WO2015115544A1 - Procede d'evaluation du risque de metastase ou de recurrence d'un cancer du colon - Google Patents

Procede d'evaluation du risque de metastase ou de recurrence d'un cancer du colon Download PDF

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WO2015115544A1
WO2015115544A1 PCT/JP2015/052521 JP2015052521W WO2015115544A1 WO 2015115544 A1 WO2015115544 A1 WO 2015115544A1 JP 2015052521 W JP2015052521 W JP 2015052521W WO 2015115544 A1 WO2015115544 A1 WO 2015115544A1
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seq
base
dna
nos
colorectal cancer
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Japanese (ja)
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博光 小見山
一博 坂本
淳司 奥澤
学 塩澤
信 赤池
洋平 宮城
敬 大津
林崎　良英
昌可 伊藤
英哉 川路
寛子 大宮
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学校法人順天堂
地方独立行政法人神奈川県立病院機構
国立研究開発法人理化学研究所
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Priority to JP2015560009A priority Critical patent/JPWO2015115544A1/ja
Publication of WO2015115544A1 publication Critical patent/WO2015115544A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates to a method for evaluating the risk of colorectal cancer metastasis or recurrence at the molecular level.
  • Colorectal cancer is a carcinoma that occurs in the large intestine (cecum, colon, rectum), and is referred to as cecal cancer, colon cancer, and rectal cancer according to the site. Colorectal cancer often metastasizes to the liver and surrounding lymph nodes, and the presence or absence of metastasis has an important effect on the prognosis of the life and greatly affects treatment options such as anticancer drugs and surgery. Therefore, colorectal cancer is finely classified by the classification method (TNM classification) based on the size of the tumor, the degree of invasion to surrounding tissues, and metastasis to lymph nodes and other organs, and the degree of progression is based on the results of the TNM classification. It is classified into stage 0 to stage 4.
  • TNM classification classification
  • stage 2 colorectal cancer treatment chemotherapy may not be added because the risk of recurrence is low. However, some cases then recur.
  • the risk of recurrence of stage 3 colorectal cancer in which metastasis to the surrounding lymph nodes is recognized is evaluated by the number and location of lymph nodes to which cancer cells have metastasized, and treatment based on the classification based on that is performed.
  • postoperative chemotherapy is recommended, and even in the same stage 3, an attempt has been made to perform different chemotherapy due to the difference in TNM classification, but prognosis does not recur even without chemotherapy.
  • Some patients do not want chemotherapy after surgery because they may be good. However, there were variations in the choice of treatment method, including some recurrence.
  • patients who have been classified into the same group and who received the same treatment may or may not relapse.
  • liver metastasis and lymph node metastasis have been difficult to identify minute liver metastasis and lymph node metastasis with the current diagnostic imaging technique in which liver metastasis and lymph node metastasis are finally diagnosed by histopathological diagnosis. Therefore, the degree of lymph node metastasis cannot be determined unless the histopathological diagnosis is performed during or after surgery.
  • RNA-seq Non-Patent Document 1
  • CAGE Cap Analysis Gene Expression: Non-Patent Document 2
  • the CAGE method is characterized in that the activity of the transcription start site can be comprehensively quantified by selecting a long capped RNA such as mRNA and sequencing the 5 'end randomly and in large quantities.
  • the present invention relates to providing a method for objectively and rapidly predicting the risk of colorectal cancer metastasis or recurrence with high accuracy.
  • the present inventors extract RNA from colon cancer tissues obtained by surgery, those with and without recurrence, those with and without liver metastasis, and those with and without lymph node metastasis, As a result of comprehensive analysis of the expression state in the vicinity of the transcription start region (Transcript Start Site; TSS) as sequence information using the CAGE analysis method, the expression level of DNA containing a specific transcription start region is significantly between the two groups. Differently, using this as an index, we found that “with recurrence” and “without recurrence”, “with liver metastasis” and “without liver metastasis”, or “with lymph node metastasis” and “without lymph node metastasis” .
  • TSS Transcript Start Site
  • the present invention relates to the following 1) to 3).
  • kit. 3 Use of one or more expression products of DNA containing a transcription initiation region as a marker for evaluating the risk of colorectal cancer metastasis or recurrence.
  • the presence or absence of recurrence risk can be determined quickly and accurately even in a patient without lymph node metastasis.
  • the presence or absence of metastasis or recurrence can be predicted, detailed follow-up and optimization of the number of follow-ups for patients who are expected to have metastasis or recurrence (high risk of recurrence) and appropriate treatment as needed Prognosis is expected to improve by implementing a method (such as chemotherapy).
  • a new classification method that can select an optimal treatment method with small variation in treatment effect can be established.
  • colorectal cancer means a carcinoma that occurs in the large intestine (cecum, colon, rectum).
  • metastasis means that colon cancer grows in lymph nodes and other organs
  • evaluation of metastasis means the presence or absence of metastasis or metastasis ability (colorectal cancer metastasizes to multiple organs and grows).
  • preferable examples include evaluating or measuring liver metastasis or lymph node metastasis.
  • risk of recurrence means the likelihood of recurrence.
  • Relapse refers to a case in which a malignant tumor reappears at the same site after the tumor has been removed from the living body for a certain period of time, and when tumor cells are separated from the primary lesion and carried to a distant tissue where they proliferate autonomously. Refers to cases. Whether or not recurrence occurs depends on the proliferative ability, viability, and migration ability of tumor cells.
  • the assessment of recurrence risk in the present invention is particularly suitable for assessing the recurrence risk of patients in stage 2 and stage 3, more preferably, the recurrence risk of patients who are in stage 2 and have not undergone postoperative chemotherapy, Suitable for assessing the risk of recurrence in patients at stage 3a or 3b who have or have not undergone postoperative chemotherapy.
  • the stage of progression shown in this specification means a stage according to the classification of the degree of progression of the Colorectal Cancer Handling Regulations 8th Edition (Colon Cancer Society (JSCCR)) or a stage corresponding thereto, and a stage corresponding thereto
  • JSCCR Cold Cancer Society
  • FIG. 1 shows the progression classification according to the 8th edition of the Colorectal Cancer Handling Regulations and the progression classification based on the UICC 7th edition
  • FIG. 2 shows a comparison table between the 8th edition of the Colorectal Cancer Handling Regulations and the TNM classification.
  • Stage 2 is the condition where the cancer has invaded beyond the intrinsic muscle layer of the large intestine to the lower serosa and the outside of the large intestine wall, but no lymph node metastasis or distant metastasis is seen
  • Stage 3 is a state where cancer has metastasized not only to the large intestine but also to lymph nodes, and stage 3 is further divided into a and b.
  • stage 3a cancer has metastasized not only to the large intestine but also to the pararectal lymph nodes or intermediate lymph nodes, but there are no more than 3 metastatic positive lymph nodes, and stage 3b has 4 or more. Or refers to those with metastasis to the main lymph nodes.
  • the biological sample used in the present invention is a colorectal cancer tissue separated from a colorectal cancer patient to be evaluated.
  • the biological sample is appropriately prepared and processed for use in measurement. For example, when the sample is subjected to measurement at the nucleic acid level, an RNA extract is prepared, and when the sample is subjected to measurement at the protein level, a protein extract is prepared.
  • RNA from a biological sample Any known method can be used as a method for extracting RNA from a biological sample. Specific examples include Ambion RiboPure kit manufactured by Life Technologies, miRNeasy manufactured by Qiagen, and RNeasy manufactured by Qiagen. Among these, the miRNeasy kit manufactured by Qiagen is preferably used.
  • nucleic acid or “polynucleotide” means DNA or RNA.
  • DNA includes not only double-stranded DNA but also single-stranded DNAs comprising a sense strand and an antisense strand constituting the DNA. Accordingly, the DNA includes double-stranded genomic DNA, single-stranded cDNA, single-stranded DNA having a sequence complementary to the DNA, and the like.
  • RNA includes any of total RNA, mRNA, rRNA, and synthetic RNA.
  • a transcription product of DNA consisting of the nucleotide sequence shown in SEQ ID NOs: 1 to 443 is 1) in stage 2 as shown in the Examples
  • specimens with recurrence and specimens with no recurrence 2 Colon in stage 3a or 3b without postoperative chemotherapy Specimens with and without recurrence in cancer patients (male / female), 3) Recurrence in colorectal cancer patients (male / female) who received postoperative chemotherapy at stage 3a or 3b Specimens with no recurrence 4)
  • stage 2 in the Examples
  • specimens with recurrence and specimens with no recurrence 2 Colon in stage 3a or 3b without postoperative chemotherapy Specimens with and without recur
  • RNA transcriptional activity of the specimens 1) to 5) is obtained from the clinical specimen-derived profile group obtained from “with recurrence” or “with metastasis”, “without recurrence” or “without metastasis”.
  • the R / Bioconductor edgeR package (Bioinformatics. 2010 Jan 1; 26 (1): 139-40) is used for the differential analysis between the clinical specimen-derived profile groups, and the threshold is set to 5% as a threshold value of FDR (false discovery rate). It has been extracted.
  • DNA consisting of a base at an arbitrary position (transcription start point) in the transcription start region in the base sequence represented by SEQ ID NOs: 1 to 443 and one or more bases downstream thereof (hereinafter referred to as “transcription in SEQ ID NOs: 1 to 443”).
  • the expression product (referred to as “the expression product of the present invention”) (referred to as “the DNA containing the starting point”) (or encoded thereby) can be a biomarker for assessing the risk of colorectal cancer metastasis or recurrence. . More details are as follows.
  • the DNA expression product is a marker that increases the expression level when the risk of recurrence is high.
  • the differential expression of the DNA is a marker that increases the expression level when the risk of recurrence is high.
  • a biomarker for assessing the risk of recurrence in colorectal cancer patients (female) who are in the expression product stage 3a or 3b of the DNA containing the transcription start point in SEQ ID NOs: 38 to 63 and have not undergone postoperative chemotherapy.
  • the DNA expression product including the transcription start point in SEQ ID NOs: 38 to 62 is a marker whose expression level increases when the recurrence risk is high
  • the DNA expression product including the transcription start point in SEQ ID NO: 63 is the recurrence risk. It is a marker that decreases the expression level when is high.
  • the DNA expression product is a marker that increases the expression level when the risk of recurrence is high.
  • the DNA expression product is a marker that increases the expression level when the risk of recurrence is high.
  • a biomarker for evaluating the risk of recurrence in colorectal cancer patients male who have undergone postoperative chemotherapy at the expression product stage 3a or 3b of the DNA containing the transcription start point in SEQ ID NOs: 218 to 253 .
  • the DNA expression product is a marker that increases the expression level when the risk of recurrence is high.
  • 4f) DNA expression product containing the transcription start site in SEQ ID NOs: 254 to 262 A biomarker for evaluating liver metastasis in colorectal cancer patients (female).
  • the expression product of the DNA is a marker that increases the expression level when the possibility of liver metastasis is high.
  • DNA expression product containing the transcription start site in SEQ ID NOS: 263 to 288 Biomarker for evaluating liver metastasis in colorectal cancer patients (male).
  • the DNA expression product containing the transcription start point in SEQ ID NOs: 263 to 287 is a marker whose expression level increases when the possibility of liver metastasis is high
  • the DNA expression product containing the transcription start point in SEQ ID NO: 288 Is a marker that decreases the expression level when the possibility of liver metastasis is high.
  • the expression product of the DNA is a marker that increases the expression level when the possibility of lymph node metastasis is high.
  • DNA expression product containing the transcription start site in SEQ ID NOs: 300 to 443 A biomarker for evaluating lymph node metastasis in colorectal cancer patients (male).
  • the DNA containing the transcription start point in SEQ ID NOs: 300 to 442 is a marker whose expression level increases when the possibility of lymph node metastasis is high
  • the expression product of the DNA containing the transcription start point in SEQ ID NO: 443 is lymphatic It is a marker that decreases the expression level when the possibility of node metastasis is high.
  • the “transfer start area” refers to an area including a transfer start point.
  • the transcription start point from a specific promoter is not limited to a single base, and may be a base present at a plurality of positions downstream of the promoter on the genome.
  • a region including a plurality of these transfer start points is referred to as a transfer start region in this specification. More specifically, the transfer start area is an area between a transfer start point located on the most 5 'side and a transfer start point located on the most 3' side among the plurality of transfer start points.
  • each of the base sequences represented by SEQ ID NOs: 1 to 443 the transcription start region is the 5 ′ end corresponding to the region defined at both ends by the base at position 1 (5 ′ end) and the 101st base from the 3 ′ end Is the base region that forms
  • each of the base sequences represented by SEQ ID NOs: 1 to 443 shows a transcription start region and 100 bases following the transcription start point located on the most 3 'side in the transcription start region.
  • such a transcription start region is also referred to as “a transcription start region shown in SEQ ID NOs: 1 to 443”.
  • Tables 1-2 The positions of the transcription start regions shown in SEQ ID NOs: 1 to 443 on the genome, and related gene information, etc. are shown in Tables 1-2, Tables 3-1 to 3-2, Tables 4-1 to 4-2, and Tables below. 5, Tables 6-1 to 6-7, Tables 7 to 9, and Tables 10-1 to 10-6.
  • the DNA for which the expression level of the expression product is measured is a base at an arbitrary position (transcription start point) in the transcription start region and one base downstream thereof in the base sequence represented by SEQ ID NOs: 1 to 443. It is DNA consisting of the above base sequence.
  • the number of bases in the downstream base sequence may be any number that can identify the expression product.
  • the number of bases include 1 base or more, 5 bases or more, 10 bases or more, 15 bases or more, 20 bases or more, 25 bases or more, 30 bases or more, 40 bases or more, 50 bases or more.
  • 10 bases or less, 15 bases or less, 20 bases or less, 25 bases or less, 30 bases or less, 40 bases or less, 50 bases or less, 100 bases or less are mentioned, for example.
  • the downstream base is not particularly necessary in the case of measurement by the CAGE method, but in the case of measurement by hybridization or PCR, any part up to about 100 bases downstream is targeted in order to ensure the accuracy.
  • the DNA has a length of at least 20 bases out of DNA consisting of the transcription start region and 100 bases downstream from it, there is a high probability that it can be identified even in an experimental system for the entire genome.
  • the DNA also includes DNA having a base sequence substantially identical to the base sequence of the DNA as long as the expression product can be a biomarker for evaluating the risk of colorectal cancer metastasis or recurrence.
  • Such expression products of the present invention can be used to assess the risk of colorectal cancer metastasis or recurrence by ascertaining the expression level by appropriately combining one or more of the expression products.
  • threshold values shown in Tables 11 to 15 below are set, DNAs that can be classified with specificity 100% and sensitivity 100%, that is, DNA that can be reliably discriminated with only one expression level, The following are mentioned.
  • 4f A DNA expression product containing the transcription start point in SEQ ID NO: 261, SEQ ID NO: 258, SEQ ID NO: 262, or SEQ ID NO: 260 in the evaluation of liver metastasis of a colon cancer patient (female).
  • 4m An expression product of DNA containing the transcription start point in SEQ ID NO: 283, SEQ ID NO: 284, or SEQ ID NO: 278 in the evaluation of liver metastasis of a colon cancer patient (male).
  • a suitable combination in the case where at least two DNA expression products are used that is, for all two combinations selected from the above transcription start regions, these expression levels are used as explanatory variables, and recurrence related to the transcription start region extraction sample.
  • a logistic regression model that estimates the presence or absence of metastasis is constructed, and the expression of DNA shown in Tables 16 to 20 below can be classified as 100% specificity and 100% sensitivity for both the transcription start region extraction sample and the verification sample. Each product is listed.
  • these can be used in combination of two sets or three sets or more as appropriate.
  • these in addition to the combination of the two DNAs, among the DNAs containing the transcription start sites in SEQ ID NOs: 1 to 443, they may be combined with an expression product of DNA other than those shown as the combination. DNA expression products consisting of any other base sequences may be combined within a range that can contribute to the evaluation.
  • the expression product of the present invention includes a transcription product and a translation product expressed from the DNA.
  • Specific examples of the transcription product include RNA generated by transcription from the DNA, preferably mRNA.
  • Specific examples of the translation product include a protein encoded by the RNA.
  • the target of the measurement or detection of the expression product is cDNA artificially synthesized from the RNA, DNA encoding the RNA, protein encoded by the RNA, molecule interacting with the protein, and interaction with the RNA. Also included are molecules that act or molecules that interact with the DNA.
  • molecules that interact with RNA, DNA or protein DNA, RNA, protein, polysaccharide, oligosaccharide, monosaccharide, lipid, fatty acid, and their phosphates, alkylated products, sugar adducts, and the like, and Any of the above-mentioned complexes can be mentioned.
  • the expression level comprehensively means the expression level and activity of the expression product.
  • RNA, cDNA or DNA is targeted as a method for measuring the expression level, nucleic acid amplification represented by PCR method, real-time RT-PCR method, SmartAmp method, LAMP method, etc. using DNA hybridizing to these primers Method, hybridization method using nucleic acids that hybridize to these as probes (DNA chip, DNA microarray, dot blot hybridization, slot blot hybridization, northern blot, hybridization, etc.), method for determining the base sequence, or these You can choose from a combination of methods.
  • the probe or primer used for the measurement that is, the primer for specifically recognizing and amplifying the expression product (transcription product) of the present invention or the nucleic acid derived therefrom, or the RNA or the nucleic acid derived therefrom is specific.
  • “specifically recognize” means that, for example, in the Northern blot method, substantially only the expression product (transcription product) of the present invention or a nucleic acid derived therefrom can be detected.
  • the detection product or product can be determined to be the transcription product or a nucleic acid derived therefrom so that only the nucleic acid is produced.
  • DNA comprising a base sequence represented by SEQ ID NOs: 1 to 443, or an oligonucleotide containing a certain number of nucleotides complementary to its complementary strand
  • the “complementary strand” refers to the other strand with respect to one strand of double-stranded DNA comprising A: T (U in the case of RNA) and G: C base pairs.
  • “complementary” is not limited to the case where the certain number of consecutive nucleotide regions are completely complementary sequences, preferably 80% or more, more preferably 90% or more, and still more preferably 95% or more. What is necessary is just to have the identity on arrangement
  • the identity of the base sequence can be determined by the algorithm such as BLAST.
  • oligonucleotide When such an oligonucleotide is used as a primer, it only needs to be capable of specific annealing and chain extension, and is usually 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more, and for example 100 bases or less. Those having a chain length of preferably 50 bases or less, more preferably 35 bases or less are mentioned. Further, when used as a probe, it only needs to be capable of specific hybridization, and has at least part or all of the sequence of DNA (or its complementary strand) consisting of the base sequence represented by SEQ ID NOs: 1 to 443, for example, A chain length of 10 bases or more, preferably 15 bases or more and, for example, 100 bases or less, preferably 50 bases or less, more preferably 25 bases or less is used.
  • oligonucleotide can be DNA or RNA, and can be synthesized or natural.
  • the probe used for hybridization is usually labeled.
  • the probe DNA is first labeled with a radioisotope, a fluorescent substance, etc., and then the obtained labeled DNA is transferred to a nylon membrane or the like according to a conventional method. Hybridize with RNA. Thereafter, a method of detecting and measuring a signal derived from the labeled product of the formed duplex of labeled DNA and RNA can be used.
  • cDNA is prepared from RNA derived from a biological sample according to a conventional method so that the target expression product of the present invention (in this case, transcription product) can be amplified.
  • a pair of prepared primers (a normal strand that binds to the cDNA ( ⁇ strand) and a reverse strand that binds to the + strand) are hybridized therewith.
  • PCR is performed according to a conventional method, and the obtained amplified double-stranded DNA is detected.
  • detection of the amplified double-stranded DNA use a method of detecting the labeled double-stranded DNA produced by performing the above PCR using a primer previously labeled with RI, a fluorescent substance, etc. Can do.
  • cDNA or DNA nucleic acid derived from the expression product of the present invention (in this case, a transcription product) is immobilized on a support.
  • cDNA or DNA nucleic acid derived from the expression product of the present invention
  • a transcription product a nucleic acid derived from the expression product of the present invention
  • an array labeled cDNA or cRNA prepared from mRNA is bound on the microarray, and the label on the microarray is detected, whereby the mRNA expression level can be measured.
  • the nucleic acid immobilized on the array may be any nucleic acid that hybridizes specifically (ie, substantially only to the target nucleic acid) under stringent conditions.
  • the expression product of the present invention transcription
  • the product may be a nucleic acid having the entire sequence or a partial sequence.
  • the “partial sequence” includes a nucleic acid consisting of at least 15 to 25 bases.
  • stringent conditions can usually include washing conditions of about “1 ⁇ SSC, 0.1% SDS, 37 ° C.”, and more stringent hybridization conditions include “0.5 ⁇ SSC, 0.1%.
  • a condition of “0.1 ⁇ SSC, 0.1% SDS, 65 ° C.” can be mentioned.
  • Hybridization conditions are described in J.JSambrook al, Molecular Cloning: lonA Laboratory Manual, Thrd Edition, Cold Spring Harbor Laboratory Press (2001).
  • examples of the method for determining the base sequence include the CAGE method, the TSS-seq method, the RNA-seq method, the DGE method, and the SAGE method, and the CAGE method is preferable.
  • a protein ie, translation product
  • DNA containing the transcription start site in SEQ ID NOs: 1 to 443, a molecule that interacts with the protein, a molecule that interacts with RNA, or a molecule that interacts with DNA is measured.
  • protein chip analysis, immunoassay (eg, ELISA, etc.), 1-hybrid method (PNAS 100, 12271-12276 (2003)) or 2-hybrid method (Biol. Reprod. 58, 302-311 (1998) )) Can be used and can be appropriately selected depending on the target.
  • an antibody against the expression product of the present invention in this case, a translation product
  • a biological sample an antibody against the expression product of the present invention
  • the polypeptide in the sample bound to the antibody is detected, and the level is detected.
  • This is done by measuring.
  • an antibody that binds to a primary antibody labeled with a radioisotope, a fluorescent substance, an enzyme, or the like as a secondary antibody is used. Labeling is performed, and signals derived from these labeling substances are measured with a radiation measuring instrument, a fluorescence detector, or the like.
  • the antibody against the translation product may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to a known method. Specifically, the polyclonal antibody is used to immunize non-human animals such as rabbits by using a protein expressed and purified in E. coli or the like according to a conventional method, or by synthesizing a partial polypeptide of the protein according to a conventional method, It can be obtained from the serum of the immunized animal according to a conventional method.
  • a monoclonal antibody is obtained by immunizing a non-human animal such as a mouse with a protein expressed and purified in Escherichia coli according to a conventional method or a partial polypeptide of the protein, and fusing the obtained spleen cells with myeloma cells. It can be obtained from the prepared hybridoma cells.
  • the expression level of the expression product of the present invention in a biological sample derived from a cancer tissue separated from a colorectal cancer patient is measured, and the risk of colorectal cancer metastasis or recurrence is evaluated based on the expression level. Is done. Specifically, the expression level of the detected expression product of the present invention is evaluated by comparing it with a control level.
  • control level refers to, for example, the expression level of the expression product in a cancer tissue isolated from a colon cancer patient without metastasis or recurrence, or a normal tissue isolated from a colon cancer patient, or Expression level of the expression product in a group of healthy people who have not developed cancer.
  • the expression level of the expression product in the cancer tissue of the target patient is close to the expression level in a cancer tissue, normal tissue, or tissue derived from a healthy person isolated from a colon cancer patient who does not metastasize or recur. If it falls within the level range, or is significantly higher (or lower) than the expression level, the colorectal cancer of the patient can be evaluated as having no metastasis, low metastatic potential, or low risk of recurrence.
  • the risk of colorectal cancer metastasis or recurrence in the present invention can be evaluated by increasing / decreasing the expression level of the expression product of the present invention.
  • a control level for example, a standard value (threshold level) based on the expression level of the expression product derived from a normal tissue, a cancer tissue isolated from a colon cancer patient without metastasis or recurrence, or a healthy tissue.
  • a standard value for example, a range of ⁇ 2SD is set as an allowable range.
  • the colorectal cancer of the patient can be evaluated as having no metastasis, low metastatic potential, or low recurrence risk. .
  • the risk of colorectal cancer metastasis or recurrence is evaluated based on the provided information in combination with other methods (CT, MRI, PET-CT, etc.) as necessary. If it is determined that there is a possibility of metastasis or recurrence, for example, chemotherapy can be performed. On the other hand, when it is determined that the possibility of metastasis or recurrence is low, it is considered unnecessary to perform these treatments.
  • the test kit for evaluating the risk of metastasis or recurrence of colorectal cancer of the present invention contains a test reagent for measuring the expression level of the expression product of the present invention in a biological sample separated from a patient.
  • a test reagent for nucleic acid amplification or hybridization containing an oligonucleotide that specifically binds (hybridizes) to the expression product (transcription product) or the like of the present invention, or the expression product (translation product) of the present invention.
  • Oligonucleotides, antibodies and the like included in the kit can be obtained by known methods as described above.
  • the test kit can contain a labeling reagent, a buffer solution, a chromogenic substrate, a secondary antibody, a blocking agent, instruments and controls necessary for the test, and the like.
  • Example 1 Transcription for distinguishing “with recurrence” and “without recurrence”, “with liver metastasis” and “without liver metastasis”, or “with lymph node metastasis” and “without lymph node metastasis” in colorectal cancer Extraction and verification of starting area (1) Obtaining specimen samples After surgical removal of the large intestine including colorectal cancer, a portion of the cancer tissue was cut out and immediately placed in a microtube and frozen in liquid nitrogen. It was stored in an ultra-low temperature chamber of degree C and used appropriately for analysis. The samples used are as follows.
  • ⁇ 1f Colorectal cancer patients who are advanced stage 2 women who have not undergone postoperative chemotherapy> ⁇ Transcription start region extraction ⁇ Evaluation sample 21 specimens (no recurrence: 19 specimens, recurrence: 2 specimens) ⁇ 1m: Colorectal cancer patients who are advanced stage 2 men who have not undergone postoperative chemotherapy> ⁇ Transcription start region extraction / 40 samples for evaluation (no recurrence: 33 specimens, recurrence: 7 specimens) ⁇ 2f: Colorectal cancer patients who are advanced stage 3a or 3b women who have not undergone postoperative chemotherapy> ⁇ Sample 6 for extracting transcription start region (no recurrence: 4 specimens, recurrence: 2 specimens) ⁇ Verification sample 3 specimens (no recurrence: 2 specimens, recurrence: 1 specimen) ⁇ 2m: Colorectal cancer patients who are men with advanced stage 3a or 3b and who have not undergone postoperative chemotherapy> -E
  • tissue pieces were appropriately frozen and stored at -80 ° C.
  • the preserved tissue piece is placed in a 2 mL microtube so that the tissue piece is 50 mg or less, QIAzol manufactured by Qiagen is added, and one zirconia bead is sealed and crushed by osmosis treatment using TissueLyser manufactured by Qiagen. did.
  • RNA was prepared for RNA according to the attached protocol using miRNeasy mini kit manufactured by Qiagen.
  • the prepared RNA was measured for ultraviolet absorption (230, 260, 280 nm) with a spectrophotometer to calculate 260/230, 260/280 ratios, and the quality of the RNA was tested.
  • electrophoresis was performed using BioAnalyzer RNA nano chip manufactured by Agilent, and the RIN value indicating the degree of RNA degradation was calculated to test the degree of RNA degradation.
  • CAGE library preparation 5 ⁇ g of purified RNA was prepared, and unamplified untagged CAGE method (advanced method for cell engineering, next-generation sequencer purpose), Juno Kanno, Satoshi Suzuki, Gakken Medical Shujunsha, 2012 09 “Chapter 3”, “Chapter 3“ Exhaustive promoter analysis (non-amplified CAGE method using Illumina sequencer) ”” was prepared).
  • the purified RNA was subjected to reverse transcription reaction and purified, and then aldehyde was formed by participation of a ribose diol with sodium periodate, and biotin hydrazide was added to add biotin to the aldehyde group.
  • RNA / cDNA double strand biotinylated with avidin magnetic beads was bound to the bead surface, and the cDNA was released and recovered by RNase H digestion and heat treatment.
  • sequencing was performed using HiSeq 2500 manufactured by Illumina.
  • the standard conditions of AMPure XP (manufactured by Beckman Coulter) used for purification, buffer replacement, etc. in this step are conditions for recovering nucleic acids having a length of 100 bases or more in the case of double strands.
  • the CAGE library produced by this process using this is composed of double-stranded DNA having a chain length of 100 bases or more.
  • Example 2 Surgical specimens other than those used in Example 1, 55 females (26 specimens with lymph node metastasis, 29 specimens without lymph node metastasis), 91 male specimens (38 specimens with lymph node metastasis, 53 no lymph node metastasis) Using each of the specimens, a CAGE library was prepared in the same manner as in Example 1, and transcription initiation regions having different activities were extracted between the group with lymph node metastasis and the group without lymph node metastasis.
  • the transcriptional activity level of the transcription initiation region represented by SEQ ID NO: 290 for females is SEQ ID NOS: 304, 309, 311, 317, 320, 343, 347, 351, 369, 372, 396, 400, 405, for males. It was confirmed that the transcriptional activity levels of the transcription initiation regions indicated by 426 and 441 differed significantly between the groups with and without metastasis (Tables 21 and 22).
  • the present invention it is possible to objectively determine or predict the risk of colorectal cancer metastasis or recurrence by examining the primary focus of colorectal cancer collected before or during surgery. This can be expected to contribute to the medical field and clinical laboratory field.

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Abstract

L'invention concerne un procédé permettant de prédire objectivement et rapidement avec une grande précision le risque de métastase ou de récurrence d'un cancer du côlon. Ce procédé comporte l'étape consistant à mesurer les taux d'expression d'un ou de plusieurs types de produits d'expression d'ADN incluant des régions d'initiation de la transcription, dans des échantillons biologiques provenant d'un tissu cancéreux prélevé sur des patients atteints d'un cancer du côlon, et permet d'évaluer le risque de métastase ou de récurrence du cancer du côlon chez ces patients.
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WO2019241899A1 (fr) * 2018-06-20 2019-12-26 Pontificia Universidad Catolica De Chile Détection non invasive du cancer gastrique par l'intermédiaire de la détection dans le sang de la méthylation du gène reprimo

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017047102A1 (fr) * 2015-09-16 2017-03-23 Riken Biomarqueur pour le cancer et son utilisation
WO2019241899A1 (fr) * 2018-06-20 2019-12-26 Pontificia Universidad Catolica De Chile Détection non invasive du cancer gastrique par l'intermédiaire de la détection dans le sang de la méthylation du gène reprimo
US11746387B2 (en) 2018-06-20 2023-09-05 Pontificia Universidad Catolica De Chile Non-invasive detection of gastric cancer by detecting the methylation of Reprimo-like in the blood

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