radiation therapy and had surgical resection (R0) between 2000 and 2009 It was used.
  • IHC validation set 2 For further validation, of 141 patients with stage I-IV colorectal cancer who underwent surgery between 2010 and 2013, 44 stage II patients were used as IHC validation set 2.
  • Immunohistochemistry Suitable antibodies were identified using the Human Protein Atlas database (www.proteinatlas.org), where exhaustive antibody-based proteomics data are available (Noda M et al, supra, and Uhlen M. et al. , Science, 2017, vol.357).
  • Mouse monoclonal anti-KRT17 antibody (M7046, Cytokeratin17, [E3], Dako, Glostrup, Denmark) was used for KRT17 staining. 4 ⁇ m thick sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase was blocked with methanol containing 0.3% hydrogen peroxide.
  • the antigen was recovered by autoclaving for 5 minutes in 10 mM citrate buffer (105°C, pH 6.0).
  • Primary anti-KRT17 antibody was incubated overnight at 4°C in a 1:40 dilution of 10 mM phosphate buffered saline containing Tween 20 (Sigma-Aldrich), followed by horseradish peroxidase (HRP) conjugated anti-mouse polymer ( Envision + System-HRP, Dako, CA, USA), followed by incubation with diaminobenzidine (Dako). All sections were counterstained with Carratsch Hematoxylin. A normal mammary gland specimen was used as a positive control for KRT17 staining. Positive was defined when at least some tumor cells or tumor cell clusters were stained in the tumor specimen. Sections were evaluated independently by two investigators with no prior knowledge of clinical data.
  • MMR mismatch repair
  • KRT17 as an independent prognostic factor in stage II colorectal cancer
  • IHC validation sets 1 and 2 were combined to enhance the power of the test.
  • KRT17 expression was significantly correlated with RFS (HR 6.60; 95% CI 2.34-18.64; P ⁇ 0.001).
  • RFS Reliable and Low Latency Cox analysis

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Abstract

Selon un mode de réalisation, la présente invention aborde le problème consistant à fournir un biomarqueur pour réaliser un pronostic chez un patient atteint d'un cancer du côlon. Selon un mode de réalisation, la présente invention concerne un biomarqueur pour réaliser un pronostic chez un patient atteint d'un cancer du côlon, le biomarqueur étant formé à partir de : la protéine KRT17 ou un fragment peptidique de celle-ci; ou un produit de transcription du gène KRT17 ou un fragment d'acide nucléique de celui-ci.
PCT/JP2020/004474 2019-02-08 2020-02-06 Biomarqueur de pronostic du cancer du côlon WO2020162523A1 (fr)

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